United States
Environmental Protection
Agency
Prevention, Pesticides
and Toxic Substances
(7101)
EPA712-C-96-158
April 1996
&EPA Ecological Effects Test
Guidelines
OPPTS 850.4600
Rhizobium-Leg u me
Toxicity
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Public Draft"

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Introduction
This guideline is one of a series of test guidelines that have been
developed by the Office of Prevention, Pesticides and Toxic Substances,
United States Environmental Protection Agency for use in the testing of
pesticides and toxic substances, and the development of test data that must
be submitted to the Agency for review under Federal regulations.
The Office of Prevention, Pesticides and Toxic Substances (OPPTS)
has developed this guideline through a process of harmonization that
blended the testing guidance and requirements that existed in the Office
of Pollution Prevention and Toxics (OPPT) and appeared in Title 40,
Chapter I, Subchapter R of the Code of Federal Regulations (CFR), the
Office of Pesticide Programs (OPP) which appeared in publications of the
National Technical Information Service (NTIS) and the guidelines pub-
lished by the Organization for Economic Cooperation and Development
(OECD).
The purpose of harmonizing these guidelines into a single set of
OPPTS guidelines is to minimize variations among the testing procedures
that must be performed to meet the data requirements of the U. S. Environ-
mental Protection Agency under the Toxic Substances Control Act (15
U.S.C. 2601) and the Federal Insecticide, Fungicide and Rodenticide Act
(7 U.S.C. 136, etseq.).
Public Draft Access Information: This draft guideline is part of a
series of related harmonized guidelines that need to be considered as a
unit. For copies: These guidelines are available electronically from the
EPA Public Access Gopher (gopher.epa.gov) under the heading "Environ-
mental Test Methods and Guidelines" or in paper by contacting the OPP
Public Docket at (703) 305-5805 or by e-mail:
guidelines@epamail.epa.gov.
To Submit Comments: Interested persons are invited to submit com-
ments. By mail: Public Docket and Freedom of Information Section, Office
of Pesticide Programs, Field Operations Division (7506C), Environmental
Protection Agency, 401 M St. SW., Washington, DC 20460. In person:
bring to: Rm. 1132, Crystal Mall #2, 1921 Jefferson Davis Highway, Ar-
lington, VA. Comments may also be submitted electronically by sending
electronic mail (e-mail) to: guidelines@epamail.epa.gov.
Final Guideline Release: This guideline is available from the U.S.
Government Printing Office, Washington, DC 20402 on The Federal Bul-
letin Board. By modem dial 202-512-1387, telnet and ftp:
fedbbs.access.gpo.gov (IP 162.140.64.19), or call 202-512-0135 for disks
or paper copies. This guideline is also available electronically in ASCII
and PDF (portable document format) from the EPA Public Access Gopher
(gopher.epa.gov) under the heading "Environmental Test Methods and
Guidelines."
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OPPTS 850.4600 Rhizobium-legume toxicity.
(a)	Scope—(1) Applicability. This guideline is intended to meet test-
ing requirements of both the Federal Insecticide, Fungicide, and
Rodenticide Act (FIFRA) (7 U.S.C. 136, et seq.) and the Toxic Substances
Control Act (TSCA) (15 U.S.C. 2601).
(2) Background. The source material used in developing this har-
monized OPPTS test guideline is 40 CFR 797.2900 Rhizobium-legume
Chronic Toxicity Test (proposed in the Federal Register of September
28, 1987 (52 FR 36339)).
(b)	Purpose. This guideline is intended for use in developing data
on the toxicity of chemical substances and mixtures ("test substances").
The guideline prescribes tests using commercially important terrestrial
plants and their nitrogen-fixing bacterial symbionts to develop data on the
phytotoxicity of test substances. EPA will use data from these tests in
assessing the hazard of a test substance to the environment.
(c)	Definitions. The definitions in section 3 of TSCA and 40 CFR
Part 792—Good Laboratory Practice Standards apply to this test guideline.
The following definitions also apply:
EC X means the experimentally-derived test substance concentration
that is calculated to affect X percent of the test effect (e.g., EC40).
Germination means the resumption of active growth by a plant em-
bryo.
Legume means a member of the pea family (leguminosae) and in-
cludes many species of great economic importance.
Nitrogen-fixation means the conversion of elemental nitrogen to ni-
trates by Rhizobium which colonize legume root nodules.
Rhizobium means a genus of symbiotic bacteria that forms nodules
on the roots of certain legumes.
Support media means the quartz sand used to support the plant.
Symbiont means either of two organisms participating in a symbiotic
relationship.
Symbiosis means the close union of two dissimilar organisms in a
mutually beneficial relationship.
(d)	Test procedures—(1) Summary of the test. Seeds of a legume
species are inoculated with their specific Rhizobium symbiont and planted
in sand irrigated with a nutrient solution. The test substance is applied
to the plant-bacteria complex via the nutrient solution or is adsorbed to
the support media, resulting in continuous exposure to the test substance
from the time the seed (or seedling, if appropriate) is planted to maturity
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of the plant. After significant leaf development has occurred (usually after
several weeks during which the Rhizobium-infected plants are irrigated at
regular intervals with the nutrient solution), all plants are harvested for
analysis. Effects are evaluated by comparing plant yield, nodule produc-
tion, and nitrogen-fixation in plants exposed to the test substance to those
plants not exposed (negative controls) to the test substance.
(2)	Application of test substance, (i) Deionized or glass-distilled
water should be used in making stock solutions of a water-soluble test
substance. Sufficient quantities of each concentration should be made as
needed to minimize storage time and disposal volume. A measured portion
of the stock solution should be added to the nutrient solution just before
beginning the test.
(ii)	A test substance that is insoluble in water, but which can be sus-
pended in an aqueous solution by a carrier, should be added with the car-
rier, to the nutrient solution. The carrier 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 carriers—however,
acetone, gum arabic, polyethylene glycol, ethanol, and other solvents have
been used extensively in testing herbicides, plant growth regulators, fun-
gicides, and other chemical substances that affect plants. Carrier controls
should be included in the experimental design and tested simultaneously
with the test substance.
(iii)	A water-insoluble test substance for which no nontoxic, water-
soluble carrier 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
weighed portion of support media should be weighed, the test substance
should be extracted with the same organic solvent, and the concentration
of the test substances should be determined before the potting containers
are filled. Solvent controls should be included in the experimental design
and tested simultaneously with the test substance.
(3)	Selection of initial test substance concentrations, (i) A prelimi-
nary test should be conducted to determine the concentrations of test sub-
stance to be used in the definitive test for each Rhizobium-legume associa-
tion. For this purpose, seed germination, the first event in the establishment
of a Rhizobium-legume symbiotic relationship, may be used.
(ii) 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 O.lx,
lx, and lOx the anticipated environmental concentration. After a given
exposure period, the effects should be assessed as the sum of the root
lengths (in millimeters) of all plants of each test concentration, relative
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to that evidenced in the controls. Should reasonable predictions of potential
environmental exposure concentrations not be possible, seeds of the same
legume should be exposed to a series of widely spaced concentrations
(e.g., 0.01, 0.1, 1.0, 10, 100, 1,000 mg/L) of the test substance. After a
given period, root lengths should be compared as previously described.
The lowest concentration tested in the series, exclusive of controls, should
be at the analytical detection limit of the test substance. The upper con-
centration, for water-soluble test substances, should not exceed 50 percent
of the saturation concentration.
(iii)	The seed-germination 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 °C) in Petri dishes, allowing ade-
quate room for linear root growth. When 65 percent of the control seeds
have germinated and developed roots at least 20 mm long, the test may
be terminated.
(iv)	No replicates are required and nominal concentrations are accept-
able.
(4) Definitive test, (i) The purpose of the definitive test is to deter-
mine whether the test substance is toxic to the selected Rhizobium-legume
association and, if so, to delineate its concentration response curves and
EC50 values for each of three variables of the test system used.
(ii)	Since the anticipated fate of the test substance involves soil or
soil water, and the mechanism of toxicity depends upon root exposure,
the test substance should be applied in nutrient solution to the support
media or coated on the support media for water-insoluble test substances
for which no nontoxic, water-soluble carrier is available. The test sub-
stance should be chemically stable in the nutrient solution.
(iii)	Seeds of legume species that are subject to attack by mold (e.g.,
clovers) may be washed with ethanol before being planted.
(iv)	Seeds should be mixed with a small amount of moist commercial
peat previously inoculated with the desired Rhizobium species. Seeds of
some legumes (e.g., Trifolium repens, white clover) may be planted imme-
diately in pots (0.3 g dry seed per pot) filled to within 2.5 cm of the
top with support media of clean, coarse (0.5 to 1.0 mm diameter) sand,
while others (e.g., Phaseolus vulgaris, bush bean), once inoculated with
Rhizobium, should be allowed to germinate for as many as 6 days in dark-
ness between moist paper towels before being planted, one seedling per
pot. The support media should be irrigated with the nutrient solution before
planting occurs. Unless it is necessary to adsorb the test substance to the
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support media, the nutrient solution should contain the desired concentra-
tion of test substance.
(v)	Six replicate pots of seed for each of at least five concentrations
of test substance, exclusive of controls, should be used in the definitive
test. For each Rhizobium-legume association tested, the concentration
range should be selected to define, as closely as possible, the concentra-
tion-response curve between the EC 10 and EC90.
(vi)	Pots should be irrigated regularly (for example, 30 min/h) with
nutrient solution, preferably using a system in which water flows from
the bottom of the pot upward. Irrigation should be suspended for 6 to
8 h before nitrogen-fixation measurement. Nutrient solution should be re-
placed with fresh solution at least once every 2 weeks. It may be necessary
to replace the nutrient solution more frequently if the test substance is
rapidly degraded under test conditions.
(vii)	Every test should include controls consisting of the same dilution
water, conditions, procedures, bacteria from the same culture, and seed
from the same lot used in the exposed groups, except that none of the
test substance should be added. If a carrier solvent is needed to dissolve
or suspend the test substance, a carrier control should also be included.
(viii)	Alternative planting methods may be required when the test sub-
stance 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 °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. Control pots should be handled identically
to the test pots but with no exposure to the test substance.
(ix)	The definitive test consists of exposing the selected Rhizobium-
legume association to at least five concentrations of the test substance,
using a minimum of six replicate pots for each concentration and control,
followed by measurements and analyses of nitrogen-fixation, nodulation
performance, and plant yield. The duration of the test should be 3 to 7
weeks from the date of planting, depending on the legume used.
(x)	Plants should be observed daily. All visible effects of the test
substance on plant growth and morphology, such as stunting, discoloration,
chlorosis, or necrosis of the leaves should be noted.
(xi)	To measure nitrogen-fixation for a small-to-moderate-sized leg-
ume species (e.g., T. repens, white clover), each pot should be enclosed
in an airtight chamber. To enhance the reduction of acetylene (C2H2), the
chambers may be flushed with an inert gas (evacuating the nitrogen-con-
taining air) before introducing the C2H2. Following exposure to C2H2 for
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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. Should be 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 with-
drawn and analyzed for C4H4 after an appropriate incubation period (as
above) in the presence of C2H2. Using the test conditions and clover and
bean species recommended, incubation periods of 5 h and 1 h, respec-
tively, are suitable for nitrogen-fixation determinations. Optimal incubation
times for other species in containers of other sizes may be different.
(xii)	Nodulation performance should be assessed by counting the cu-
mulative number of root nodules on the plants from each treatment group.
Yield should be recorded as the total dry (70 °C, 48 h) biomass (tops
and roots) per pot.
(xiii)	The assignment of pots to test substance concentrations should
be random. In addition, 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.
(xiv)	Irradiation measurements should be taken at the top of the plant
canopy and the mean, maximum, and minimum values determined over
the plant-growing area. These measurements should be taken daily, but
should be taken at least at the start and completion of the test. If the test
is conducted in a greenhouse facility, hourly measurements of irradiation
should be recorded and presented as daily total irradiance including rep-
resentative hourly curves for clear-sky conditions and cloudy days.
(xv)	Temperature should be monitored continuously at the top of the
plant canopy, while humidity should be measured at least once during each
light and dark period.
(xvi)	For chamber-grown plants, measurements of carbon dioxide
concentrations should be made at the top of the plant canopy on a continu-
ous basis.
(5) Analytical measurements—(i) Test substance. Stock solutions
of test substance should be diluted with glass distilled or deionized water
to obtain the test solutions. Standard analytical methods, if available,
should be used to establish concentrations of these solutions and should
be validated before beginning the test. An analytical method is not accept-
able if likely degradation products of the test substance, such as hydrolysis
and oxidation products, give positive or negative interference. The pH of
these solutions should also be measured before use.
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(ii) Numerical. Entire plants (tops and roots) should be dried and
weighed, and numbers of root nodules should be counted for the definitive
test. Means and standard deviations of ethylene production (from acetylene
reduction assay), plant yields, and nodulation should be calculated and
plotted for each treatment and control. Appropriate statistical analyses
should provide a goodness-of-fit determination for the concentration re-
sponse curves.
(e) Test conditions—(1) Test species—(i) Selection. (A) A species
of the genus Trifolium (e.g., T. repens, white clover) is the preferred leg-
ume for this test. The specific complementary species of Rhizobium should
be obtained from a reliable source of bacterial cultures.
(B) 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 alternative species
should be provided.
(ii) Seed selection. Information on seed lot, the seed year, or growing
season collected and germination percentage should be provided by the
source of the seed. Only untreated seed (not treated with fungicides,
repellents, etc.) taken from the same lot and year or season of collection
should be used in a given test. In addition, all seed of a 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.
(2) Facilities—(i) Apparatus. (A) Greenhouse or environmental
chambers should provide adequate environmental controls to meet the car-
bon dioxide, humidity, irradiation, photoperiod, and temperature specifica-
tions. 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.
(B) Laboratory facilities for testing should include nonporous floor
covering, absorbent bench covering with nonporous backing, and adequate
disposal facilities to accommodate plant nutrient, test, and wash solutions
containing the test substance at the end of each run, and any bench cover-
ing, lab clothing, or other contaminated materials.
(ii) Containers and support media. (A) For each run at least 36
to 42 potting containers (6 per concentration of test substance, 6 for the
control, and 6 if a carrier control is necessary) will be needed. Containers
used in each experiment should be of equal size and volume and possess
the same configuration. Potting containers should be filled with support
media to within 2.5 cm 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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(B) Six or seven 25-L carboys (one per concentration of test substance
and one for controls; another if a carrier control is necessary) will serve
as reservoirs from which nutrient solution will be delivered, under air pres-
sure, to the appropriate tray of potting containers. An automatic system
design is recommended (see the following Figure 1.).
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Figure 1.—Cross-Sectional Diagram of a Representative Tray
Unit and the Nutrient Solution Reservoir for Irrigating Potted Plants
PLEXIGLAS
DRAIN
HOLE
_ X'l'TA	EPOXY^*n^Kfc»rNO. 5 RUBBER -	_ _
V ^	STOPPER	51x41 cm y
BOARD GLASS TUBE*
^SCREEN
TABLE TOP
FLEXIBLE
RUBBER
TUBING
,NO. 13 RUBBER
STOPPER
TRAY
AIR
.HEAVY
TUBE
15-L 	
NUTRIENT
SOLUTION
THREADED CAP
•30-L
CARBOY
BLACK PLASTIC
(C) Each series of six replicate pots (per test concentration, control,
and if applicable, carrier control) may be placed in a large tray into which
the appropriate nutrient solution will be delivered (see Figure 1. in para-
graph (e)(2)(ii)(B) 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 ac-
commodate the pots, keeping them upright and properly spaced.
(iii) Cleaning and sterilization. (A) Potting and receiving containers,
nutrient storage containers, and support media should be cleaned before
use. All equipment should be washed according to good standard labora-
tory procedures to remove any residues remaining from manufacturing or
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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. Disposal
should conform to existing regulations.
(iv) Nutrient media. (A) The recommended medium for growth and
establishment of the Rhizobium-legume association consists of the follow-
ing:
Chemical
Amount
(mg/L)
K2SO4 		901
KH2PO4		272
CaCI2 		416
MgS0.,7H20 		493
Fe 		8.3
H3BO3 1 		2.9
MnCI24H20 1 		1.8
ZnS0,7H20 1 		0.22
H2MoO |H20 1 		0.02
CuSO |6H20 1 		0.03
1 A single stock solution containing 1,000x concentrations of each of these trace
elements should be prepared in advance, so that in use, 1 ml_ of stock solution per
liter of nutrient solution yields the required concentration of each.
For certain legumes (e.g., Phaseolus vulgaris, bush bean), growth in this
medium will be enhanced by the addition, after 2 weeks, of 50 mL of
a nitrate supplement (10.2 g of KNO3 + 28.3 g of Ca(N03)2-4H20 per
liter) to the 15 L of nutrient solution.
(B)	The pH of the nutrient medium should be maintained within a
range of 4 to 7.
(C)	Nutrient solution levels in 25-L carboys should be maintained
at 15-L throughout the experiment by replacing transpired water with dis-
tilled water, and by complete replacement with fresh solution at least twice
a week.
(D)	Nutrient solutions should be transported by inert tubing from the
carboys to the trays of pots at regular intervals. A timer-activated air pump
is recommended for maintaining a controlled irrigation schedule.
(3) Test parameters. Environmental conditions should be maintained
as follows:
(i) If a growth chamber is used, the carbon dioxide concentration
should be 350 ±50 ppm.
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(ii)	Irradiation, measured at 1 m from the source, at 350 ±59
|iEinstein/m2/sec. at 400 to 700 rim.
(iii)	Photoperiods of 16 h light and 8 h darkness.
(iv)	Optimum temperature for growth and nitrogen-fixation for the
species used. For example, the optimum range for clover is 15 to 25 °C.
(v)	Relative humidity in growth chambers should approach 70 ±5 per-
cent during light periods and 90 ±5 percent during dark periods.
(vi)	pH range of 5 to 8.
(f) Reporting. (1) The final report should include, but not necessarily
be limited to, the following information.
(i)	Name and address of the facility performing the study and the
dates on which the study was initiated and was completed, terminated,
or discontinued.
(ii)	Objectives and procedures stated in the approved protocol, includ-
ing any changes in the original protocol.
(iii)	Statistical methods used for analyzing the data.
(iv)	The test substance identified by name, CAS registry number or
code number, source, lot or batch number, strength, purity, and composi-
tion or other appropriate characteristics.
(v)	Stability of the test and, if used, control substances under the con-
ditions of administration.
(vi)	A description of the methods used, which should include the fol-
lowing:
(A)	Description of greenhouse or environmental chamber conditions,
including type, size, and carbon dioxide concentration (applicable to cham-
bers), temperatures, humidity, photoperiod, and lighting intensity.
(B)	Description of nutrient solutions including source of any unusual
components.
(C)	Description of delivery system including a diagram if the design
is complex.
(D)	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.
(E)	Frequency, duration, and methods of observation.
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(vii)	A description of the test system used, including the scientific
names and sources of the test species (legume and bacterial), and histories
of the species (e.g., percentage of plants germinating, seed size class, and
culture history of Rhizobium strain used).
(viii)	The number of total weight (for smaller species) of seeds tested
per concentration, number of replicates, description of carriers, any seed
sterilization procedures used, and times of exposure.
(ix)	Concentration of the test substance in nutrient solution and in
the support media when the test substance is soluble in water or solubilized
with a carrier; the concentration of carrier solvent in nutrient solution when
carrier is used; the quantity of test substance per unit weight of root sup-
port media when the substance is coated on the sand.
(x)	pH of the nutrient solution when fresh and when replaced. The
reported results should include:
(A)	The results of the preliminary test and measurements. Species
and concentrations of test substance used, and observed effects on seed
germination, should be stated.
(B)	For the definitive test, the species, concentrations of test substance
used, and the following:
(7) Mean plant yield (in grams (dry weight) per pot), cumulative nod-
ule count (per pot), and C2H4 production (nanomoles per gram (dry
weight) per hour and nanomoles per pot per hour) for untreated controls
and for each concentration of the test substance used.
(2) Visible effects, if any, of the test substance on the intact plants
(tops, roots, and nodules).
(xi)	A description of all circumstances that may have affected the
quality or integrity of the data.
(xii)	The name of the sponsor, study director, principal investigator,
names of other scientists or professionals, and the name of all supervisory
personnel involved in the study.
(xiii)	A description of the transformations, calculations, or operations
performed on the data, a summary and analysis of the data, and a statement
of the conclusions drawn from the analysis. Results of the analysis of data
should include the concentration response curves with 95-percent con-
fidence limits, the results of a goodness-of-fit test (e.g., X2 test), and
EC50's.
(xiv)	The signed and dated reports of each of the individual state-
ments or other professionals involved in the study including each person
who, at the request or direction of the testing facility or sponsor, conducted
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an analysis or evaluation of data or specimens from the study after data
generation was completed.
(xv)	The locations where all specimens, raw data, and the final report
are stored.
(xvi)	The statement prepared and signed by the quality assurance unit.
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