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
i

NOTICE

This guideline is one of a series of test guidelines established by the United States 

Environmental Protection Agency’s Office of Chemical Safety and Pollution Prevention 

(OCSPP) for use in testing pesticides and chemical substances to develop data for 

submission to the Agency under the Toxic Substances Control Act (TSCA) (15 U.S.C. 2601, 

et seq.), the Federal Insecticide, Fungicide and Rodenticide Act (FIFRA) (7 U.S.C. 136, et 

seq.), and section 408 of the Federal Food, Drug and Cosmetic (FFDCA) (21 U.S.C. 346a). 

Prior to April 22, 2010, OCSPP was known as the Office of Prevention, Pesticides and Toxic 

Substances (OPPTS). To distinguish these guidelines from guidelines issued by other 

organizations, the numbering convention adopted in 1994 specifically included OPPTS as 

part of the guideline’s number. Any test guidelines developed after April 22, 2010 will use 

the new acronym (OCSPP) in their title.

The OCSPP harmonized test guidelines serve as a compendium of accepted scientific 

methodologies and protocols that are intended to provide data to inform regulatory decisions 

under TSCA, FIFRA, and/or FFDCA. This document provides guidance for conducting the 

test, and is also used by EPA, the public, and the companies that are subject to data 

submission requirements under TSCA, FIFRA, and/or the FFDCA. As a guidance 

document, these guidelines are not binding on either EPA or any outside parties, and the 

EPA may depart from the guidelines where circumstances warrant and without prior notice. 

At places in this guidance, the Agency uses the word “should.” In this guidance, the use of 

“should” with regard to an action means that the action is recommended rather than 

mandatory. The procedures contained in this guideline are strongly recommended for 

generating the data that are the subject of the guideline, but EPA recognizes that departures 

may be appropriate in specific situations. You may propose alternatives to the 

recommendations described in these guidelines, and the Agency will assess them for 

appropriateness on a case-by-case basis.

For additional information about these test guidelines and to access these guidelines 

electronically, please go to http://www.epa.gov/ocspp and select “Test Methods & 

Guidelines” on the left side navigation menu. You may also access the guidelines in 

http://www.regulations.gov grouped by Series under Docket ID #s: EPA-HQ-OPPT-2009-

0150 through EPA-HQ-OPPT-2009-0159, and EPA-HQ-OPPT-2009-0576.
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OCSPP 850.4600: 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 
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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 Rhizobiumlegume 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 (

oC)) 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 
Page 3 of 23

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/cm3). 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 multiconcentration 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 enduse 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 
Page 4 of 23

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 
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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. 
Page 6 of 23

(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 (

oC), 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.
Page 7 of 23

(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: fieldgarden 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 
Page 8 of 23

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.
Page 9 of 23

(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 
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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.
Page 11 of 23

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 
Page 12 of 23

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 oC, so the 

recommended test temperature for this species is 20 ± 5 oC. 

(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 (μmol/m2/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.
Page 13 of 23

(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/m3) 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.
Page 14 of 23

(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-tomoderate-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, 
Page 15 of 23

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 oC) 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 timeweighted 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.
Page 16 of 23

(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.

  

C

C X

I

100 %

 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 

(μlimit) from the control mean response (μcontrol) is greater than or equal to a 50 reduction, 

compared to the control (i.e., H0: μcontrol - μlimit > δ0). The alternative hypothesis (HA) is 

that this difference is less than a 50% reduction, compared to the control (HA: μcontrol - μlimit

< δ0). An example of a parametric two-sample comparison test of this is the Student’s ttest. 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).

   100  0  xcontrol  p Equation 2

where:

δ0 = difference between two parameters, defined in this case as a p percent 

reduction from the control sample mean;

control x = 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.
Page 17 of 23

(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. 
Page 18 of 23

Table 2.—Summary of Test Conditions for Rhizobium-legume Toxicity Test1

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 oC 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 μmol/m2/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. 
Page 19 of 23

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.
Page 20 of 23

(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 
Page 21 of 23

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 
Page 22 of 23

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.
Page 23 of 23

(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-offit 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.