&EPA
United States
Environmental Protection
Agency
Office of Chemical Safety
and Pollution Prevention
(7101)
EPA712-C-004
January 2012
Ecological Effects
Test Guidelines
OCSPP 850.4600:
Rhizobium-Legume
Toxicity
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NOTICE
This guideline is one of a series of test guidelines established by the United States
Environmental Protection Agency's Office of Chemical Safety and Pollution Prevention
(OCSPP) for use in testing pesticides and chemical substances to develop data for
submission to the Agency under the Toxic Substances Control Act (TSCA) (15 U.S.C. 2601,
et seq.), the Federal Insecticide, Fungicide and Rodenticide Act (FIFRA) (7 U.S.C. 136, et
seq.), and section 408 of the Federal Food, Drug and Cosmetic (FFDCA) (21 U.S.C. 346a).
Prior to April 22, 2010, OCSPP was known as the Office of Prevention, Pesticides and Toxic
Substances (OPPTS). To distinguish these guidelines from guidelines issued by other
organizations, the numbering convention adopted in 1994 specifically included OPPTS as
part of the guideline's number. Any test guidelines developed after April 22, 2010 will use
the new acronym (OCSPP) in their title.
The OCSPP harmonized test guidelines serve as a compendium of accepted scientific
methodologies and protocols that are intended to provide data to inform regulatory decisions
under TSCA, FIFRA, and/or FFDCA. This document provides guidance for conducting the
test, and is also used by EPA, the public, and the companies that are subject to data
submission requirements under TSCA, FIFRA, and/or the FFDCA. As a guidance
document, these guidelines are not binding on either EPA or any outside parties, and the
EPA may depart from the guidelines where circumstances warrant and without prior notice.
At places in this guidance, the Agency uses the word "should." In this guidance, the use of
"should" with regard to an action means that the action is recommended rather than
mandatory. The procedures contained in this guideline are strongly recommended for
generating the data that are the subject of the guideline, but EPA recognizes that departures
may be appropriate in specific situations. You may propose alternatives to the
recommendations described in these guidelines, and the Agency will assess them for
appropriateness on a case-by-case basis.
For additional information about these test guidelines and to access these guidelines
electronically, please go to http://www.epa.qov/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)0) 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-moculated 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-legumQ
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 (°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-legumQ 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 ICso 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
1C 10 to ICgo) 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
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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 ICso 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. ICso 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 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
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Rhizobium-legumQ 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)(l) 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)(7j 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)(7j of this
guideline) or added in solvent stock solution to nutrient medium (see
paragraph (e)(4)(i)(B)(%) 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 arable, 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.
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(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., 1C50), 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
ICso 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 (ICio).
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 (°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.
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(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)(%) 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
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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-legumQ 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.
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(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
.PLEXIGLAS
LID
DRAIN
HOLE
BOARD GLASS TUBE
"•SCREEN
TABLE TOP
FLEXIBLE
RUBBER.
TUBING
,NO. 13 RUBBER
STOPPER
THREADED CAP
30-L
CARBOY
AIR
BLACK PLASTIC
15-L
NUTRIENT
SOLUTION
(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-legumQ association is described in Table 1.
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Table 1.—Preparation of nutrient medium
Concentration of
Substance Substance (mg/L)
Potassium sulfate (K2SO4) 901
Potassium phosphate (KH2PO4) 272
Calcium chloride (CaCI2) 416
Magnesium sulfate heptahydrate (MgSO4 • 7H2O) 493
Ferric chloride hexahydrate (FeCI3 • 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 (MnCI2 • 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 (KNOs)
and 28.3 grams of calcium dinitrate tetrahydrate (Ca(NC>3)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 (HC1) 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 °C 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
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inert gas to flush the chambers and acetylene gas (C2H2) to fill chambers to
promote production of ethylene gas (€4114). 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 °C, so the
recommended test temperature for this species is 20 ± 5 °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 (|imol/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 (HC1) 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.
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(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.
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(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 €2^2 Following exposure
to C^2 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 C^2 as
described in this paragraph. Using the test conditions and clover and bean
species recommended, incubation periods of 5 hours and 1 hour,
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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 °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.
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(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 .
Equat.on1
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 ICso 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 ICso, compared to the controls, is operationally defined as a 50% reduction
from the control sample mean (Equation 2). The null hypothesis (Ho) stated in terms of
true population parameters is that the difference of the "limit" treatment mean response
delimit) from the control mean response (|iCOntroi) is greater than or equal to a 50 reduction,
compared to the control (i.e., HQ: |icontroi - Hiimit > So). The alternative hypothesis (HA) is
that this difference is less than a 50% reduction, compared to the control (HA: ^control - Hiimit
< 5o). An example of a parametric two-sample comparison test of this is the Student's t-
test. 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:
5o = difference between two parameters, defined in this case as a p percent
reduction from the control sample mean;
* control = 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.
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(4) Multiple-dose definitive test—
(i) Concentration-response curve, slope and ICso. For dose-response tests
statistical procedures are employed to calculate the ICso 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 ICso, 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 ICio
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
ICio 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.
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Table 2.—Summary of Test Conditions for Rhizobium-\egume Toxicity Test1
Test duration
Substrate
Nutrients
Temperature
Relative humidity
Carbon dioxide
Light quality
Light intensity
Photoperiod
Watering
Test chamber (pot) size
Number of organisms per test chamber
Number of replicate chambers per test treatment
Test treatment levels
Test substance application method
Measures of effect (Measurement endpoints)
Three to seven weeks (establishment of effective
root nodulation in controls, dependent on
Rhizobium-legume complex tested)
Quartz sand, 0.5 - 1 .0 mm diameter
Watered with nutrient solution
Optimal for the species used; 20 ± 5 °C for clover
70/90% (day/night) ± 5% (for growth chamber
studies)
350 ± 50 ppm
fluorescent or representative of natural sunlight
350 ± 50 umol/m^/s at the top of the canopy
16-hours light: 8-hours dark
Bottom watering as needed, using nutrient solution
Varies with plant species selected
0.3 g dry seed per pot (for clover); 1 seedling per
pot (for bush beans)
6 (minimum)
Unless performing limit test, minimum of 5 treatment
levels plus appropriate controls
Root exposure (via watering with nutrient solution or
sorbed onto the substrate)
IC50 values for nitrogen fixation as nanomoles
C4H4/g/h, number of nodules per plant, and dry
weight per plant.
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.
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Table 3.—Test validity elements for the Rhizobium-\egume 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)0) of
the OCSPP 850.4000 guideline.
(2) Guideline deviations. Provide a statement of the guideline or protocol followed.
Include a description of any deviations from the test guideline or any occurrences which
may have influenced the results of the test.
(3) Test substance.
(i) Identification of the test substance: common name, IUPAC and CAS names,
CAS number, structural formula, source, lot or batch number, chemical state or
form of the test substance, and its purity (i.e. for pesticides, the identity and
concentration of active ingredient(s)).
(ii) Storage conditions of the test chemical or test substance and stability of the test
chemical or test substance under storage conditions if stored prior to use.
(iii) Methods of preparation of the test substance and the treatment 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.
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(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
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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
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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
C4HVg/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 ICso values for nitrogen fixation, root
nodules per plant, and dry weight per plant.
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(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 ICso 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.
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