United States
Environmental Protection Agency
   Office of Chemical Safety
   and Pollution Prevention
   (7101)
EPA 712-C-004
January 2012

Ecological Effects
Test Guidelines

OCSPP 850.4600:

Rhizobium-Legume Toxicity

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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 850.4600: Rhizobium-legume toxicity.
(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 40 CFR 797.2900 Rhizobium-legume Chronic Toxicity 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.  The guideline prescribes tests using commercially important terrestrial plants and their nitrogen-fixing bacterial symbionts to develop data on the phytotoxicity of test substances (see paragraphs (j)(1) of this guideline).  The Environmental Protection Agency will use data from these tests in assessing the hazard and risk a test substance may present in the terrestrial environment.  This guideline should be used in conjunction with OCSPP 850.4000 (Background and special considerations for conducting ecological effects tests with terrestrial and aquatic plants, cyanobacteria, and terrestrial soil core microcosms), which provides general information and overall guidance for non-target plant test guidelines.
(c) Definitions.  The definitions in OCSPP 850.4000 apply to this test guideline. In addition, the more specific definitions in this paragraph also apply:
	Inoculum refers to a specific Rhizobium symbiont obtained from a culture and added to legume seeds to result in a plant-bacteria complex.
	Legume refers to a member of the pea family (Leguminosae) and includes many species of great economic importance.
	Nitrogen-fixation refers to the conversion of elemental nitrogen to nitrates by Rhizobium spp. bacteria which colonize legume root nodules.
	Rhizobium refers to a genus of symbiotic bacteria that forms nodules on the roots of certain legumes.
	Support media refers to the quartz sand used to support the test plant during the test.
	Symbiont means either of two organisms participating in a symbiotic relationship.
	Symbiosis refers to the close union of two dissimilar organisms in a mutually beneficial relationship.
(d) General considerations--
      (1) Summary of the test.  Seeds of a legume species are inoculated with their specific Rhizobium symbiont (forming a plant-bacteria complex) and planted in sand irrigated with a nutrient solution.  The test substance is applied to the plant-bacteria complex via the nutrient solution or is adsorbed to the sand, resulting in continuous exposure to the test substance from the time the seed (or seedling, if appropriate) is planted to maturity of the plant.  After significant leaf development has occurred (usually after several weeks during which the Rhizobium-inoculated plants are irrigated at regular intervals with the nutrient solution), all plants are harvested for analysis.  Effects are evaluated by comparing plant yield, nodule production, and nitrogen-fixation in plants exposed to the test substance to those plants not exposed (negative controls) to the test substance.
      (2) General test guidance.  The general guidance in OCSPP 850.4000 applies to this test guideline except as specifically noted herein.
	(3) Range-finding test.
            (i) 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, Rhizobium-legume complexes are exposed to a series of widely-spaced concentrations of the test substance.  For range-finding purpose, seed germination, the first event in the establishment of a Rhizobium-legume symbiotic relationship, may be used.  If the concentration of test substance to which the Rhizobium-legume association is likely to be exposed in nature can be predicted, seeds of the selected legume should be treated with concentrations that are 0.10, 10, and 100 times the anticipated environmental concentration.  Should reasonable predictions of potential environmental exposure concentrations not be possible, seeds should be exposed to a series of widely spaced concentrations (e.g., 0.01, 0.1, 1.0, 10, 100, 1,000 milligrams per liter (mg/L)) of the test substance.  Nominal concentrations of the test substance are acceptable.  After a given exposure period, the effects should be assessed as the sum of the root lengths (in millimeters (mm)) of all plants of each test concentration, relative to that evidenced in the controls.
            (ii) The seed-germination range-finding test consists of exposing a minimum of 15 seeds of one legume species (representing the plant host in the selected Rhizobium-legume association) to each concentration of test substance and to the control.  Seeds, placed between sheets of filter paper moistened with varying concentrations of test substance, should be incubated in darkness at room temperature (approximately 22 degrees Celsius ([o]C)) in Petri dishes, allowing adequate room for linear root growth.  When 65 percent (65%) of the control seeds have germinated and their radicle roots are at least 20 mm long per seedling, the test may be terminated.
      (4) Definitive test.  For the Rhizobium-legume complex tested, the goal of the definitive test is to determine for each measure of effect (plant yield, nodule production, and nitrogen fixation) its concentration-response curve and IC50 values (with 95% confidence intervals and standard errors).  If possible the slopes of the concentration-response curves, associated standard errors, and the 95% confidence intervals should also be determined.  However, at a minimum, the full-concentration-response curve (concentration range covers IC10 to IC90) is determined for the most sensitive measure of effect (plant yield, nodule production, or nitrogen fixation).  A minimum of five concentrations or dose levels of the test substance plus appropriate control(s) is used.  Adding one or two additional test concentrations to ensure coverage in the lower tail of the concentration-response curve is recommended.  Analytical confirmation of the test concentrations is performed as described in OCSPP 850.4000.  A summary of test conditions for an acceptable definitive test is given in Table 2 of this guideline.
      (5) Limit test.  In some situations, it is only necessary to ascertain that the plant yield, nodule production, and nitrogen fixation IC50 values for the Rhizobium-legume complex tested are all above a certain limit concentration.  In the Rhizobium-legume limit test, at least six replicates, containing the specified number of seeds or seedlings (see paragraph (e)(6) of this guideline), are exposed to a single "limit concentration," with the same number of organisms in appropriate controls.  The multiple-concentration definitive test may be waived if for all measures of effect (plant yield, nodule production, and nitrogen fixation) the "limit treatment" response is statistically less than a 50% decrease from the control response (i.e. IC50 values are greater than the limit concentration).  For most industrial chemicals, 1,000 milligrams per liter (mg/L) or the limits of water solubility or dispersion are considered appropriate as the limit concentration.  For pesticides, the limit concentration is based upon the maximum label rate, e.g. the maximum recommended amount of active ingredient (a.i.) used per land area.  Results are reported in grams or pounds of a.i. per acre.  The assumptions used to calculate the limit concentration involve direct application to a 3-centimeter (cm) deep column of soil with a bulk density of 1.5 grams per cubic centimeter (g/cm[3]).  Alternatively, the limit concentration should be three times the estimated environmental concentration.  Except for the number of treatment groups and endpoint determinations, 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 test (Table 2).  Acceptable limit tests like definitive tests include analytical confirmation of the limit dose.
(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 a.i. and/or the one most commonly used should be tested.  If there is more than one TEP with different inert substances, a TEP representative of each different inert substance should be tested in the range-finding test and at a minimum the most sensitive one tested in the definitive or limit test.  In addition, if an adjuvant is recommended for use on a TEP label, the adjuvant is added with the TEP at the label rate to constitute the test substance.  If a TEP is not available (i.e. new pesticide), the technical grade a.i. is used with a representative formulation, which should include any adjuvant that will be recommended for use on the label.  If products are applied in a tank mixture, dosages of each a.i. should be reported with identification and formulation for each product in the tank mix.  OCSPP 850.4000 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 limit and definitive tests last 3 to 7 weeks from the date of planting to effective root nodulation in the controls.  The specific duration depends on the Rhizobium-legume complex, and amount of inoculum.  Duration can be determined based on time to effective root nodulation documented in a preliminary or pre-definitive study, or the definitive study could be conducted with sufficient numbers of controls to pull plants periodically (at week 3 and weekly thereafter until nodulation is indicated) to check for effective nodulation.
	(3) Test organism--
            (i) Species.  A species of the genus Trifolium (e.g., T. repens, white clover) is the preferred legume for this test.  As an alternative, other legume species (e.g., Phaseolus vulgaris, bush bean) of economic or ecologic importance to the region of impact may be selected for testing.  The rationale for selecting an alternative legume species from that of the genus Trifolium should be provided.  When selecting inoculum, consult the seed packet or supplier and the inoculant manufacturer for the specific complementary species of Rhizobium to use for the selected test legume.
            (ii) Source.  Within a given test, all seeds of the test species are taken from the same lot and year or season of collection and the Rhizobium sp. bacteria used for inoculation are from the same culture.  Fresh inoculum should be obtained from a reliable source of bacterial cultures.
            (iii) Condition.
                  (A) All the seeds for a given species used in a test should be of the same size class, and that size class which contains the most seed should be selected and used in a given test.  Any damaged seed should be discarded.  For a satisfactory test, healthy seeds should be used with reported germination rate for a lot number of 65% or better for T. repens or for other species the minimum acceptable USDA seed germination standards described in paragraph (e)(5)(ii) of this guideline.
			(B) Inoculum should be used before the date of expiration.
            (iv) Care and handling.  Seeds should be stored in a dessicator and refrigerated until needed.  Only untreated seed (not treated with fungicides, repellents, etc.) should be used.  Seeds of legume species that are subject to attack by mold (e.g., clovers) may be washed with ethanol before being planted.  Inoculant should be kept refrigerated and stored according to the inoculant package instructions to preserve the bacteria.  Heat and direct sunlight can kill Rhizobia, even when packaged.
	(4) Administration of test substance--
            (i) Preparation of exposure treatments--
                  (A) Exposure technique.  Since the anticipated fate of the test substance involves soil or soil water, and the mechanism of toxicity depends upon root exposure, the test substance should be applied in nutrient solution to the support media (paragraph (e)(4)(i)(B) of this guideline) or coated on the support media (paragraph (e)(4)(i)(C) of this guideline) for water-insoluble test substances for which no nontoxic, water-soluble vehicle is available.  Unless it is necessary to adsorb the test substance to the support media, the nutrient solution should contain the desired concentration of test substance.  However for pesticides, because a TEP is typically tested along with any labeled adjuvants (see paragraph (e)(1) of this guideline), solvents are generally not necessary and the test substance is applied directly to the nutrient solution (see paragraph (e)(4)(i)(B)(1) of this guideline).
                  (B) Test substance applied in nutrient solution.  Test substance is added either directly to nutrient medium (see paragraph (e)(4)(i)(B)(1) of this guideline) or added in solvent stock solution to nutrient medium (see paragraph (e)(4)(i)(B)(2) of this guideline).  The stability of the test substance in the nutrient solution under the conditions of the test should be investigated as part of the method validation (see OCSPP 850.4000).
                        (1) Direct addition to nutrient solution.  Test substances that are soluble in water should be dissolved in the nutrient solution immediately prior to its use.  Sufficient quantities of each concentration should be made as needed to minimize storage time and disposal volume.
                        (2) Dissolved in nontoxic solvent.  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 immediately prior to its use.  The vehicle should be soluble in water, nontoxic to plants, and 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 other solvents have been used extensively.  Vehicle controls should be included in the experimental design and tested simultaneously.  OCSPP 850.4000 contains additional information on the preparation of stock solutions.
                  (C) Test substance applied to support media.  Water-insoluble test substances for which no nontoxic, water-soluble vehicle is available should be dissolved in an appropriate volatile solvent.  The stock solution of the test substance should be mixed with the support media, placed in a rotary vacuum apparatus and evaporated, leaving a uniform coating of the test substance on the support media.  A portion of support media should be weighed, the test substance should be extracted with the same organic solvent, and the concentration of the test substance determined before the potting containers are filled.  When the test substance is administered via the support medium, no test substance is added to the nutrient solutions.

            (ii) Treatment levels.
                  (A) For the Rhizobium-legume test complex, five treatment levels are tested at a minimum.  For scientifically sound estimates of a given point estimate (e.g., IC50), 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 mg/L).  While a twofold progression is preferred, threefold and fourfold progressions are acceptable.  If a fourfold series progression is used, the rationale for using this large of interval between concentrations and the effect on the accuracy and reproducibility of the point estimate or NOEC should be provided.  For an acceptable study, the lowest test treatment level should be lower than the IC50 values for plant yield, nodule production, and nitrogen-fixation.  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 most sensitive effect measure is acceptable for calculation of the 10% effect or inhibition concentration (IC10).  Recommend adding one or two additional test concentrations in the lower tail of the concentration-response curve of the most sensitive endpoint to ensure the full-concentration response curve is captured.
                  (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 and test design).
            (iii) Introduction of test organisms.
                  (A) Seeds are mixed with a small amount of moist commercial peat previously inoculated with the desired Rhizobium species.  At a minimum use the recommended manufacturer's inoculant rate (two to three times the normal inoculant rate is sometimes recommended for new soils).  Newly inoculated seeds are planted immediately in pots filled with support media (quartz sand).  The support media should be irrigated with the nutrient solution (containing test substance, if this is the selected application route) before planting occurs.
                  (B) 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 approximately 22 degrees Celsius ([o]C), and the barrier should be positioned such that 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.
                  (C) The assignment of pots to test substance concentrations should be random.  Placement of groups of pots (six per group, all within each group receiving nutrients and test substance from the same source) should be randomized, to the extent possible, in the greenhouse or growth chamber.  A group of pots constitutes a replicate and should be maintained as a group throughout the study duration.
	(5) Controls.
            (i) Every test includes negative controls consisting of the same support medium, nutrient solution, environmental conditions, handling procedures, bacteria from the same culture, and seed from the same lot used in the exposed groups, except that no test substance is added.  A vehicle (solvent) control (either nutrient solution or support media) is also included if a solvent is used.  Specifically a nutrient solution vehicle control is included if a vehicle is used to dissolve or suspend the test substance in the nutrient solution (see paragraph (e)(4)(i)(B)(2) of this guideline) and a support medium vehicle control is included if a vehicle is used to apply the test substance to the support media (see paragraph (e)(4)(i)(C) of this guideline).  However for pesticides, because a TEP is typically tested, vehicles are generally not used.
            (ii) Minimum seed germination.  A test is not acceptable if seed germination in either the negative or vehicle control is less than 65% for Trifolium repens or is less than 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: field-garden bean (70%), pea (80%), soybean (75%), vetch (75%), alfalfa--clover (70%), rape (75%).  Refer to regulation for additional legume crops.
            (iii) A test is not acceptable if at test termination mean negative or vehicle control survival of emerged seedlings is less than 90%.
            (iv) A test is not acceptable if control plant condition indicates visible phytotoxic or developmental symptoms during the test.
            (v) If the control(s) is contaminated with the test substance, the study is not acceptable and should be repeated.
            (vi) The coefficient of variation for the growth parameters in the control should generally be less than 20%.  If the Rhizobium-legume complex selected for testing, or testing facility handling and processing will result in higher variability recommend increasing the number of replicates to ensure detection of differences at the level the given study design with 20% variation is able to detect.
      (6) Number of test organisms and replicates.
            (i) The minimum number of replicates per treatment level and control(s) is six.  For smaller seeds (e.g., Trifolium repens), 0.3 grams of dry seed per pot is used.  In this case to evaluate the germination rate in test controls, the number of seeds in a representative number of samples (e.g., 6) of 0.3 grams of dry seed should be counted to provide an estimate of the mean and standard deviation of the number of seeds planted per pot.  For larger seeds (e.g., Phaseolus vulgaris), one seed per pot is planted.
            (ii) Placement of pots within the greenhouse should follow a random or randomized block design.  To minimize spatial differences, which can have a significant impact upon plant growth, the placement of replicates should be randomized during the test (at least every three to five days).
            (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 six 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 or environmental growth chambers.  Rhizobium-legume tests are conducted under controlled conditions in greenhouses or environmental growth chambers.  Greenhouse or environmental growth chambers 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 the test substance.
            (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.  Containers used in an experiment should be of equal size and volume and possess the same configuration.  The volume of the pot should be large enough so that growth is not restricted due to space or nutrient limitations during the test.
            (iii) Support medium.  Support medium consists of clean, coarse (0.5 to 1.0 millimeter diameter) quartz sand and should not have been used previously.  Potting containers should be filled with support media to within 2.5 centimeters of their tops.  Perlite, vermiculite, native soils, etc. should not be used for root support.  A cellulose sponge plug in the pot drain hole will prevent the loss of sand during drainage.
            (iv) Large holding trays.  Each series of six replicate pots (per test concentration, control, and if applicable, vehicle control) may be placed in a large tray into which the appropriate nutrient solution will be delivered (see Figure 1 of this guideline).  Trays should be constructed of an inert material to which adsorption of the test substance will be minimal, e.g., glass, Teflon, polyethylene, or linear high-density polypropylene.  Each tray may be covered with a Plexiglas sheet bearing six holes to accommodate the pots, keeping them upright and properly spaced.
Figure 1. -- Cross-Sectional Diagram of a Representative Tray Unit and the Nutrient Solution Reservoir for Irrigating Potted Plants

            (v) Nutrient solution delivery system.
                  (A) 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 as depicted in Figure 1 to deliver nutrient medium.  The test substance may be in the nutrient solution (see paragraph (e)(4)(i)(B) of this guideline) or on the support medium (see paragraph (e)(4)(i)(C) of this guideline).  Six or seven 25-liter carboys (one per treatment concentration of test substance and one for negative controls; another if a vehicle control is included) serve as reservoirs from which nutrient solution may be delivered, under air pressure, to the appropriate tray of potting containers.  Each carboy contains 15 liters of nutrient solution.
                  (B) Nutrient solutions should be transported by inert tubing from the carboys to the trays of pots at regular intervals (for example, 30 min/h).  A timer-activated air pump is recommended for maintaining a controlled irrigation schedule.  Irrigation should be suspended for 6 to 8 hours before nitrogen-fixation measurement.
                  (C) Nutrient solution levels in the 25-liter carboys should be maintained at 15-liters throughout the experiment by replacing transpired water with reagent water, and by complete replacement with fresh nutrient solution twice per week.  If test substance is applied in the nutrient solution it may be necessary to replace the nutrient solution more frequently if the test substance is rapidly degraded under test conditions.  The time to renewal (renewal cycle) should be shorter than the time it takes for the concentration of the test substance to decline to less than 80% of the initial measured concentration.  The stability of the test substance in the nutrient solution under the conditions of the test should be investigated as part of the method validation (see OCSPP 850.4000).
            (vi) Cleaning and disposal.
                  (A) Potting and receiving containers, nutrient storage containers, and support media should be cleaned before use.  All equipment should be washed to remove any residues remaining from manufacturing or prior use.  A dichromate solution should not be used for cleaning pots or other containers.
                  (B) Support media should be discarded at the end of the test.  Nutrient media should be discarded after use.  Disposal should conform to existing regulations.
            (vii) Nutrient medium.
                  (A) The recommended medium for growth and establishment of the Rhizobium-legume association is described in Table 1.

                   Table 1. -- Preparation of nutrient medium
Substance
                       Concentration of Substance (mg/L)
Potassium sulfate (K2SO4)
                                      901
Potassium phosphate (KH2PO4)
                                      272
Calcium chloride (CaCl2)
                                      416
Magnesium sulfate heptahydrate (MgSO4 :: 7H2O)
                                      493
Ferric chloride hexahydrate (FeCl3 :: 6H2O)
                                      8.3
1 mL of micronutrient stock solution per liter of nutrient solution

Micronutrient stock solution: add reagent grade chemicals to reagent water (final volume to be 1 liter)
Borric acid (H3BO3)  -  2.9 grams

Manganese chloride tetrahydrate (MnCl2 :: 4H2O)  -  1.8 grams

Zinc sulfate heptahydrate (ZnSO4 :: 7H2O)  -  0.22 grams

Molybdenum (VI) acid monohydrate (H2MoO4 :: H2O)  -  0.02 grams

Copper sulfate hexahydrate (CuSO4 :: 6H2O)  -  0.03 grams

                  (B) The medium is prepared by adding the substance listed to reagent water (deionized, distilled or reverse osmosis) to give the desired final concentrations of the substance.
                  (C) For certain legumes (e.g., Phaseolus vulgaris, bush bean), growth in this medium will be enhanced by the addition, after 2 weeks, of 50 milliliters of a nitrate supplement (10.2 grams of potassium nitrate (KNO3) and 28.3 grams of calcium dinitrate tetrahydrate (Ca(NO3)2 :: 4H2O) per liter) per 15 liters of nutrient solution.
                  (D) The pH of the nutrient medium should be maintained near 7.  The pH may be adjusted in stock solutions to match the pH of the nutrient medium if the pH change does not affect the stability of the test substance in the stock solution or test solution.  Hydrochloric acid (HCl) and sodium hydroxide (NaOH) may be used for this adjustment if warranted.  The pH should not be adjusted after the addition of the test substance or stock solution into the test medium.
			(E) Store medium in the dark at approximately 4 ºC until use.
            (viii) Environmental monitoring equipment.  Equipment for determination of test environmental conditions (e.g., pH meter, photosynthetically active radiation (PAR) light sensor, etc.)
            (ix) Ethylene production, collection, and measurement.  Airtight chambers, one for each pot used in the test, fit with lines or ports to supply and evacuate gas from the chamber and to collect gas samples are used.  The airtight chamber should be large enough to fit a single pot with the fully grown test plant(s) for small legume species such as T. repens, white clover, or large enough to fit the roots of the plant for large legume species such as P. vulgaris, bush bean.  Gases used include an inert gas to flush the chambers and acetylene gas (C2H2) to fill chambers to promote production of ethylene gas (C4H4).  Gas chromatogram equipment for measuring ethylene gas.
      (8) Environmental conditions.  The test conditions should approximate those optimal conditions for the species and varieties tested.  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)(vi) 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 specifications may be relaxed on a case-by-case basis.  The test conditions should be uniform throughout the growth chamber, growth room or greenhouse.
            (i) Temperature.  Optimum temperature for growth and nitrogen-fixation for the species used.  For example, the optimum range for clover is 15 to 25 [o]C, so the recommended test temperature for this species is 20 +- 5 [o]C.
            (ii) Humidity.  Relative humidity in growth chambers 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.  The photoperiod should be 16 hours light and 8 hours darkness assuming an average wavelength of 400 to 700 nanometers (nm).  Luminance, measured at the top of the canopy, at 350 +- 50 micromoles per square meter per second (umol/m[2]/s), is desirable.  For greenhouses, artificial lighting may be used to lengthen short-day periods or to supplement natural sunlight on overcast days.  Additional information on the use of lighting in plant toxicity tests can be found in the references given in the OCSPP 850.4000 guideline.  Care should be taken to ensure that plants are not affected from the heat generated from supplemental lighting.
            (iv) Watering.  Nutrient solution is used to water the plants on a regular schedule throughout the study duration (see paragraph (e)(7)(v) of this guideline).
            (v) Nutrient media pH.  Prior to use, the pH of the nutrient medium should be adjusted to approximately 7.0.  Adjustment of pH can be accomplished by adding acid or base prior to the addition of the test substance.  The pH may be adjusted in stock solutions to match the pH of the nutrient medium if pH change does not affect the stability of the test substance in the stock solution or test solution.  Hydrochloric acid (HCl) and sodium hydroxide (NaOH) may be used for this adjustment if warranted.  The pH should not be adjusted after addition of the test substance or stock solution into the test medium.  Test solution pH may vary from the nutrient medium after the addition of the test substance and/or vehicle (if used).  Any such changes should be recorded but not adjusted.
            (vi) Carbon dioxide concentration in air.  If a growth chamber is used, the carbon dioxide concentration should be 350 +- 50 parts per million (678 +- 97 milligrams per cubic meter (mg/m[3]) of air).
      (9) Observations--
            (i) Measurement of test substance.  Analytical confirmation of the concentration of test substance in the nutrient medium (if test substance addition was by the nutrient medium) or on the sand support medium (if test substance addition was by support medium) should be performed as described in OCSPP 850.4000.  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, 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.  Where test substance was introduced by addition to the 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.  The analytical methods used to measure the amount of test substance in a sample are validated before beginning the test, as described in OCSPP 850.4000.
            (ii) Environmental conditions--
                  (A) Air temperature and relative humidity.  The air temperature and relative humidity during the study should be recorded, preferably continuously, but alternatively as maximum and minimum values over each 24-hour period at representative locations throughout the area in which the test plants are growing.
                  (B) Carbon dioxide air concentrations.  For chamber-grown plants, measurements of carbon dioxide concentrations should be made at the top of the plant canopy on a continuous basis.
                  (C) Light intensity.  Light intensity (luminance) 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.
                  (D) Watering.  Records should be kept noting the frequency upon which nutrient solution was applied to trays, failure of system to deliver nutrient medium, and any observations of moisture stress should be made and recorded daily.  Dates of addition and amounts of reagent water added to a carboy to maintain volume at 15 liters should be recorded.  Dates of nutrient solution replacement should be recorded.
                  (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 each nutrient solution treatment concentration and control(s) are measured in nutrient solution at the start of each renewal period prior to use, and at replacement in the used solution.
            (iii) Measures of effect--
                  (A) Appearance and condition.  Observations of plants should be made at day 3 or 4, 7, 10, and 14during the first two weeks and at least once every seven days after throughout the duration of the study.  The number of emerged seedlings, seedling and plant mortality, and all abnormalities (visible effects of the chemicals on plant growth and morphology including stunting of growth, discoloration, chlorosis and/or necrosis of the leaves, or morphological abnormalities) should be noted and recorded.  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) Nitrogen-fixation.  Nitrogen-fixation measurements are made at the end of the exposure period.  To measure nitrogen-fixation for a small-to-moderate-sized legume species (e.g., T. repens, white clover), each pot should be enclosed in an airtight chamber.  The number of surviving plants in each pot should be recorded.  To enhance the reduction of acetylene (C2H2), the chambers may be flushed with an inert gas (evacuating the nitrogen-containing air) before introducing the C2H2.  Following exposure to C2H2 for a period of time sufficient to yield a linear production of ethylene (C4H4), gas samples should be withdrawn and analyzed for C4H4 as an index of nitrogen-fixation, using gas chromatography.  Nitrogen fixation is expressed as nanomoles of C4H4 per gram dry weight per hour (nanomoles C4H4/g/h).  Also recommend calculating the number of nanomoles of C4H4 per plant per hour (nanomoles C4H4/plant/h) (for pots with more than one surviving plant calculate the total number of nanomoles of ethylene gas for the pot per hour and then divide by the number of surviving plants in the pot).  Should the Rhizobium-legume association selected for the test use a larger species of legume (e.g., P. vulgaris, bush bean), plant roots may be removed, washed with distilled water, and placed in an airtight plastic jar.  Gas samples should be withdrawn and analyzed for C4H4 after an appropriate incubation period in the presence of C2H2 as described in this paragraph.  Using the test conditions and clover and bean species recommended, incubation periods of 5 hours and 1 hour, respectively, are suitable for nitrogen-fixation determinations.  Optimal incubation times for other species in containers of other sizes may be different.  The optimal incubation times should be tested and documented prior to the definitive test.
                  (C) Root nodule count.  At test termination, nodulation performance should be assessed by counting and recording the cumulative number of root nodules on the plants in each pot and treatment group.  For each pot divide the total number of nodules by the number of surviving plants in the pot to determine the number of nodules per plant.
                  (D) Dry weight.  At test termination, plant yield in each pot and treatment group is determined and recorded.  Yield is measured as the total dry biomass (tops and roots) per pot.  Dry weight (48 hours at 70 [o]C) is used because it is less variable than wet weight.  Total pot yield is divided by the number of surviving plants to give yield per plant per pot.
(f) Treatment of results--
      (1) Summary statistics--
            (i) Environmental conditions.  Calculate descriptive statistics (mean, standard deviation, coefficient of variation, minimum, maximum) for temperature, humidity, carbon dioxide and light intensity by test position.  Calculate descriptive statistics (mean, standard deviation, coefficient of variation, minimum, maximum) for medium pH by treatment level.
            (ii) Test substance concentration.  For each treatment level compare the test substance concentration at the start and end of each renewal period.  If the test substance was not stable calculate a rate of decline of the test substance; a time-weighted mean concentration should be calculated under these circumstances (e.g., exponential decay calculate the area under the exponential decay concentration curve divided by the total exposure days).  Calculate descriptive statistics such as the time-weighted mean, standard deviation, minimum, maximum, and coefficient of variation for each test vessel and treatment level.
            (iii) Appearance and condition.  The number of emerged seedlings and number of mortalities at each observation time for each treatment group and pot should be tabulated.  The number of surviving plants in a pot at test termination should be tabulated by treatment group and pot.  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.
            (iv) Nitrogen fixation.  For each treatment level and control(s) the mean, standard deviation, and coefficient of variation for nitrogen fixation (nanomoles C4H4/g/h) at test termination are calculated and plotted.
            (v) Number of root nodules.  For each treatment level and control(s) the mean, standard deviation, and coefficient of variation for number of root nodules per plant at test termination are calculated and plotted.
            (vi) Dry weight.  For each treatment level and control(s) the mean, standard deviation, and coefficient of variation for dry weight per plant at test termination are calculated and plotted.
      (2) Percent inhibition.  For nitrogen fixation, number of root nodules, and dry weight calculate the percent inhibition (%I) at each treatment level using Equation 1.
		Equation 1
      where:
            C = the control mean response value (nitrogen fixation, number of root nodules, or dry weight); and
            X = the treatment mean response value (nitrogen fixation, number of root nodules, or dry weight, respectively).  Stimulation is reported as negative %I.
      (3) Limit test.  To ascertain that the nitrogen-fixation, number of root nodules, and dry weight IC50 values occur above the "limit" concentration, a one-sided test which compares the difference between two sample groups to a fixed value (or difference) is performed for each of these response variables.  For a comparison of sample means, the difference defining the IC50, compared to the controls, is operationally defined as a 50% 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 50 reduction, compared to the control (i.e., H0: ucontrol - ulimit  > δ0).  The alternative hypothesis (HA) is that this difference is less than a 50% 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.  A multiple-concentration definitive test is performed if inhibition is equal to or greater than 50% at the limit concentration (i.e., the null hypothesis is not rejected) for any of the effect measures (nitrogen-fixation, number of root nodules, and dry weight).
		Equation 2
		where:
            δ0 = difference between two parameters, defined in this case as a p percent reduction from the control sample mean;
             = control sample mean response (e.g., nitrogen-fixation, number of root nodules, dry weight); and
            p = percent reduction from the control sample mean, which is 50 in the case of the IC50.
	(4) Multiple-dose definitive test--
            (i) Concentration-response curve, slope and IC50.  For dose-response tests statistical procedures are employed to calculate the IC50 value (standard error and 95% confidence interval) for nitrogen fixation, number of root nodules, and dry weight.  If a concentration-response curve model was fit to the data to determine the IC50, the model parameters (e.g., slope) and their uncertainty estimates (e.g., standard error) should be recorded.  The response values for each pot, not the mean response for each treatment level, should be used in fitting the model.
            (ii) Statistical methods.  Statistical procedures for modeling continuous toxicity data are available and should be used.  Additional discussion about endpoints and statistical procedures is found in the OCSPP 850.4000 guideline.
            (iii) NOEC.  Determine the NOEC and LOEC for nitrogen fixation, number of nodules, and dry weight.  If a NOEC value can not be determined for a given response measure, the concentration at which there is a 10% inhibition (i.e., an IC10 value for yield or growth rate) is estimated and used in place of the given NOEC.  The standard error and 95% confidence interval should also be calculated for the IC10 value.  Methods, assumptions, and results of the statistical approaches used should be recorded (see OCSPP 850.4000 for discussion on NOEC testing and reporting).
(g) Tabular summary of test conditions.  Table 2 lists the important conditions that should prevail during the definitive test.  Except for the number of test concentrations, Table 2 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.

	Table 2. -- Summary of Test Conditions for Rhizobium-legume Toxicity Test[1]
Test duration
Three to seven weeks (establishment of effective root nodulation in controls, dependent on Rhizobium-legume complex tested)
Substrate
Quartz sand, 0.5 - 1.0 mm diameter
Nutrients
Watered with nutrient solution
Temperature
Optimal for the species used; 20 +- 5 [o]C for clover
Relative humidity
70/90% (day/night) +- 5% (for growth chamber studies)
Carbon dioxide
350 +- 50 ppm
Light quality
fluorescent or representative of natural sunlight
Light intensity
350 +- 50 umol/m[2]/s 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
Number of organisms per test chamber
0.3 g dry seed per pot (for clover); 1 seedling  per pot (for bush beans)
Number of replicate chambers per test treatment
6 (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)
Measures of effect (Measurement endpoints)
IC50 values for nitrogen fixation as nanomoles C4H4/g/h, number of nodules per plant, and dry weight per plant.
[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 occurred or one or more performance objectives in Table 3 were not met.  This list should not be misconstrued as limiting the reason(s) that a test could be found unacceptable or invalid.  However, except for the conditions listed in Table 3 and in OCSPP 850.4000, it is unlikely 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.
	Table 3. -- Test validity elements for the Rhizobium-legume Toxicity Test
--------------------------------------------------------------------------------
1. Seed germination percentage in controls was not at least 65% for Trifolium repens or for other legume species was not at least at the minimum acceptable USDA seed germination standards for vegetable crops and other available standards for agronomic crops described in paragraph (e)(5)(ii) of this guideline.
2. Mean survival of emerged control plants in either the negative or solvent control [if a vehicle was used] was not at least 90% for the duration of the study.
3. Control plant condition indicates visible phytotoxic or developmental symptoms during the test.
3. For any species, all organisms in a test were not from the same source.
4. All test containers were not identical and did not contain the same amount of substrate from the same source and the same amount of bacterial inoculum from the same source.
--------------------------------------------------------------------------------
5. Untreated (negative controls) and appropriate solvent controls [if a solvent was used] were not included in a 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 concentrations used in the range-finding and definitive test, or limit test.
            (iv) If a vehicle (solvent) 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 concentration(s) used in the treatments and solvent control.
	(4) Plant-bacteria association test species.
      (i) Plant scientific and common name, family, and variety.
	(ii) Bacterial scientific and common name, family, and strain.
	(iii) History of the legume seed: source, name of supplier, seed year or growing season collected, batch or lot number, seed treatment(s), and storage conditions.
	(iv) Test date of legume seed germination rating and germination percentage provided by supplier.
	(v) Seed size class.
	(vi) History of the Rhizobium bacteria: source, name of supplier, serial number, culture history and storage method, date of expiration and the package label listing of legumes for which the culture is to be used, and storage method.
	(vii) Cell count concentration in the inoculum and amount of peat and Rhizobium inoculum used per pot.
	(viii) Description of handling and processing of seeds before use in the test: storage, seed treatment(s) (e.g., sterilization), inoculation with Rhizobium sp., any pre-planting germination period.
	(ix) Date and description of planting.
      (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) The type of growth chamber or greenhouse.
	(ii) The description of pots: type, material, and dimensions.
	(iii) The number of seeds or total weight (for smaller species) of seeds per pot.  For smaller seeded species provide the results of the number of seeds counted in representative samples of the same total weight of seeds applied to pots, and descriptive statistics (mean and standard deviation of seeds per total weight).
	(iv) Number of pots per replicate, and number of replicates per treatment concentration and controls.
	(v) Description of the support medium: source, type, material.
	(vi) Volume of support medium per pot.
	(vii) Description of nutrient medium preparation.
	(viii) For the definitive test describe the methods for preparing the test treatments: exposure medium (nutrient solution or support medium), description of stock solution preparation and preparation of nutrient solution treatment concentrations (if test substance addition was to nutrient medium) or method of application and treatment to support medium (if test substance addition was to support media), and the test substance concentrations (concentration of the test substance in nutrient solution or the quantity of test substance per unit weight of root support material when it is coated on the support material).
	(ix) Methods used to determine the placement of potting containers in the test trays and the assignment of test concentrations to particular trays of pots to ensure randomization of exposure.
	(x) Date of test initiation and duration of test.
	(xi) Description of the nutrient solution delivery system and the frequency of nutrient solution delivery and replacement with fresh nutrient solution.
	(xii) Description of addition of reagent water to nutrient solution between replacement with fresh nutrient solution: source, frequency, volume.
	(xiii) Culture practices during the test such as pest control.
	(xiv) The photoperiod and light source.
	(xv) Methods and frequency of environmental monitoring performed during the definitive or limit study for air temperature, carbon dioxide concentration in air, relative humidity, light intensity, and nutrient solution pH.
	(xvi) Description of the methods, frequency, and duration of observations on plant appearance and condition and determination of nitrogen fixation (ethylene production).
	(xvii) For the definitive, or limit test, all analytical procedures should be described (e.g., nitrogen fixation, test substance, carbon dioxide air concentration).  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, relative humidity, light intensity, nutrient solution pH) in tabular form (provide raw data for measurements not made on a continuous basis), and descriptive statistics (mean, standard deviation, minimum, maximum).
	(ii) Analytical results in tabular form (provide raw data) of concentration of test substance in nutrient solution when the test substance addition was to the nutrient medium or concentration of test substance in substrate when the test substance addition was to the substrate medium and descriptive statistics (mean, standard deviation, minimum, maximum).
	(iii) For preliminary range-finding tests, if conducted, the number of emerged seedlings, survival of emerged seedlings, the sum of the root lengths (mm) of all plants of each test concentration and controls 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).
	(iv) For a limit test, tabulate for the limit concentration and the control by replicate, the number of seeds or total weight of seeds exposed at test initiation, the number of emerged seedlings, the number of surviving and number of dead emerged seedlings at each observation time, and the nitrogen fixation as nanomoles C4H4/g/h and nanomoles C4H4/plant/h, root nodules per plant, and total dry weight per plant at test termination (provide the raw data).
	(v) For the definitive test, tabulation by treatment and replicate of the number of seeds or total weight of seeds exposed at test initiation, the number of emerged seedlings, the number of surviving and number of dead emerged seedlings at each observation time, and the nitrogen fixation as nanomoles C4H4/g/h and nanomoles C4H4/plant/h, root nodules per plant, and total dry weight per plant at test termination (provide the raw data).
	(vi) For the limit and definitive tests, tabulation by each treatment level and control(s) the mean, standard deviation, and coefficient of variation for nitrogen fixation nanomoles C4H4 /g/h and nanomoles C4H4/plant/h, root nodules per plant and dry weight per plant, and percent reduction in nitrogen fixation and root nodules and dry weight per plant to controls at test termination.
	(vii) For the limit and definitive test, descriptions of the appearance and the growth and development of the plants (tops, roots, and nodules), indicating any abnormalities and visual (morphological) signs of phytotoxicity including: time of onset, duration, severity (e.g. rank), and number affected at each treatment level and control(s) (provide the raw data).  A description of the phytotoxicity rating system used, if any, should be included.
	(viii) Graphs of the concentration-response data for percent emerged, percent of emerged seedling survival, and nitrogen fixation, root nodules per plant, and total dry weight per plant at test termination.
	(ix) For a limit test, provide the results of hypothesis tests for nitrogen fixation, root nodules, and dry weight.
	(x) For the limit test, provide a description of the statistical methods used including software package, and the basis for the choice of method.
	(xi) For the definitive study and for those effect measures (nitrogen fixation, root nodules, and dry weight) with data sufficient to fit a concentration-response relationship, tabulation of the slope of the dose-response curve and its standard error and 95% confidence limits and any goodness of fit results.
	(xii) For the definitive test, provide table of IC50 values for nitrogen fixation, root nodules per plant, and dry weight per plant.
	(xiii) For the definitive test, a tabulation of the NOEC and LOEC for each measure of effect (nitrogen fixation, root nodules per plant, dry weight per plant).
	(xiv) Description of statistical method(s) used for point estimates, including software package, for determining IC50 values, fitting the concentration-response model, and the basis for the choice of method.  Provide results of any goodness-of-fit tests.
	(xv) Description of statistical method(s) used for NOEC and LOEC determination, including software package, and the basis for the choice of method.
(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.


[Federal Register Volume 86, Number 216 (Friday, November 12, 2021)]
[Notices]
[Pages 62855-62858]
[FR Doc No: 2021-24620]

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SECURITIES AND EXCHANGE COMMISSION

[Release No. 34-93528; File No. SR-NYSEArca-2021-93]

Self-Regulatory Organizations; NYSE Arca, Inc.; Notice of Filing
and Immediate Effectiveness of Proposed Rule Change To Reflect a
Modification to the Permitted Components of the Tracking Baskets of the
Putnam Focused Large Cap Growth ETF, Putnam Focused Large Cap Value
ETF, Putnam Sustainable Future ETF, and Putnam Sustainable Leaders ETF

November 5, 2021.
    Pursuant to Section 19(b)(1) \1\ of the Securities Exchange Act of
1934 (``Act'') \2\ and Rule 19b-4 thereunder,\3\ notice is hereby given
that, on October 29, 2021, NYSE Arca, Inc. (``NYSE Arca'' or the
``Exchange'') filed with the Securities and Exchange Commission
(``Commission'') the proposed rule change as described in Items I and
II below, which Items have been prepared by the Exchange. The
Commission is publishing this notice to solicit comments on the
proposed rule change from interested persons.
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    \1\ 15 U.S.C. 78s(b)(1).
    \2\ 15 U.S.C. 78a.
    \3\ 17 CFR 240.19b-4.
---------------------------------------------------------------------------

I. Self-Regulatory Organization's Statement of the Terms of Substance
of the Proposed Rule Change

    The Exchange proposes to reflect an amendment to the Application
and Exemptive Order governing the Putnam Focused Large Cap Growth ETF,
Putnam Focused Large Cap Value ETF, Putnam Sustainable Future ETF, and
Putnam Sustainable Leaders ETF, which are listed and traded on the
Exchange under NYSE Arca Rule 8.601-E. The proposed rule change is
available on the Exchange's website at www.nyse.com, at the principal
office of the Exchange, and at the Commission's Public Reference Room.

II. Self-Regulatory Organization's Statement of the Purpose of, and
Statutory Basis for, the Proposed Rule Change

    In its filing with the Commission, the self-regulatory organization
included statements concerning the purpose of, and basis for, the
proposed rule change and discussed any comments it received on the
proposed rule change. The text of those statements may be examined at
the places specified in Item IV below. The Exchange has prepared
summaries, set forth in sections A, B, and C below, of the most
significant parts of such statements.

A. Self-Regulatory Organization's Statement of the Purpose of, and the
Statutory Basis for, the Proposed Rule Change

Purpose
    The Exchange adopted NYSE Arca Rule 8.601-E for the purpose of
permitting the listing and trading, or trading pursuant to unlisted
trading privileges (``UTP''), of Active Proxy Portfolio Shares, which
are securities issued by an actively managed open-end investment
management company.\4\

Commentary .01 to Rule 8.601-E requires the Exchange to file separate
proposals under Section 19(b) of the Act before listing and trading any
series of Active Proxy Portfolio Shares on the Exchange. Pursuant to
this provision, the Exchange submitted a proposal to list and trade
shares (``Shares'') of Active Proxy Portfolio Shares of the Putnam
Focused Large Cap Growth ETF, Putnam Focused Large Cap Value ETF,
Putnam Sustainable Future ETF, and Putnam Sustainable Leaders ETF \5\
(each a ``Fund'' and, collectively, the ``Funds'') on the Exchange
under NYSE Arca Rule 8.601-E. The Shares are listed and traded on the
Exchange pursuant to their listing rule.\6\
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    \4\ See Securities Exchange Act Release No. 89185 (June 29,
2020), 85 FR 40328 (July 6, 2020) (SR-NYSEArca-2019-95). Rule 8.601-
E(c)(1) provides that ``[t]he term ``Active Proxy Portfolio Share''
means a security that (a) is issued by a investment company
registered under the Investment Company Act of 1940 (``Investment
Company'') organized as an open-end management investment company
that invests in a portfolio of securities selected by the Investment
Company's investment adviser consistent with the Investment
Company's investment objectives and policies; (b) is issued in a
specified minimum number of shares, or multiples thereof, in return
for a deposit by the purchaser of the Proxy Portfolio and/or cash
with a value equal to the next determined net asset value (``NAV'');
(c) when aggregated in the same specified minimum number of Active
Proxy Portfolio Shares, or multiples thereof, may be redeemed at a
holder's request in return for the Proxy Portfolio and/or cash to
the holder by the issuer with a value equal to the next determined
NAV; and (d) the portfolio holdings for which are disclosed within
at least 60 days following the end of every fiscal quarter.'' Rule
8.601-E(c)(2) provides that ``[t]he term ``Actual Portfolio'' means
the identities and quantities of the securities and other assets
held by the Investment Company that shall form the basis for the
Investment Company's calculation of NAV at the end of the business
day.'' Rule 8.601-E(c)(3) provides that ``[t]he term ``Proxy
Portfolio'' means a specified portfolio of securities, other
financial instruments and/or cash designed to track closely the
daily performance of the Actual Portfolio of a series of Active
Proxy Portfolio Shares as provided in the exemptive relief pursuant
to the Investment Company Act of 1940 applicable to such series.''
    \5\ On May 13, 2021, the Commission published the notice of
filing and immediate effectiveness relating to the listing and
trading of shares of the Putnam Focused Large Cap Growth ETF, Putnam
Focused Large Cap Value ETF, Putnam Sustainable Future ETF, and
Putnam Sustainable Leaders ETF. See Securities Exchange Act Release
No. 91895 (May 13, 2021), 86 FR 27126 (May 19, 2021) (SR-NYSEArca-
2021-39) (the ``Notice'').
    \6\ See id. The Putnam ETF Trust filed an application for an
order under Section 6(c) of the 1940 Act for exemptions from various
provisions of the 1940 Act and rules thereunder (File No. 812-
15203), dated February 19, 2021 (the ``Application''). On May 10,
2021, the Commission issued an order (the ``Exemptive Order'') under
the 1940 Act granting the exemptions requested in the Application
(Investment Company Act Release No. 34266, May 10, 2021).
Investments made by the Funds will comply with the conditions set
forth in the Application and the Exemptive Order. See Notice, 86 FR
at 27127-28 n. 9.
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    The Application and Exemptive Order incorporate by reference the
terms and conditions of the exemptive order granted to Fidelity Beach
Street Trust (``Beach Street''), Fidelity Management & Research Company
(``FMR''), Fidelity Distributors Corporation (``FDC''), as such order
may be amended from time to time, and the purpose of this filing is to
reflect an amendment to such relief, as described below.
The Fidelity Exemptive Relief
    Beach Street, FMR, and FDC filed a ninth amended application for an
order under Section 6(c) of the 1940 Act for exemptions from various
provisions of the 1940 Act and rules thereunder (the ``Prior Fidelity
Application'').\7\ On December 10, 2019, the Commission issued an order
(the ``Prior Fidelity Exemptive Order'') under the 1940 Act granting
the exemptions requested in the Prior Fidelity Application.\8\ The
Exchange previously submitted a proposal to list and trade shares of
Active Proxy Portfolio Shares of the Fidelity Women's Leadership ETF
and Fidelity Sustainability U.S. Equity ETF (the ``Fidelity Funds''),
which are subject to the Prior Fidelity Exemptive Order.\9\
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    \7\ See File No. 812-14364, dated November 8, 2019.
    \8\ See Investment Company Act Release No. 33712, December 10,
2019.
    \9\ On April 14, 2021, the Commission published the notice of
filing and immediate effectiveness relating to the listing and
trading of shares of the Fidelity Funds. See Securities Exchange Act
Release No. 91514 (April 8, 2021), 86 FR 19657 (April 14, 2021) (SR-
NYSEArca-2021-23) (the ``Fidelity Notice'').
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    Under the Prior Fidelity Exemptive Order, the Fidelity Funds are
required to publish a basket of securities and cash that, while
different from a fund's portfolio, is designed to closely track its
daily performance (the ``Fidelity Proxy Portfolio''). The Prior
Fidelity Application stated that the Fidelity Proxy Portfolio is
comprised of (1) select recently disclosed portfolio holdings
(``Strategy Components''); (2) liquid ETFs that convey information
about the types of instruments in which the fund invests that are not
otherwise fully represented by Strategy Components (``Representative
ETFs''); and (3) cash and cash equivalents. As set forth in the
Fidelity Notice, investments made by the Fidelity Funds will comply
with the conditions set forth in the Prior Application and the Prior
Exemptive Order.\10\
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    \10\ See Fidelity Notice, 86 FR at 19658, n. 8.
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    On October 30, 2020, and as amended on April 2, 2021, June 11, 2021
and June 30, 2021, Beach Street, FMR, FDC, and Fidelity Covington Trust
sought to amend the Prior Fidelity Exemptive Order to, among other
things, permit the Fidelity Funds to include select securities from the
universe from which a fund's investments are selected such as a broad-
based market index in the fund's Proxy Portfolio.\11\ On August 5,
2021, the Commission issued an order granting the relief requested (the
``Updated Fidelity Exemptive Order'').\12\ The Exchange subsequently
submitted a proposal with respect to the listing and trading of the
Fidelity Funds to reflect the conditions set forth in the Updated
Fidelity Exemptive Order.\13\
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    \11\ See File No. 812-15175.
    \12\ See Investment Company Act Release No. 34350, August 5,
2021. Although the Updated Fidelity Exemptive Order permits the use
of Creation Baskets that include instruments that are not included,
or are included with different weightings, in a fund's Proxy
Portfolio, that aspect of the Updated Fidelity Exemptive Order is
not part of this proposed rule change.
    \13\ See Securities Exchange Act Release No. 93108 (September
23, 2021), 86 FR 53993 (September 29, 2021), (SR-NYSEArca-2021-81).
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The Funds
    As set forth in the Exchange's previous rule filing to list and
trade Shares of the Funds, under the Exemptive Order, the Funds are
required to publish a Tracking Basket \14\ of securities and cash that,
while different from a Fund's portfolio, is designed to closely track
its daily performance. Like the Fidelity Funds, the Tracking Basket for
each of the Funds will be comprised of Strategy Components,
Representative ETFs, and cash and cash equivalents.
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    \14\ The Funds use the term ``Tracking Basket'' to mean the
Proxy Portfolio for purposes of Rule 8.601-E(c)(3). See Notice, 86
FR at 27128 n. 12.
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    As set forth in the Notice, investments made by the Funds will
comply with the conditions set forth in the Application and the
Exemptive Order, which incorporate by reference the terms and
conditions of the exemptive order granted to Beach Street, FMR, and
FDC, as such order may be amended from time to time.\15\ Accordingly,
the Exchange proposes to update the listing rule for the Shares to
reflect the amendment of the Prior Fidelity Exemptive Order and the
updated conditions set forth in the Updated Fidelity Exemptive Order as
they relate to the Funds. Specifically, the Exchange proposes to
reflect the condition in the Updated Fidelity Exemptive Order that
permits the Funds to include select securities from the universe from
which a Fund's investments are selected such as a broad-based market
index in the Fund's Tracking Basket.
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    \15\ See Notice, 86 FR at 27127-28 n. 9; Application at 2 n. 1;
Exemptive Order at 1 n. 1.
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    Except for the change noted above, all other representations made
in the Exchange's previous rule filing to list and trade Shares of the
Funds remain unchanged and will continue to constitute continuing
listing requirements for the Funds. The Funds will also continue to
comply with the requirements of Rule 8.601-E.
2. Statutory Basis
    The Exchange believes that the proposed rule change is consistent
with Section 6(b) of the Act,\16\ in general, and furthers the
objectives of Section 6(b)(5) of the Act,\17\ in particular, in that it
is designed to prevent fraudulent and manipulative acts and practices,
to promote just and equitable principles of trade, to remove
impediments to and perfect the mechanism of a free and open market and
a national market system, and, in general, to protect investors and the
public interest.\18\
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    \16\ 15 U.S.C. 78f(b).
    \17\ 15 U.S.C. 78f(b)(5).
    \18\ The Exchange represents that, for initial and continued
listing, the Funds will be in compliance with Rule 10A-3 under the
Act, as provided by NYSE Arca Rule 5.3-E.
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    The proposed rule change is designed to perfect the mechanism of a
free and

open market and, in general, to protect investors and the public
interest. The proposed revision is intended to allow the Funds to
comply with the Updated Fidelity Exemptive Order, which is incorporated
by reference in the Application and Exemptive Order. Specifically, the
proposed rule change would permit the Funds to include select
securities from the universe from which a Fund's investments are
selected such as a broad-based market index in such Fund's Tracking
Basket. The Exchange also believes that the proposed change is designed
to perfect the mechanism of a free and open market and, in general, to
protect investors and the public interest because it would permit each
Fund to make certain adjustments to the components of its Tracking
Basket, thereby continuing to promote competition among various ETF
products. Except for the changes noted above, all other representations
made in the respective rule filings remain unchanged and, as noted,
will continue to constitute continuing listing requirements for the
Funds.

B. Self-Regulatory Organization's Statement on Burden on Competition

    The Exchange does not believe that the proposed rule change will
impose any burden on competition that is not necessary or appropriate
in furtherance of the purpose of the Act. As noted, the purpose of the
filing is to reflect an amendment to the Prior Fidelity Exemptive
Order, the terms and conditions of which are incorporated by reference
in the Application and Exemptive Order governing the listing and
trading of the Funds. To the extent that the proposed rule change would
continue to permit listing and trading of other types of actively-
managed ETF that have characteristics different from existing actively-
managed and index ETFs, the Exchange believes that the proposal would
benefit investors by continuing to promote competition among various
ETF products.

C. Self-Regulatory Organization's Statement on Comments on the Proposed
Rule Change Received from Members, Participants, or Others

    No written comments were solicited or received with respect to the
proposed rule change.

III. Date of Effectiveness of the Proposed Rule Change and Timing for
Commission Action

    Because the foregoing proposed rule change does not: (i)
Significantly affect the protection of investors or the public
interest; (ii) impose any significant burden on competition; and (iii)
become operative for 30 days from the date on which it was filed, or
such shorter time as the Commission may designate, it has become
effective pursuant to Section 19(b)(3)(A) of the Act \19\ and Rule 19b-
4(f)(6) thereunder.\20\
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    \19\ 15 U.S.C. 78s(b)(3)(A).
    \20\ 17 CFR 240.19b-4(f)(6). In addition, Rule 19b-4(f)(6)(iii)
requires a self-regulatory organization to give the Commission
written notice of its intent to file the proposed rule change, along
with a brief description and text of the proposed rule change, at
least five business days prior to the date of filing of the proposed
rule change, or such shorter time as designated by the Commission.
The Exchange satisfied this requirement.
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    A proposed rule change filed under Rule 19b-4(f)(6) \21\ normally
does not become operative prior to 30 days after the date of the
filing. However, pursuant to Rule 19b-4(f)(6)(iii),\22\ the Commission
may designate a shorter time if such action is consistent with the
protection of investors and the public interest. The Exchange has asked
the Commission to waive the 30-day operative delay so that the proposed
rule change may take effect upon filing. The Exchange states that the
proposed rule change raises no novel regulatory issues because the
proposed rule change would reflect an aspect of exemptive relief that
the Commission has already granted. The Exchange also notes that
similar proposals to amend the listing rules of other shares that NYSE
Arca also lists and trades pursuant to Rule 8.601-E currently are in
effect.\23\ For these reasons, the Commission believes that waiver of
the 30-day operative delay is consistent with the protection of
investors and the public interest. Accordingly, the Commission waives
the 30-day operative delay and designates the proposal operative upon
filing.\24\
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    \21\ 17 CFR 240.19b-4(f)(6).
    \22\ 17 CFR 240.19b-4(f)(6)(iii).
    \23\ See Securities Exchange Act Release No. 92449 (July 20,
2021), 86 FR 40102 (July 26, 2021) (SR-NYSEArca-2021-61) and No.
93108 (September 23, 2021) (SR-NYSEArca-2021-81).
    \24\ For purposes only of waiving the 30-day operative delay,
the Commission has considered the proposed rule's impact on
efficiency, competition, and capital formation. See 15 U.S.C.
78c(f).
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    At any time within 60 days of the filing of the proposed rule
change, the Commission summarily may temporarily suspend such rule
change if it appears to the Commission that such action is necessary or
appropriate in the public interest, for the protection of investors, or
otherwise in furtherance of the purposes of the Act. If the Commission
takes such action, the Commission shall institute proceedings to
determine whether the proposed rule change should be approved or
disapproved.

IV. Solicitation of Comments

    Interested persons are invited to submit written data, views, and
arguments concerning the foregoing, including whether the proposed rule
change is consistent with the Act. Comments may be submitted by any of
the following methods:

Electronic Comments

     Use the Commission's internet comment form (http://www.sec.gov/rules/sro.shtml); or
     Send an email to [email protected]. Please include
File Number SR-NYSEArca-2021-93 on the subject line.

Paper Comments

     Send paper comments in triplicate to: Secretary,
Securities and Exchange Commission, 100 F Street, NE, Washington, DC
20549-1090.

All submissions should refer to File Number SR-NYSEArca-2021-93. This
file number should be included on the subject line if email is used. To
help the Commission process and review your comments more efficiently,
please use only one method. The Commission will post all comments on
the Commission's internet website (http://www.sec.gov/rules/sro.shtml).
Copies of the submission, all subsequent amendments, all written
statements with respect to the proposed rule change that are filed with
the Commission, and all written communications relating to the proposed
rule change between the Commission and any person, other than those
that may be withheld from the public in accordance with the provisions
of 5 U.S.C. 552, will be available for website viewing and printing in
the Commission's Public Reference Room, 100 F Street NE, Washington, DC
20549 on official business days between the hours of 10:00 a.m. and
3:00 p.m. Copies of the filing also will be available for inspection
and copying at the principal office of the Exchange. All comments
received will be posted without change. Persons submitting comments are
cautioned that we do not redact or edit personal identifying
information from comment submissions. You should submit only
information that you wish to make available publicly. All submissions
should refer to File Number SR-NYSEArca-2021-93 and should be submitted
on or before December 3, 2021.

    For the Commission, by the Division of Trading and Markets,
pursuant to delegated authority.\25\
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    \25\ 17 CFR 200.30-3(a)(12).
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Jill M. Peterson,
Assistant Secretary.
[FR Doc. 2021-24620 Filed 11-10-21; 8:45 am]
BILLING CODE 8011-01-P