
United States
Environmental Protection Agency
   Office of Chemical Safety
   and Pollution Prevention
   (7101)
EPA 712-C-002
January 2012


Ecological Effects
Test Guidelines



OCSPP 850.4800:

Plant Uptake and Translocation 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 850.4800: Plant uptake and translocation 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 material used in developing this harmonized OCSPP test guideline is the OPPT guideline under 40 CFR 797.2850 Plant Uptake and Translocation Test.
(b) Purpose.  This guideline is intended for use in developing data on the uptake and translocation, by terrestrial plants, of chemical substances and mixtures ("test chemicals" or "test substances") subject to environmental effects test regulations.  This guideline describes tests using commercially important terrestrial plants to develop data on the quantity of test substances incorporated in plant tissues and the potential for entry into food chains with resultant indirect human and/or wildlife exposure.  The test design is a composite of prior plant experiments in which the uptake of heavy metals, pesticides, or organic chemicals has been investigated (see paragraphs (j)(2) to (j)(12) of this guideline).  Test substances of concern may be transported to various sites as gases or dust, solubilized in preparation or in irrigation water, or may be encountered in soils.  It is well known that plants readily take up, translocate, accumulate, and metabolize chemicals that are non-essential for plant growth and development.  Such uptake and incorporation often represents the first step in the transport of these substances within terrestrial food webs.  The uptake and translocation test addresses this concern of entry into food webs.  This test is not concerned with toxicity to plants; it deals with hazard to animals and the potential for indirect human exposure.  The Environmental Protection Agency will use data from this test to assess the hazard or risks a test substance may present in the terrestrial environment.  This guideline should be used in conjunction with the OCSPP 850.4000 guideline, Background and special considerations for conducting ecological effects tests with terrestrial and aquatic plants, cyanobacteria, and terrestrial soil core microcosms.
(c) Definitions.  The definitions in the OCSPP 850.4000 guideline apply to this test guideline.  In addition, the following more specific definitions also apply:
	Mass balance refers to a quantitative accounting of the distributions of test substance in plant components, support media, and test solutions.  For gas fumigation with the test substance, it also refers to a quantitative determination of uptake as the difference between the quantity of gas entering an exposure chamber, the quantity leaving the chamber, and the quantity adsorbed to the chamber walls.
	Translocation is the transference or transport of chemical from the site of uptake to other plant components.
	Uptake refers to a process by which substances are transferred into a terrestrial plant.

(d) General considerations--
      (1) Summary of the test.  In preparation for the test, seeds are planted in 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 surface to 1 to 3 plants per pot, depending on the species, and discarding the pinched off seedlings.  Potting mixtures of sand, glass beads or soil are subirrigated with nutrient solution.  The test substance is applied to the plants via nutrient solution or adsorbed to the support media to produce a root exposure scenario; or applied to the plants by either spraying or dusting the foliage or exposing the plants to gas in a fumigation chamber to produce a foliar exposure scenario.  An alternative root exposure scenario involves application of the test substance to soil after the seeds have been planted but prior to germination.  Plants are then harvested when there is either adequate material for analysis or until fruit or seeds are mature.  If plants are fumigated, either rates of uptake and surface adsorption should be calculated, or the plants may be harvested and analyzed for test chemical and residues.  The primary endpoints for the plant uptake and translocation test are the concentrations of free parent test substance, metabolites and soluble residues, and bound residues in pooled plant organs and pooled whole plants.  For gaseous test substances, in addition to or in lieu of these measurements, calculated measurements of photosynthesis, transpiration, and stomatal conductance before, during, and after exposure to the test substance are made.
      (2) General test guidance.  The general guidance in the OCSPP 850.4000 guideline applies to this test guideline, except as specifically noted herein.
      (3) Range-finding test.  A range-finding test is usually conducted to establish the appropriate test concentrations to be used for the definitive test.
            (i) Because of the different mechanisms involved in root and leaf uptake, and to more closely define the chemical concentrations to be used in the uptake test, the definitive early seedling growth test (see the OCSPP 850.4230 guideline) is recommended as the range-finding test for tests with industrial chemicals.  The exposure technique used in the early seedling growth test (root or foliar exposure) should reflect the anticipated fate of the test substance.  The concentration selected as the upper limit for the uptake and translocation test should be near to but below the threshold of visible injury (e.g., NOEC).  Short exposure periods to gas in fumigation chambers are not expected to promote visible injury or gross reductions in growth but may alter stomatal resistance, transpiration, or photosynthesis.  Absorption and adsorption rates may be calculated and gas concentrations for definitive testing selected based on the calculated sorption rates.
            (ii) Alternatively, a seed germination or root elongation test or other appropriate phytotoxicity test (see paragraph (j)(1) of this) may be used to establish the appropriate upper concentration for testing.  For tests with pesticides, which are applied to surface soil, the seedling emergence and seedling growth test (see the OCSPP 850.4100 guideline) is recommended as the range-finding test.
      (4) Definitive test.  The purpose of the definitive uptake and translocation test is to determine the propensity for a test substance to accumulate in plants or plant parts.  The test substance should be added to the hydroponic or nutrient solution, coated on glass beads, or applied to the soil surface (for the root uptake test); or sprayed, dusted, or gassed directly on the foliage (for the foliage uptake test).  Two different plant species are used.  The test consists of one run for each of the two specified plant species.  For a particular chemical, a run is defined as exposure of the plant species to three concentrations of the test chemical with a minimum of six replicate pots per treatment level and appropriate controls.  Exposure is followed by extraction and analysis for parent compound, metabolites, and bound residues in plant tissues for solid and liquid test substances and by calculating these values and/or rates of absorption and adsorption for gas test substances.  Analytical confirmation of the test concentrations in exposure media is performed as described in the OCSPP 850.4000 guideline.  The elements of an acceptable or valid definitive test are given in Table 3 of this guideline.
(e) Test standards--
      (1) Test substance.  For industrial chemicals, the substance to be tested should be technical grade unless the test is designed to test a specific formulation, mixture, or end-use product.  For pesticides the substance to be tested is the typical end-use product (TEP).  If there is more than one TEP with the same inert substances, the one with the highest percent active ingredient (a.i.) and/or the one most commonly used should be tested.  If there is more than one TEP with different inert substances, a TEP representative of each different inert substance should be tested in the range-finding test and at a minimum the most sensitive one tested in the definitive test.  In addition, if an adjuvant is recommended for use on a TEP label, the adjuvant is added with the TEP at the label rate to constitute the test substance.  If a TEP is not available (i.e. new pesticide), the technical grade a.i. is used with a representative formulation, which should include any adjuvant that will be recommended for use on the label.  The OCSPP 850.4000 guideline lists the type of information that should be known about the test substance before testing, and discusses methods for preparation of test substances.
      (2) Test duration.  The duration of the exposure period, for solid and liquid test substances, should be equal to the length of time required for the particular test species to achieve sufficient biomass for analysis or until fruit or seeds are mature.  Carrot, lettuce, onion, cabbage, and ryegrass may be harvested whenever there is adequate plant material for chemical analysis.  Cucumber, corn, soybean, tomato, and oats should be grown until fruit or seed are mature.  The test duration for gaseous test substances should be similar, or may be reduced to the length of time required to make the specified gas exchange measurements if these measurements are used as the test endpoints.
	(3) Test organism--
            (i) Species.  
                  (A) The test is performed using two plant species of potentially differing sensitivity, such as a monocotyledonous and a dicotyledonous species.  It is further suggested that the test plants selected should be of different growth forms, e.g., a root crop and a leaf crop.  Plant species and groups recommended for testing are listed in Table 1.
                  (B) A listing of some additional plant taxa that have been used in toxicity tests can be found in the reference in paragraph (j)(1) of this guideline.  Use of these alternative species should be submitted to and reviewed by the Agency prior to conduct of the study.  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 1. -- Species and families acceptable for the plant uptake and translocation 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

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
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
            (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.
            (iii) Growth stage at test initiation.  
                  (A) Except for surface soil applications, newly germinated plants are used at test initiation for root (applied in nutrient solution or sorbed to support medium) and foliage exposures.  The plants should be exposed from seedlings through maturation when monitoring residues in fruit or seeds.
                  (B) Where the test substance is applied or deposited on the soil surface, the test is initiated using seeds which have been planted but have not yet germinated.
            (iii) Condition.  Healthy seeds should be used to generate seedlings.  Within a given test, all test organisms of a given species, including the controls, 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 from those used in the test and discarded.
            (iv) Care and handling.  
            (A) Seeds should be stored in a dessicator and refrigerated until needed.
                  (B) Pesticide treated seeds should be avoided.  The Agency should be consulted prior to test initiation if seed treatments other than a weak hypochlorite solution (recommended by Environment Canada), captan, or thiram are used.  Captan and thiram seed treatments are the only approved pesticide seed treatments (non-systemic with most other pesticides; see paragraph (j)(9) of this guideline).  Steam sterilization of soil is recommended as a non-pesticide alternative for killing pathogens, fungi, and insects in soil media.  When unapproved pesticide seed treatments are used in a study, the test should be designed to demonstrate no synergistic or antagonistic interactions occur between or among the seed treatment and test substance.
                  (C) 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 potting containers with sand or glass beads to within 2.5 centimeters (cm) of the top and to sow seeds directly on the support medium.
	(D) When soil is used, for pesticides, the recommended planting procedure is to fill potting container with soil medium (see the OCSPP 850.4100 or OCSPP 850.4150 guidelines) to within 2.5 cm of the top and sow seeds in the soil.
	(E) A sufficient number of seeds should be planted (e.g. 30) to provide more seedlings than called for in the test.  After 50 percent (50%) of the seeds have germinated, the seedlings should be thinned, by pinching the stem at the support surface, to the desired number (typically, one to four per pot, depending upon the species).  Any plants subsequently emerging are also pinched off.
	(F) Alternate planting methods may be required 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 length 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 inert, non phytotoxic, pliable putty should be used to seal the holes around the stems.  Control pots should be handled identically 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) Exposure pathway.  The choice of exposure technique is dependent upon the expected route of exposure of plants in the environment and the form of the test substance.
                  (A) Root exposure.  
                        (1) If the anticipated fate of the test substance is incorporation into soil or soil-water, and the mechanism of concern is root uptake, the test substance should be applied in nutrient solution to the root support media, or coated on sand or glass beads (for non-water-soluble test substances).
                        (2) For pesticides where the test substance is expected to be applied or deposited on the soil surface, the test substance should be applied to the soil surface prior to germination of seed but after the seeds have been planted.
                  (B) Foliar exposure.  With a test substance whose anticipated mode of exposure to plants is surface deposition by atmospheric transport or irrigation water, the appropriate testing method may be foliar application (accomplished by spraying, dusting, or fumigating the plants), allowing subsequent movement into the rooting zone with watering.
            (ii) Preparation of exposure treatment--
                  (A) Root exposure.  Except for test substances that are purposely applied to surface soil (such as pesticides), root exposure is accomplished by adding the test substance to the nutrient medium (or hydroponic solution) used to subirrigate the plants, as described in paragraphs (e)(4)(ii)(A)(1) and (e)(4)(ii)(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)(ii)(A)(3) of this guideline.  The test substance is repeatedly applied through the subirrigation scenario, while the test substance is only applied once when it is sorbed on the support medium.  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.  Methodology for surface soil applications for pesticides are described in paragraph (e)(4)(ii)(A)(4) of this guideline.
                        (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 850.1000 and OCSPP 850.4000 guidelines contain additional information on preparation of stock solutions.
                        (2) Test substances that are insoluble in water but which can be placed in aqueous suspension with 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 required 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 and tested simultaneously.  The OCSPP 850.4000 guideline 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.
                        (4) For the surface soil exposure scenario, the test substance is applied to the soil surface after planting the seeds and prior to germination.  For pesticides, this is typically accomplished using a properly calibrated sprayer after the seeds have been planted.  At test initiation, test substance is applied to surface soil immediately after the seeds have been planted using a properly calibrated sprayer.  The amount of water used in the spray as a carrier is equivalent to the recommendation on the pesticide label.  For example, vegetation within a given pot (test container) is sprayed with x milligrams of TEP mixed into 10 milliliters of water, where the 10 milliliters of water per the pot area is equivalent to the minimum number of gallons of water per acre specified on the label.  For a satisfactory test, all pots in all treatment levels and controls have the same equivalent volume of water applied (e.g., in the example just given this is 10 milliliters).  Additional information can be found in the OCSPP 850.4100 guideline.
                  (B) Foliar exposure.  Foliar exposure is accomplished by spraying the plants, as described in paragraph (e)(4)(ii)(B)(1) of this guideline by dusting the plants, as described in paragraph (e)(4)(ii)(B)(2) of this guideline or by exposing the plants to gas in a fumigation chamber, as described in paragraph (e)(4)(ii)(B)(3) of this guideline. The test substance is applied weekly via spraying or dusting, but only applied once when the fumigation scenario is used.
                        (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)(ii)(A)(1) 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 at weekly intervals.  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 as quantity per pot area (i.e., micrograms per square meter).  Shoots of control plants should also be sprayed, in an identical manner, with 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 leaves in single drops.
                        (2) Water-insoluble test substances, existing as solids, may be prepared for testing by grinding or other reduction methods to particles of smaller than 200 micrometers (um) in diameter.  These test substances should be applied at weekly intervals.  Plants should be placed in an exhaust hood, a plastic sleeve fitted over the top of the pot, and a specific quantity of test substance sprinkled uniformly over them.  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 substance 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 for use as needed or stored under pressure.  The bottled gas may be 100% pure 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 that required for chemical analysis.  Plants should be fumigated whenever they have reached sufficient size for measurement of photosynthesis and transpiration rates, assuming equivalent detection sensitivity of carbon dioxide, water vapor, and chemical analyzers.  The appropriate size is a function of the gas exchange system and constitutes an area of expert judgment.
            (iii) Treatment levels.  
                  (A) For this determination, at least three concentrations of the test substance, exclusive of controls, are tested.  Recommended concentrations would be a descending geometric progression from the upper concentration tested (i.e. 100, 50, 25 milligrams per liter (mg/L)).  The concentration selected as the upper limit for the uptake and translocation test should be near to but below the threshold of visible injury (e.g., NOEC).  Short exposure periods to gas in fumigation chambers are not expected to promote visible injury or gross reductions in growth but may alter stomatal resistance, transpiration, or photosynthesis.  Absorption and adsorption rates may be calculated and the upper limit for the uptake and translocation test for definitive testing selected based on the calculated sorption rates.
                  (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 pesticide treatments are used in the test for pest control, for a satisfactory test there should be a negative control and a control with only the treatments used for infestation purposes.
            (iv) Introduction of test organisms.  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 1-4 plants of similar size and condition, such that there is no crowding or competition among the plants at harvest or maturity.  Within a given test, all test organisms of a given species, including the controls, must be from the same source and seed lot.  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.
      (5) Controls.  
            (i) 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. 
            (ii) 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)(ii) 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 following minimum acceptable USDA seed germination (control) standards 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%), cabbage (75%), lettuce (55%), mustard -- all types (75%), soybean (75%), sugar beet (55%), small grains -- wheat, oats, barley, rice (80%), ryegrass (75%), and rape (75%).  Refer to regulation for additional vegetable crops.
      (6) Number of test organisms and replicates.  
            (i) For each species, the minimum number of replicates is six pots per concentration, with each pot containing from one to four seedlings.  If techniques other than radioisotopes are used to determine uptake, more replicates may be needed to provide sufficient plant materials for analysis.  The number of seedlings per replicate depends on the species tested, the size of the containers, and the size to which the plants will grow.  When plants are grown hydroponically, one plant per pot will probably be the preferred method.  The number of plants selected should provide sufficient biomass for analytical procedures.  A greater number of plants may be required depending on species tested, duration of test, and analytical procedures.  Too many plants in a container may actually reduce the growth and biomass; therefore, more replicate pots rather than increasing plants per pot may be required to obtain sufficient biomass.
            (ii) A randomized complete block design is recommended for this test, with blocks delineated within the growth chambers or over greenhouse benches and randomization of treatments occurring within the blocks.  If there exists inadequate space within chambers for blocking because of very large pots and plants, total randomization within chambers is acceptable.  This design is also appropriate for the growth of plants to be used for foliar exposure with gas.  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).
            (iii) 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) Greenhouse, environmental chamber, or growth room.  Greenhouses, environmental chambers, or growth rooms should provide adequate environmental controls to meet the carbon dioxide, humidity, irradiation, 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.  Environmental 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) the desired number of test plants for at least 28 days or up to maturity.  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.  Potting containers should be covered with opaque polyethylene bags to exclude light and minimize volatilization of test chemical.
            (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 use.  Dichromate solution should not be used for cleaning beads or pots.
                  (B) Quartz sand rooting medium, but not 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.  
                  (A) For tests with industrial chemicals, an artificial support medium, consisting of quartz sand or glass beads, is used.  Alternatively, a hydroponic system may be used, in which 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 root support medium in this test.  Additional information is found in the OCSPP 850.4320 guideline.
                  (B) For tests with pesticides, a natural soil (free of chemical contamination) or a synthetic soil may be used.  Additional information on natural and synthetic soil is found in the OCSPP 850.4100 and OCSPP 850.4150 guidelines.
            (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.  Hydroponic solutions should be aerated and sand or glass filled potting containers should be periodically filled with nutrient solution and drained to provide aeration.  For root exposure tests, the test substance should be added to the nutrient solution or directly to substrate.  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.  For tests with pesticides, any commonly recommended fertilizer can be added to the soil at recommended rates.
      (8) Environmental conditions.  Controlled environmental conditions are necessary 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 not as critical and 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.
            (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 micromoles per square meter per second (umol/m[2]/s), measured at the top of the canopy, is desirable.  Artificial lighting may be used to lengthen short-day periods or to supplement natural sunlight on overcast days.  A photoperiod of 16 hours light and 8 hours darkness, assuming an average wavelength of 400 to 700 nanometers (nm), should be used for all species except soybean which should be provided with 11 hours light and 13 hours darkness prior to flowering.
            (iv) Watering.  Nutrient media (containing test substance, if this is the selected application route) is used to subirrigate the plants, 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, for example 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 the one described in the OCSPP 850.4600 guideline.  For soil applications, top watering is used at the first watering after the test substance has been applied to initiate the capillary movement of water for bottom watering (subirrigation).
            (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.  Additionally, analytical confirmation of the amount of test substance in plant tissues is determined at test termination as discussed in (e)(9)(iv)(A) of this guideline.  Analytical methods, validated before test initiation, are used to measure the amount of test substance in a sample matrix, 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.  The amount of test substance to be applied to a replicate should be weighed prior to application and the weight recorded.  The sleeve placed around the pot during application should be weighed before and after application if there will be difficulties expected with test substance not landing in the pot or on the plants or remaining on the sleeve.
                  (E) Gas application.  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) 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.
                  (B) Carbon dioxide.  In environmental chambers, carbon dioxide measurements should be made at the top of the plant canopy, preferably on a continuous basis.
                  (C) Light intensity.  Light intensity should be determined daily at representative locations throughout the area in which the test plants are growing, and should be measured at the top of the foliage.  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 references given in the OCSPP 850.4000 guideline.
                  (D) Watering and evapotranspiration.  The amount of nutrient solution depleted each week should be recorded, to observe changes in evapotranspiration rates which may indicate stress.  Furthermore, these data will be used to compute chemical uptake per volume of water transpired for the uptake test.
                  (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) Appearance and condition.  Plants are observed on a weekly basis for any abnormalities and phytotoxic symptoms.  All abnormalities (visible effects including stunting of growth, discoloration, chlorosis and/or necrosis of the leaves, decreased moisture content, or morphological abnormalities, etc.) should be recorded.  Uniform scoring procedures should be used to evaluate any observable toxic response.  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.  Any lack of effects by the test substance should also be recorded.  Observations of plants should be made at least once every seven days.
            (iv) Uptake and translocation.  When sufficient plant biomass for analysis has been obtained, or when fruits or seeds are mature, the plants are harvested and the various plant organs are analyzed to establish the mass balance of the test substance.  Carrot, lettuce, onion, cabbage, and ryegrass may be harvested whenever there is adequate plant material for chemical analysis.  Cucumber, corn, soybean, tomato, and oats should be grown until fruit or seed are mature.  An alternative method can be used for the gaseous exposure system, where direct calculation of uptake rates can be made based upon measurements of photosynthesis, transpiration and stomatal conductance, obviating the need for chemical analysis of the plant tissues.
                  (A) Plant residue measurements--
                        (1) Preparation of plant material.  The entire plant should be harvested, rinsed with a minimum amount of reagent water (which is returned to the nutrient solution), and separated into its respective organs as follows: carrot -- root peels, peeled roots, and tops; cucumber -- fruit, vines plus leaves, and roots; corn -- kernels, husk plus cob, stalk plus leaves, and roots; lettuce -- tops and roots; onion -- bulb and tops; ryegrass -- tops and roots; soybean -- grain, chaff plus tops, and roots; oats -- grain, chaff plus tops, and roots; tomato -- fruit, vines, and roots; cabbage -- head and roots. Plants from two pots in each treatment may be pooled, giving three replicate sample pools per treatment.
                              (a) Fresh weight, dry weight, and percent moisture.  After the fresh weights of the plant organs are obtained, they should be subsampled for percent moisture determinations by drying, at 70 degrees Celsius ([o]C) for 24 hours in a forced-air drying oven, and weighing.  Percent moisture determined from these subsamples is used to correct for dry weight of the fresh samples.
                              (b) Extraction.  Each sample is then homogenized and extracted in organic and aqueous solvents.  A suggested extraction procedure appropriate for many organic chemicals is as follows: plant material (1 gram (g)) is homogenized with 1 g of solvent-washed anhydrous sodium sulfate in 4 milliliters (mL) of hexane or acetonitrile.  The homogenate is then filtered or centrifuged, the solid residue rinsed with an appropriate organic solvent, and the filtrate or supernatant combined with the rinse.  The solid residue should be extracted by sequentially (one) homogenizing in water, (two) centrifuging and decanting the supernatant, (three) extracting of the pellet with 6 Normal (N) hydrochloric acid at 60 [o]C for 10 hours, (four) subsequently digesting with 10 N potassium hydroxide, and (five) combining supernatants.
                        (2) Analytical methods.  If radioisotopes are used, the amount of test substance in each extract should be determined by liquid or solid scintillation; otherwise, the amount of test substance should be determined by standard methods.  The organic extract should be evaporated under vacuum to a sufficiently small volume for thin layer chromatography (TLC) and co-chromatographed on silica gel plates with known standards of the parent test substance.  If radioisotopes were used, the chromatographs could be scanned for radioactive substances on a radiochromatogram scanner.  Alternatively, zones may be removed from the plates, extracted, and the quantity of test substance from each zone determined by liquid scintillation spectrometry or gas liquid chromatography (GLC) methodology.  The unextractable test substance in the remaining residue may be determined by oxidizing the residue in a complete combustion oxidizer.
                  (B) Gaseous test substances--
                        (1) Direct calculation of uptake rates.  A gas exposure system yields requisite data for a direct calculation of uptake rates.  At steady state, test substance uptake may be determined by a mass balance calculation.  Correction for adsorption to surfaces of the exposure chamber should be made by operating the system without plants.  Pots filled with hydroponic solution or support media should be included in the system adsorption calibration.  Consequently, chemical analysis of plant tissues exposed to gaseous chemicals may not be required in order to demonstrate and quantitate uptake rates.
                        (2) Physiological rates.  Altered rates of net photosynthesis, transpiration, and stomatal conductance are anticipated as a result of test substance uptake.  Rates of these physiological processes before, during, and after exposure to the gaseous chemical should be determined.  Data required for these calculations are available as a consequence of maintaining the specified environmental conditions within the fumigation chamber.
                        (3) Plant surface area mass measurements.  For each pot measure the plant surface area mass.
                  (C) Root support medium measurements.  At test completion, the root support material should be washed in organic and then aqueous solvent and analyzed for test substance before discarding.
(f) Treatment of results--
      (1) Data summary--
            (i) For liquid and solid test substances.  
                  (A) Means and standard deviations of the fresh weight and dry weight mass of plant organs and by summation, the mass of the whole plant are calculated for each treatment (includes controls).
                  (B) Means and standard deviations of the concentration of free parent test substance, metabolites and soluble residues, and bound residues in plant organs and whole plants by treatment.  Concentrations should be expressed in appropriate weight units per grams of dry plant material.
			(C) Mean evapotranspiration rate per plant.
                  (D) Initial and final total concentration of the test substance (total residues: parents plus degradates) in the support media.
                  (E) Mean and standard deviations of the mass balance of test substance by treatment.
            (ii) For gaseous test substances.  
                  (A) Measurements as described in paragraphs (f)(1)(i)(A) through (f)(1)(i)(E) of this guideline for solid and liquid test substances; alternatively, the measurements described in paragraphs (f)(1)(ii)(B) through (f)(1)(ii)(D) of this guideline.
                  (B) Mass of leaves and stems and surface area (one side of leaves) in the exposure system.
                  (C) Steady state rates of photosynthesis, transpiration, and test substance uptake before, during, and after fumigation.
                  (D) Stomatal conductance or leaf diffusion resistance before, during, and after fumigation.
      (2) Definitive test.  Mass balance of the test substance and evapotranspiration rate of the plants is subjected to analysis of variance.  An analysis of variance, followed by a means separation test, is performed to determine which test substance treatments resulted in residues from uptake/translocation or evapotranspiration rates that are significantly different from those in the control(s).
(g) Tabular summary of test conditions.  Table 2 lists the important conditions that should prevail during the definitive 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 2. -- Summary of Test Conditions for Plant Uptake and Translocation Test[1]
Test duration
To provide sufficient biomass (or to allow gas exchange measurements), or until fruit or seeds are mature.
Substrate
Quartz sand or glass beads.  Hydroponic system may also be used.  (For pesticides, natural or synthetic soil may also be used.)
Nutrients
Watered with nutrient solution (half-strength modified Hoagland's medium)
Temperature 
25/20 [o]C (daytime/nighttime) +- 3 [o]C (applicable to growth chambers)
Relative humidity
70/90% (daytime/nighttime) +- 5%  (applicable to growth chambers)
Carbon dioxide
350 +- 50 ppm (applicable to growth chambers)
Light quality
Fluorescent or representative of natural sunlight
Light intensity
350 +- 50 umol/m[2]/sec 
Photoperiod
16 hours light: 8 hours dark for all species except soybean with 11 hours light: 13 hours dark prior to flowering
Watering
Bottom watering as needed, using nutrient solution
Test chamber (pot) size
Varies with plant species selected.
Number of organisms per test chamber
Typically, 1 - 4 seedlings of one species per pot
Number of replicate chambers per test treatment
6 (minimum) 
Number of organisms per test treatment
6 - 24 (minimum)
Test treatment levels
Minimum of 3 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) or soil surface exposure.
Measures of effect (Measurement endpoints)
Mass balance on test substance, including concentrations of free parent test substance, metabolites and soluble residues, and bound residues in plant organs and whole plants. (Alternatively, for gaseous test substances, measurements of photosynthesis, transpiration, and stomatal conductance, and mass of plant surface area).
[1] Constraints on environmental conditions are mainly applicable to growth chamber studies 

(h) Test validity elements.  This test would be considered to be unacceptable or invalid if one or more of the conditions in Table 3 did not occur or one or more performance objectives in Table 3 were not met.  This list should not be misconstrued as limiting the reason(s) that a test could be found unacceptable or invalid.  However, except for the conditions listed in Table 3 and in 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 report, all departures from the guideline should be identified, reasons for these changes given, and any resulting effects on test endpoints noted and discussed.
	
 Table 3. -- Test validity elements for the plant uptake and translocation test
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1. For any species, all organisms in a test are from the same source.
2. All test chambers used for a particular species are identical and contain the same amount of substrate from the same source.
3. An untreated (or negative) control [and solvent (or vehicle) control, when a solvent was used] is included in the test.
4. 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)(ii)(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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5.  There are no signs of stress from competition or crowding of plants.
(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)), radiolabeling if any, location of label(s), and radiopurity.
	(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.
	(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 determining percent germination.
	(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) Height and condition of seedlings selected for the test.
      (5) Test system and conditions.  Description of the test system and conditions used in the definitive 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) The method for calibrating the application equipment.
	(x) Number of applications and dates applied.
	(xi) Culture practices during the test such as pest control, and irrigation practices (type and watering schedule or regime).
	(xii) The photoperiod and light source.
	(xiii) Methods and frequency of environmental monitoring performed during the definitive test for air temperature, humidity, and light intensity.
	(xiv) For the definitive 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) Tabulation of the watering schedule, the volume of nutrient solution (or hydroponic solution) used and depleted each week by replicate within a treatment and control and the evapotranspiration rates calculated from the data.
	(iii) For the gas exposure system, tabular summary of 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 (provide raw data for measurements not made on a continuous basis).
	(iv) For the 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.
	(v) For the definitive test, tabulation of the fresh weight, dry weight, and percent moisture of plant organs (e.g., carrot -- root peels, peeled roots, and tops; cucumber -- fruit, vines plus leaves, and roots; oats--grain, chaff plus tops, and roots) by replicate and treatment (provide the raw data).  For gaseous test substances, in addition to or in lieu of these measurements, tabulation of the plant surface area mass by replicate and treatment.
	(vi) For the definitive test, tabulation of the concentrations of parent test substance, metabolites, soluble residues and bound residues in plant organs and whole plants by replicate (pooled replicate, if applicable) and treatment.  For gaseous test substances, in addition to or in lieu of these measurements, tabulation of the calculated measurements of photosynthesis, transpiration, and stomatal conductance before, during, and after exposure to test chemicals by replicate and treatment (provide raw data).
	(vii) For the definitive test, tabulation of the mass balance of test substance by replicate and treatment.
	(viii) For the definitive test, means and standard deviations of mass balance of test substance by treatment.
	(ix) For the definitive test, plots of the means and standard deviations of mass balance of test substance by treatment.
	(x) For the definitive test, analysis of variance, F-test, means, and standard deviation about the mean for the measured endpoints.  
	(xi) Methods of statistical analyses, including software used, should be reported.
(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) Beall, M.L., Jr. and Nash, R.G.  1969.  Crop seedling uptake of DDT, dieldrin, endrin, and heptachlor from soil.  Agronomy Journal 61:571-575.
	(3) Bennett, J.H., Hill, C., and Gates, D.M.  1973.  A model for gaseous pollutant sorption by leaves.  Journal of the Air Pollution Control Association 23:957-962.
	(4) Chou, S.F., Jacobs, L.W., Penner, D., and Tiedje, J.M.  1978.  Absence of plant uptake and translocation of polybrominated biphenyls (PBBs).  Environmental Health Perspectives 23:9-12
	(5) Cole, L.K., Sanborn, J.R., and Metcalf, R.L.  1976.  Inhibition of corn growth by aldrin and the insecticide's fate in the soil, air, crop, and wildlife of a terrestrial model ecosystem.  Environmental Entomology 5:583-589.
	(6) Downs, R.J.  1975.  Controlled Environments for Plant Research.  Columbia Univ. Press, NY.
	(7) Downs, R.J. and Helmers, H.  1975.  Environment and Environmental Control of Plant Growth. Academic Press, NY. 
	(8) Fuhr, F. and Mittelstaedt, W.  1980.  Plant experiments on the bioavailability of unextracted (carbonyl-14C) methabenzthiazuron residue from soil.  Journal of Agriculture and Food Chemistry 28:122-125.
	(9) Hatzios, K.K. and D. Penner.  1985.  Interactions of herbicides with other agrochemicals in higher plants.  Rev. Weed Sci. 1:1-63.
	(10) Kelly, J.M., Parker, G.R., and McFee, W.W.  1980.  Heavy metal accumulation and growth of seedlings of five forest species as influenced by soil cadmium level.  Journal of Environmental Quality 8:361-364. [
	(11) Tibbits, T.W. and Kozlowski, T.T. (eds.).  1979.  Controlled Environment Guidelines for Plant Research. Academic Press, NY
	(12) Wickliff, C., Evans, H.J., Carter, K.R. and Russell, S.A., 1980.  Cadmium effects on the nitrogen fixation system of red alder. Journal of Environmental Quality 9:180-184.
