
United States
Environmental Protection Agency
   Office of Chemical Safety
   and Pollution Prevention
   (7101)
EPA 712-C-010
January 2012


Ecological Effects
Test Guidelines



OCSPP 850.4230:

Early Seedling Growth Toxicity Test




                                       
                                       
                                       
                                       
                                       
                                       
                                       
                                       
                                       
                                       
  
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  NOTICE
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  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.
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  	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
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  	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 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 EC10 and EC50 values for seedling survival and IC10 and IC50 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), (j)(7) and (j)(10) 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 EC50 and EC10 values (with 95% confidence intervals and standard errors) for seedling survival; and the IC50 and IC10 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 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 EC10 and EC90 (or IC10 and IC90)) 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 EC10 (seedling survival) and IC10 (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 EC10, EC50, IC10 and IC50 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. IC10 >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)(1) of this guideline).  At a minimum ten plant species are tested, and 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

Family
Species
Common name
Dicots
Solanaceae
Lycopersicon esculentum or Solanum lycopersicum
Tomato

Cucurbitaceae
Cucumis sativus
Cucumber

Asteraceae
Lactuca sativa
Lettuce

Fabaceae[1]
Glycine max
Soybean

Brassicaceae
Brassica oleracea
Cabbage

Apiaceae
Daucus carota
Carrot
Monocots
Poaceae
Avena sativa
Oat

Poaceae
Lolium perenne
Perennial ryegrass

Poaceae
Zea mays
Corn

Liliaceae
Allium cepa
Onion
[1] Inoculation with Rhizobium japonicum is unnecessary.
                  (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
Species
Common name
Chenopodiaceae
Beta vulgaris
Sugar beet or table beet
Asteraceae
Helianthus annuus
Sunflower
Fabaceae
Pisum sativum
Pea
Brassicaceae
Brassica rapa
Field mustard, Canola
Brassicaceae
Brassica napus
Rape, Turnip
Fabaceae
Phaseolus vulgaris
Garden bean
Malvaceae
Gossypium spp.
Cotton
Polygonaceae
Fagopyrum esculentum
Buckwheat
            (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 ([o]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 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)(1) and (e)(4)(i)(A)(2) 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)(3) 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 arabic, 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 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)(1) of this guideline, by dusting the plants, as described in paragraph (e)(4)(i)(B)(2) of this guideline or by exposing the plants to gas in a fumigation chamber, as described in paragraph (e)(4)(i)(B)(3) 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)(2) 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 (um) 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 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., EC50, EC10), 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 EC10 value for seedling survival and the IC10 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 (EC10 or IC10).  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 EC10 or IC10 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).
      (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. 
            (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 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.  Dichromate 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 [o]C while temperature during the night should be 20 +- 3 [o]C.  Temperature in greenhouses is more difficult to control.  Under these circumstances, temperature during the day should be 25 +- 6 [o]C while temperature during the night should be 20 +- 6 [o]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 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 (umol/m[2]/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/m[3])) 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.  
                  (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.
            
            (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 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 [o]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.  
      	(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. 
		Equation 1
      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 EC10 and the length and weight IC10 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 IC10 or EC10, compared to the controls, is operationally defined as a 10% reduction from the control sample mean (Equation 2).  The null hypothesis (H0) stated in terms of true population parameters is that the difference of the "limit" treatment mean response (ulimit) from the control mean response (ucontrol) is greater than or equal to a 10 reduction, compared to the control (i.e., H0: ucontrol - ulimit  > δ0).  The alternative hypothesis (HA) is that this difference is less than a 10% reduction, compared to the control (HA: ucontrol - ulimit < δ0).  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).
		Equation 2
		where:
            δ0 = difference between two parameters, defined in this case as a p% reduction from the control sample mean;
             = 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 EC10 and IC10.
	(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 EC10 and EC50 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 IC10 and IC50 values for each of shoot, root and entire plant length and weight at test termination (see references in paragraph (j)(4) and (j)(11) of this guideline).  Standard error and 95% confidence interval (CI) values for the calculated IC 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.

	Table 3. -- Summary of Test Conditions for Early Seedling Growth Toxicity Test[1]
Test duration
At least 14 days after 50% of the control plants have germinated
Substrate
Quartz sand or glass beads.  Hydroponic system may also be used.
Nutrients
Watered with nutrient solution (e.g., half-strength modified Hoagland's medium)
Temperature 
25/20 [o]C (day/night) +- 3 [o]C
Relative humidity
70/90% (day/night) +- 5%
Carbon dioxide
350 +- 50 ppm at the top of the canopy
Light quality
Fluorescent or representative of natural sunlight
Light intensity
350 +- 50 umol/m[2]/sec at the top of the canopy
Photoperiod
16 hours light: 8 hours dark
Watering
Bottom watering as needed, using nutrient solution
Test chamber (pot) size
Varies with plant species selected.  Six-inch diameter plastic pots are typical. 
Number of organisms per test chamber
8 - 10 seedlings of one species per pot
Number of replicate chambers per test treatment
4  -  5
Number of organisms per test treatment
40 (minimum) 
Test treatment levels
Unless performing limit test, minimum of 5 treatment levels plus appropriate controls
Test substance application method
Root exposure (via watering with nutrient solution or sorbed onto the substrate) or foliar exposure (via spray, dust or gas)
Measures of effect (measurement endpoints)
EC10, EC50, NOEC and LOEC based upon survival
IC10, IC50, NOEC and LOEC for each of roots, shoots and entire plant length
IC10, IC50, NOEC and LOEC for each of roots, shoots and entire plant weight
Visual phytotoxicity reported 
1 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).
	Table 4. -- Test validity elements for the early seedling growth toxicity test
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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.
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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.

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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)(1) 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.
            
      (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.
            (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.
      	(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 EC10 and EC50 values for survival and IC10 and IC50 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 D.J. Versteeg, 1992. A statistical procedure for modeling continuous toxicity data. Environmental Toxicology and Chemistry 11:1485-1494.
	(5) Downs, R.J. and Helmers, H., 1975.  Environment and Environmental Control of Plant Growth. Academic Press, NY. 
	(6) Gulley, D.D. et al, 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.
	(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.
