United Slatas
Environmental Protection
Agency
Prevention, Pesticides
and T oxic Substances
(7101)
EPA 712-G-96-154
April 1996
&EPA Ecological Effects Test
Guidelines
OPPTS 850.4200
Seed Germination/Root
Elongation T oxicity T est
"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, et seq.).
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 850.4200 Seed germination/root elongation toxicity test.
(a) Scope—(1) Applicability. This guideline is intended to meet test-
ing requirements of both the Federal Insecticide, Fungicide, and
Rodenticide Act (FJFRA) (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 797.2750 Seed Germination
Root Elongation Toxicity Test.
(b) Purpose. This guideline is intended for use in developing data
on the acute toxicity of chemical substances and mixtures ("chemicals")
subject to environmental effects test regulations. This guideline prescribes
test procedures and conditions using seed of commercially important ter-
restrial plants to develop data on the phytotoxicity of chemicals. The EPA
will use data from these tests in assessing the hazard of a chemical to
the environment.
(c) Definitions. The definitions in section 3 of TSCA and in 40 CFR
Part 792—Good Laboratory Practice Standards (GLP) apply to this test
guideline. The following definitions also apply to this test guideline.
(1) ECX means the experimentally derived chemical concentration
that is calculated to affect X percent of the test criterion.
(2) Embryo means the young sporophytic plant before the start of
germination.
(3) Germination means the resumption of active growth by an em-
bryo. The primary root should attain a length of 5 mm for the seed to
be counted as having germinated.
(4) Hypocotyl means that portion of the axis of an embryo or seedling
situated between the cotyledons (seed leaves) and the radicle.
(5) Radicle means that portion of the plant embryo which develops
into the primary root.
(6) Test solution means the test chemical and the dilution water in
which the test chemical is dissolved or suspended.
(d) Test procedures—(1) Summary of the test (i) Seed should be
separated into appropriate size classes, and that size class containing the
most seed should be used exclusively for the test. Fresh test solutions
should be added to Petri dishes that have been completely filled with either
precleaned quartz sand, 200 Jim glass beads, or other inert material. The
seed should then be positioned on the substrate allowing adequate room
for anticipated growth. It is recommended that the radicle end of the seed
be aligned in the direction of this growth. Petri dish lids should be used
to hold the seed in place, and the dishes sealed with tape. Deionized or
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glass-distilled water should be added to the substrate prior to positioning
the seed for those chemicals that are insoluble in water and that should
be sorbed to the substrate.
(ii) The dishes should be placed in a seed germinator or other growth
facility at a slight angle to facilitate linear root growth. Seed should be
incubated in the dark until at least 65 percent of the control seed have
germinated and developed roots that are at least 20 mm long.
(iii) The number of seed that germinate should be counted, and root
lengths measured. Concentration response curves, EC 10s, and EC50s for
seed germination and root elongation should be determined and reported
for each of the species tested.
(2) Chemical application, (i) Test chemicals that are soluble in water
should be dissolved in deionized or glass-distilled water and added to the
substrate in the petri dishes at the start of the test.
(ii) Test chemicals that are insoluble in water but which can be placed
in aqueous suspension with a carrier should be suspended in deionized
or glass-distilled water with the carrier and then added to the Petri dishes.
The carrier should be soluble in water, relatively nontoxic to plants, and
should be used in the minimum amount required to dissolve or suspend
the test chemical. There are no preferred carriers; however, acetone, gum
arabic, polyethylene glycol, ethanol, and others have been used extensively
in testing herbicides, plant growth regulators, fungicides, and other chemi-
cals that affect plants. Tests of the carrier effect should be included in
the test experimental design and conducted simultaneously as controls.
(iii) Water-insoluble chemicals for which no nontoxic water-soluble
carrier is available, should be dissolved in an appropriate volatile solvent.
The solution and substrate should be placed in a rotary vacuum apparatus,
and evaporated, leaving a uniform coating of test chemical on the sub-
strate, A weighed portion of the substrate should be extracted with the
same organic solvent and the chemical assayed before the containers are
filled. Solvent controls should be included in the experimental design and
tested simultaneously. Deionized or glass-distilled water should be added
to the treated substrate prior to positioning the seed on the substrate.
(3) Range-Finding test, (i) A range-Finding test should be conducted
to establish if definitive testing is necessary and to determine test solution
concentrations for the definitive test.
(ii) The seed should be exposed to a chemical concentration series
(e.g., 0.01, 0.1, 1.0, 10, 100, and 1,000 mg/L. The lowest concentration
in the series, exclusive of controls, should be at the detection limit of
the chemical. The upper concentration, for water soluble compounds,
should be the saturation concentration.
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(iii) The test consists of one run for each of the recommended plant
species or selected alternates, A minimum of 15 seeds per species should
be exposed to each chemical concentration and control The test period
may be ended when at least 65 percent of the control seed have germinated
and developed roots that are at least 20 mm long. The exposure period -
may be shortened if data suitable to establish the test solution concentra-
tion series for the definitive test can be obtained in less time and if the
definitive test is to be conducted. No replicates are required and nominal
concentrations of the chemical are acceptable unless definitive testing is
not required as specified in paragraph (d)(3)(iv) of this guideline.
(iv) Definitive testing is not necessary if the highest chemical con-
centration tested results in less than a 50 percent inhibition of germination
or reduction in root growth or if the lowest concentration tested (analytical
detection limit) results in greater than a 50 percent inhibition of germina-
tion or reduction in growth,
(v) Graphical analysis of the range-finding data facilitates selection
of chemical concentrations for the definitive test.
(4) Definitive test, (i) The purpose of the definitive test is to deter-
mine the concentration-response curves, the EC 10s and EC50s for seed
germination and root elongation for each species tested, with the minimum
amount of testing beyond the range-finding test,
(ii) The seed of each species tested should be exposed to at least
6 concentrations of the chemical chosen in a geometric series in which
the ratio is between 1.5 and 2.0 (e.g., 2, 4, 8, 16, 32, and 64 mg/L), The
concentration ranges should be selected to determine the concentration re-
sponse curves between the EC 10 and EC50 for both germination and root
elongation. Test solutions or substrate extracts should be analyzed to deter-
mine chemical concentration prior to use. Selection of seed from the size
class lot to be exposed to each test concentration should be unbiased.
(iii) At least three replicates, each with at least 10 seed per species
should be tested for each concentration and control.
(iv) livery test should include controls consisting of the same dilution
water, conditions, procedures and seed from the same lot used in the expo-
sure group, except that none of the chemical is added. If a carrier (solvent)
is needed to suspend or disperse the chemical, a separate carrier control
should also be used.
(v) The test period may be ended when at least 65 percent of the
control seed have germinated and developed roots that are at least 20 mm
long. When both conditions are satisfied, the mean number of seed germi-
nating and mean root length per treatment (and control) can be determined.
If the test chemical concentration series does not bracket the EC 10 through
EC50, for both germination and root elongation, the test should be repeated
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(at a higher or lower concentration series). Concentration response curves,
EC 10s and ECSOs for germination and root elongation should be deter-
mined for each species tested and reported along with their 95 percent
confidence limits.
(vi) Any abnormal seedling development or appearance such as le-
sions, enhanced root growth (measured), discoloration, swelling, loss of
turgor, etc., should also be reported.
(vii) A randomized complete block design is recommended for the
definitive test with blocks delineated within the seed germinator or growth
chamber. If, for any reason, blocking is not feasible, total randomization
within chambers is acceptable.
(viii) Temperature in the germination facility should be recorded
hourly. The pH of the test solutions should be recorded at the initiation
of the definitive test.
(5) Analytical measurements—(i) Test chemical. Stock solutions
should be diluted with glass-distilled or deionized water to prepare test
solutions. Standard analytical methods should be used (if available) to es-
tablish concentrations of these solutions and should be validated before
beginning the test. An analytical method is not acceptable if likely deg-
radation products of the chemical, such as hydrolysis and oxidation prod-
ucts, give positive or negative interference. The pH of these solutions
should also be measured prior to use.
(ii) Numerical. The number of seeds that germinate should be count-
ed and root lengths measured for each definitive test species. All root elon-
gation measurements for a given species should be made sequentially be-
fore proceeding to the next species. Root length should be measured from
the transition point between the hypocotyl and root to the tip of the root.
Means and standard deviations should be calculated and plotted for each
treatment and control. Appropriate statistical analyses should provide a
goodness-of-fit determination for the concentration response curves.
(e) Test conditions—(1) Test species, (i) Test plants recommended
for use include:
(A) Lycopersicon esculentum (tomato).
(B) Cucumis sativus (cucumber).
(C) Lactuca saliva (lettuce).
(D) Glycine max (soybean).
(E) Brassica oleracea (cabbage).
(F) Avcna sativa (oat).
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(G) Lolium perenne (perennial ryegrass).
(H) Allium cepa (common onion).
(I) Daucus carota (carrot).
(J) Zea mays (corn).
(ii) Other species of economic or ecological importance to the region
of impact may also be appropriate for testing. A minimum of 10 species
should be tested.
(lii) Information on seed lot, the seed year or growing season col-
lected, and germination percentage should be provided by the supplier 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 from the size class which contains the most seed. Damaged seed should
be discarded. Standard seed dockage sieves should be used to size seed.
(2) Facilities—(i) Apparatus. (A) Seed germinator, or other con-
trolled environment chamber capable of maintaining a uniform testing tem-
perature of 25 + 1 °C is required. In addition, the facilities should include
work areas for sizing, counting, and exposing seed for root measurement.
If possible, these areas should be isolated from other activities. A fume
hood may be needed when testing substances potentially hazardous to
human health. Apparatus for distilling and deionizing water are needed
unless reagent grade water is used. Refrigeration facilities to hold the seed
in cold storage (5 °C) in moisture-proof containers at seed moisture con-
tents of less than 10 percent are also needed,
(B) Disposal facilities should be adequate to accommodate spent
glassware, sand, beads, and test solutions at the end of each run and any
bench covering, lab clothing, or other contaminated materials,
(ii) Containers and support media. A minimum of 210 Petri dishes
and sufficient sand or glass beads, or other inert substrate to fill them
are needed. Large (200 mm) glass Petri dishes are recommended. Perlitc,
vermiculite, or native soils should not be used as substrates.
(iii) Cleaning and sterilization. (A) All glassware and the substrate
should be cleaned before each test following standard good laboratory
practice. The substrate should be washed in 7.5 N nitric acid and rinsed
with a mild base followed by washes of glass-distilled or deionized water.
The pH of the washed substrate should be near neutral. If the glass beads
are to be reused, they should be heated to 100 °C for 8 to 12 hours prior
to acid washing. A dichromate solution should not be used for cleaning
beads or Petri dishes. Sand and plastic Petri dishes should not be reused.
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(B) If fungal or other microbial contamination interferes with seed
germination so that germination is less than 65 percent in the controls,
glassware should be sterilized and/or the seed surface sterilized prior to
use, e.g., the seed may be soaked for 10 minutes in a 10 percent sodium
hypochlorite solution, then rinsed and soaked for 1 hour in glass-distilled
water.
(3) Test parameters. Environmental conditions should be controlled
to maintain incubation temperature at 25 ± 1 °C in complete darkness. In-
cubation conditions may have to be adjusted to meet germination and root
length criteria in the controls if species other than the 10 recommended
for use are tested
(f) Reporting. The sponsor should submit all data developed during
the test that are suggestive or predictive of phytotoxicity to the Agency,
In addition to the general reporting requirements prescribed in 40 CFR
part 792, the following should be reported:
(1) Information on the source and history of the seed, germination
percentage reported by the supplier, and the seed size class used for test-
ing.
(2) The number of seed of each species per treatment, the number
of replicates, carriers, incubation conditions, and seed sterilization proce-
dures.
(3) The concentration of the chemical added to each treatment dish
and its pH (pfl is optional).
(4) If the range-finding test showed that the highest concentration of
the chemical tested (not less than 1,000 mg/L) had no effect on the test
species, report the results by species and concentration and a statement
that the chemical is of minimum phytotoxic concern,
(5) If the range-finding test showed greater than 50 percent inhibition
of germination or root elongation at a test concentration at the analytical
detection limit, the results by species and concentration and a statement
that the chemical is phytotoxic below the analytical detection limit.
(6) For each species included in the definitive test, means and stand-
ard deviations for germination and root length in each treatment. In addi-
tion, concentration response curves with 95 percent confidence limits de-
lineated, goodness-of-fit determination, and EClOs and EC50s identified.
(7) Methods and data records of all chemical and numerical analyses
including method validation and reagent blanks.
(8) The data records of the incubation temperature, germination
counts, and root length measurements.
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