United States
Environmental Protection
Prevention, Pesticides
and Toxic Substances
April 1996
&EPA Ecological Effects Test
OPPTS 850.4400
Aquatic Plant Toxicity
Test Using Lemna spp.,
Tiers I and II

Public Draft"

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
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:
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, 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

OPPTS 850.4400 Aquatic plant toxicity test using Lemna spp., tiers
I and II.
(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 are 40 CFR 797.1160 Lemna Acute Tox-
icity Test and OPP 122-2 Growth and Reproduction of Aquatic Plants
(Tier I) and 123-2 Growth and Reproduction of Aquatic Plants (Tier 2)
(Pesticide Assessment Guidelines, Subdivision J—Hazard Evaluation;
Nontarget Plants) EPA report 540/09-82-020, 1982.
(b)	Purpose. This guideline prescribes test procedures and conditions
using the freshwater aquatic plants Lemna gibba and L. minor to develop
data on the phytotoxicity of chemicals (see paragraphs (g)(1), (g)(2), and
(g)(3) of this guideline). The Environmental Protection Agency will use
data from these tests in assessing the hazard of a chemical to the environ-
(c)	Definitions. The definitions in section 3 of TSCA and 40 CFR
part 792—Good Laboratory Practice Standards are applicable to this guide-
line. The following definitions also apply:
Axenic means a culture of Lemna fronds free from other organisms.
Colony means an aggregate of mother and daughter fronds attached
to each other.
ECX means the experimentally derived chemical concentration that
is calculated to effect X percent of the test criterion.
Frond means a single Lemna "leaf-like" structure.
Frond mortality means dead fronds which may be identified by a
total discoloration (yellow, white, black, or clear) of the entire frond.
LOEC is the lowest test concentration of a material used in this test
that has an adverse effect.
MATC is the hypothetical toxic threshold concentration lying in a
range bounded by the NOEC and LOEC. GmMATC is the geometric mean
of these values.
NOEC is the highest test concentration of a material used in this test
that does not have an adverse effect.
Static-renewal test means a test method in which the test solution
is periodically replaced at specific intervals during the test.

(d) Test procedures—(1) Summary of the test, (i) In preparation
for the test, containers are filled with appropriate volumes of nutrient me-
dium and/or the test solutions. The test is started by introducing Lemna
fronds into each of the containers. Nutrient medium and test solutions may
need to be replaced on day 3 or 5, or as needed to prevent nutrient limita-
tion or depletion of the test chemical. Periodic renewal (static-renewal)
will help to maintain constant exposure concentrations of the test chemical
over the test period for compounds that are unstable in water. In a
14-day test, renewal may be necessary every 3 to 5 days.
(ii) Colonies should be inspected for changes in frond number and
appearance at the beginning of the exposure period (day 0), on days 3,
5, and at the end of the exposure period (day 7). On day 7, the total
number of living and/or dead fronds are counted. Any frond which is visi-
ble as a bud when viewed under a hand lens or dissecting microscope
should be counted. Concentration response curves are plotted for total
frond number, growth rate (as number of fronds per day) and mortality
(percentage of dead fronds to total number of fronds). EC5s, EC50s, and
EC90s are determined from the curves.
(2) 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. Water solubility of the test chemical
(as well as other physical chemical characteristics, e.g. volatility) should
be determined before definitive testing. A validated analytical method
should also be developed prior to any toxicity testing.
(ii)	The recommended procedure is to expose Lemna to a chemical
concentration series (e.g., 0.1, 1.0, 10, 100, and 1,000 mg/L plus controls.
For pesticide testing under FIFRA, one concentration equivalent to the
maximum recommended label rate is sufficient (Tier I, 40 CFR part 158).
A minimum of three replicates of three to five plants consisting of three
to four fronds each should be added to each test chamber. Plants of similar
size should be selected, and the number of plants and number of fronds
should be identical or as near identical as possible in each test chamber.
A total of at least 12, but no more than 16 fronds, per test chamber are
recommended (e.g. three 4-frond plants and one 3-frond plant could be
used, for a total of 15 fronds). Plants are exposed to equal volumes of
each chemical concentration for a period of 7 days. It has been shown
that sufficient numbers of plants are produced within 7 days to provide
adequate results (see paragraph (g)(2) of this guideline).
(iii)	The lowest chemical concentration in a test series, exclusive of
controls, should be the lowest concentration which can be analytically
quantified; except for pesticide testing under FIFRA. The highest con-
centration should be at least 1,000 mg/L; except for pesticide testing under
FIFRA. Replicates are not needed and nominal concentrations of the chem-
ical are acceptable for range-finding. If testing a pesticide under FIFRA

at the maximum labeled dosage, a minimum of three replicates is required.
If the calculated 50 percent inhibition value is greater than 1,000 mg/L
(greater than the maximum label rate if a pesticide is tested under FIFRA)
or is less than the analytical detection limit, definitive testing is not nec-
essary. However, replicates and measured concentrations of the appropriate
dose are needed to substantiate this result.
(3) Definitive test, (i) The purpose of the definitive test is to deter-
mine the EC5s, EC50s, EC90s, LOECs, and NOECs for Lemna growth
based on total frond number, growth rate, and/or frond mortality with a
minimum amount of testing beyond the range-finding test. Other end-ponts
(optional) include dry weight and chlorophyll and pheophytin pigment
analyses, under paragraphs (g)(2) and (g)(3) of this guideline.
(ii)	At least five concentrations of chemical, exclusive of controls,
should be used in the definitive test and chosen in a geometric series in
which the ratio is between 1.5 and 2.0 (e.g. 2, 4, 8, 16, 32, 64 mg/L).
The concentration range should be selected to define the concentration re-
sponse curve between the EC5 and EC90. For each concentration and con-
trol at least three replicate containers should be used, each containing
150 mL of test solution, or enough test solution to result in a volume-
to-vessel size ratio of 2:5, and three to five plants consisting of three to
four fronds each should be used. See paragraph (d)(2)(i) of this guideline
for a discussion on plant size and frond numbers to be used in the defini-
tive test. Fewer replicates, each containing a greater number of colonies,
may be used; however, test containers and solution volumes will have to
be adjusted accordingly. The range of chemical concentrations tested
should result in the highest concentration affecting at least 90 percent of
the fronds and the lowest concentration affecting no more than 5 percent
of the fronds compared with the controls or the selected test concentrations
should bracket the expected EC50 value.
(iii)	Every test should include controls consisting of the same nutrient
medium, number of fronds, environmental conditions, and procedures as
the test containers except that none of the chemical is added. If a solvent
or carrier is used to dissolve or suspend the test chemical, additional con-
trols containing the solvent or carrier should also be included in the test
to determine any effect of the solvent or carrier on the plants. The upper
limit of carrier volume is 0.5mL/L and the same amount of carrier should
be added to each test concentration.
(iv)	Positive controls using zinc chloride as a reference chemical
should be run periodically. The purpose of a running a positive control
with a reference chemical is to determine if the test Lemna fronds are
responding to a known chemical in the expected manner. If the fronds
respond to subsequent reference chemical tests consistently, it is assumed
that the Lemna will respond to other chemicals consistently. Changes in
Lemna response caused by factors such as poor nutrition, genetic drift,

and contaminants, may not be detected by negative controls, yet may still
influence test results. A minimum of three concentrations of the reference
chemical are run at or near the expected median effect level.
(v)	The colonies should be transferred to test solution on days 3 and
5. No more than 20 percent of the test substance should be lost by vola-
tilization (nor by processes such as hydrolysis, photolysis, etc.) between
replacements. Flow-through procedures may be necessary for volatile sub-
stances.The colonies may have to be transferred more frequently for highly
volatile test substances in order to maintain 80 percent of the initial test
substance concentration. Transfer should be done in a clean, draft-free area
as quickly as possible to minimize contamination of the colonies.
(vi)	Observations of frond numbers and appearance should be made
of the colonies on day 0, 3, 5, and 7. A dissecting microscope will facili-
tate observations.
(vii)	Concentration response curves should be plotted. These curves
can provide the basis for determining the EC5s, EC50s, and EC90s for
total frond number, growth rate, and plant necrosis.
(viii)	Any change in frond development or appearance such as in-
crease in number (a frond is counted regardless of size as long as it is
visible adjacent to the parent frond), decrease in size, necrosis, chlorosis,
etc., should be reported. Any additional observations such as sedimentation
of test solution, sinking of fronds, or other abnormalities should also be
(ix)	Other end-points that can be assessed in this test and that have
been shown to be indicative of growth inhibition are chlorophyll values
(chlorophyll a, b, c, and pheophytin a pigments) and biomass (dry weight
at 60 °C) at the end of the test (refer to paragraphs (g)(2) and (g)(3) of
this guideline).
(x)	A randomized complete block design is recommended for the de-
finitive test with blocks delineated within the test chamber. If, for any
reason, blocking is not feasible, total randomization within chambers is
(4) Analytical measurements—(i) Chemical. Stock solutions or
growth media should be prepared just prior to use and diluted with water
of high quality such as glass-distilled, deionized water, or ASTM Type
I to obtain the test solutions. Volume increases should be limited to pre-
vent dilution of the nutrients. 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 chemical, such as hydrolysis and
oxidation products, give positive or negative interference. The pH of the
test solutions should also be measured prior to and after use. Chlorophyll

and pheophytin pigment analyses should be based on accepted procedures
under paragraphs (g)(2) and (g)(3) of this guideline.
(ii) Numerical. The number of fronds is counted at the end of the
definitive test. Means and standard deviations are calculated and plotted
for each treatment and control. Appropriate statistical analyses are used
to provide a goodness-of-fit determination for the concentration response
(e) Test conditions—(1) Test species. The test species to be used
in these tests are L. gibba G3 and L. minor. Axenic cultures may be ob-
tained from laboratory cultures or commercial sources. A stock culture
grown from a single isolated plant should be used to inoculate all the
flasks used in a given test.
(2)	Stock cultures. Axenic stock cultures should be grown in the
aquariums for 2 weeks (with necessary transfers) prior to being used in
a test. Plants used in a test should be randomly selected from the culturing
tank. Inocula should be taken from cultures which are less than 2 weeks
(3)	Facilities—(i) Apparatus. (A) A controlled environment growth
chamber or an enclosed area capable of maintaining the specified number
of test chambers and test parameters (see paragraph (e)(4) of this guide-
line) is needed.
(B) Laboratory facilities for the mixing and diluting of nutrient me-
dium and a source of distilled or deionized water are needed. An autoclave
(for sterilizing glassware and media) and a sterile transfer hood (for main-
tenance of an axenic Lemna culture) are also necessary. Disposal facilities
should be adequate to accommodate spent test solutions and plant materials
as well as any bench covering, lab clothing, or other contaminated mate-
(ii)	Containers and support media. Test containers may be
250-mL glass beakers or Erlenmeyer flasks, large enough to hold 150 mL
of test solution and the Lemna colonies without crowding for the duration
of the test. All containers should be of the same type and size. Even though
at least three replicates are used, larger containers may sometimes be nec-
essary to hold additional colonies and test solution volume. The ratio of
test solution volume to container volume should not exceed 2:5. For each
test concentration and control the same number of replicates should be
(iii)	Cleaning and sterilization. All glassware and equipment should
be cleaned following good laboratory practice. The Nytex screen or
inoculating loops used for transferring the Lemna should be disposed of
after use or thoroughly cleaned and sterilized before reuse.

(iv)	Nutrient media. M-Hoagland's or 20X-AAP nutrient media are
recommended for maintaining Lemna cultures and for use as the diluent
in the preparation of various concentrations of test solutions under para-
graph (g)(1) of this guideline. Water of high quality (ASTM Type I for
example) can be used to make the nutrient medium. The medium should
be prepared prior to each transfer of Lemna cultures and for the preparation
of new test solutions during the course of a test. If prepared in advance
it should be refrigerated. The pH of the medium should be adjusted to
between 4.8 to 5.2 by the addition of 0.1N or 1 N NaOH for culture main-
tenance prior to addition of the test chemical for M-Hoagland's medium.
If 20X-AAP medium is used, the pH should be adjusted to 7.5 ±0.1 with
0.1 N NaOH or HC1 (see paragraph (g)(1) of this guideline). Nutrient
media should not contain organic metabolites such as sucrose. Chelating
agents, such as EDTA, are present in the 20X-AAP medium to ensure
that trace nutrients will be available to the Lemna fronds. M-Hoagland's
medium (which contains no EDTA) should be used for test solution prepa-
ration if it suspected that the chelator will interact with the test chemical.
(v)	Carriers. Stock solutions of substances of low aqueous solubility
may be prepared by use of organic solvents, emulsifiers, or dispersants
of low phytotoxicity to plants. When a solvent or carrier is used, a second
set of controls should contain the same concentration of the solvent or
carrier as that used in the highest concentration of the test substance. The
concentration of the solvent or carrier should not exceed 0.5 mL/L and
the same amount of carrier should be added to each test concentration
(see paragraph (g)(1) of this guideline).
(4) Test parameters. Environmental conditions should be maintained
as specified below:
(i)	Temperature at 25 ±2 °C.
(ii)	The pH of the nutrient medium between 4.8 and 5.2 for
M-Hoagland's medium, 7.5 ±0.1 for 20X-AAP medium. Test solution pH
may vary from the nutrient medium after the addition of the test chemical
and/or carrier (if used). Any such changes should be recorded but not ad-
(iii)	Continuous warm-white fluorescent lighting should be used to
provide a light intensity in the range of 4,200 and 6,700 lx (394 and
620 fc), as measured adjacent to each test chamber at the surface of the
test solution. The light intensity at each position in the incubation area
should be measured and should not differ by more than 15 percent from
the selected light intensity.
(f) Reporting. Reporting requirements of 40 CFR part 792—Good
Laboratory Practice Standards apply to this guideline. The following data
should also be reported.

(1)	Source of Lemna and taxonomic verification.
(2)	Description of test chambers, type of lights, size of beakers or
flasks used, number of concentrations and replicates per concentration,
number of colonies per replicate, solution volumes, physical parameters
of growth chambers (e.g. temperature, and light intensity).
(3)	The pH and concentration of the test chemical in the test solutions
prior to use and discarding on day 3, 5, and 7.
(4)	Number of fronds per test concentration and control at the end
of the test, the percent inhibition and/or stimulation of growth rate, and
percent frond mortality for each test concentration compared to controls.
(5)	If the range-finding test showed that the highest concentration of
the chemical tested (not less than 1,000 mg/L or the maximum pesticide
label application rate) had no effect on Lemna, report the results and meas-
ured concentrations and a statement that the chemical is not phytotoxic
at concentrations less than 1,000 mg/L.
(6)	If the range-finding test showed greater than a 50 percent effect
with a test concentration below the analytical detection limit, report the
results and a statement that the chemical is phytotoxic below the analytical
detection limit.
(7)	Charts of growth in the no-treatment and solvent controls, on each
counting day and at the end of the test, for each toxicity test.
(8)	Means and standard deviations for frond number, growth rate, and
percent frond mortality in each test concentration. In addition, concentra-
tion response curve(s) with 95 percent confidence limits delineated, good-
ness-of-fit determination, and EC5s, EC50s, EC90s, LOECs, and NOECs
identified (see paragraph (g)(4) of this guideline).
(9)	Methods and data records from chemical and numerical analyses
including validation methods and quality assurance procedures.
(g) References. The following references should be consulted for ad-
ditional background material on this test guideline.
(1)	American Society for Testing and Materials. ASTM E1415-91.
Standard guide for conducting static toxicity tests with Lemna gibba G3.
In 1991 Annual Book of ASTM Standards, vol. 11.04: Pesticides; resource
recovery; hazardous substances and oil spill response; waste disposal; bio-
logical effects, pp 1137-1146 (1991).
(2)	Cowgill, U.M. and D.P. Milazzo, The culturing and testing of
two species of duckweed, pp 379-391 in U.M. Cowgill and L.R. Williams
(eds.). Aquatic Toxicology and Hazard Assessment, 12th volume, ASTM
STP 1027 ASTM, Philadelphia, PA (1989).

(3)	Taraldsen, J.E. and T.J. Norberg-King, New method for determin-
ing effluent toxicity using duckweed (Lemna minor). Environmental Toxi-
cology and Chemistry 9:761-767 (1990).
(4)	Bruce, R.D. and D J. Versteeg. A statistical procedure for model-
ing continuous toxicity data. Environmental Toxicology and Chemistry
11:1485-1494 (1992).