8211

8211 DUCKWEED*
8211 A. Introduction
1. Organism Characteristics
Lemna minor L. (Figure 8211:1) (also known as common
duckweed) is a small flowering aquatic macrophyte (a monocot)
widely distributed in quiescent fresh water and estuaries ranging
from tropical to temperate zones.
1
It is the most common species
of the family Lemnaceae in the United States and many other
parts of the world. It is morphologically simple, consisting only
of frond and root. The frond size is approximately 2 to 4 mm and
root length is up to 50 mm. The plant is colonial (up to 8 fronds),
multiplies sexually and asexually, and has a maximum growth
rate far exceeding those of other flowering plants. Duckweed is a
food for waterfowl and small animals, and provides food, shelter,
and shade for fish and other aquatic organisms. It also serves as
a habitat for various invertebrates.
2. Test Applications
Duckweed is an ideal organism for testing the aquatic phyto-
toxicity of herbicides, industrial and municipal wastewaters, and
leachates or slurries of waste and soil and other contaminants.
2
Because many ambient waters and effluents are colored and/or
turbid, they are difficult to test directly for toxicity via most algal
tests without filtering, which can alter sample integrity. How-
ever, duckweed is a floating plant that can be used to test
unfiltered colored or turbid samples. In addition, some samples
may contain labile, volatile, or sorptive materials and require
either renewal or flow-through methods. Algal testing may be
inappropriate for these types of tests, but the duckweed toxicity
test described herein can be easily modified to apply in either
method.
The duckweed toxicity test is useful, especially for determin-
ing phytotoxicity at the air–water interface, where surface-active
substances, oil and grease, and toxic organic compounds may
be concentrated. The test also is useful for determining the
toxicity of metals,
3
organic compounds,
4,5
industrial and munic-
ipal effluents,
6–9
and herbicides.
10
As a higher plant, duckweed
has a sensitivity pattern nearer to most vascular plants than
to algae. However, as monocots, duckweeds are insensitive to
auxin-stimulating herbicides (e.g., 2,4-D). The test is generally
described as fast, simple, sensitive, and cost-effective.
2
This is
essentially true when compared to growth tests with higher
plants in soil.
However, because common duckweed is a floating plant, the
test may underestimate the toxicity of a substance that adsorbs
on particulates and precipitates during a static test. Also, sub-
stances that concentrate at the air–water interface tend to affect
duckweed more than other aquatic plants (e.g., algae or sub-
merged plants). Gently shaking or stirring test vessels to increase
mixing may overcome or lessen these problems. Alternatively,
renewal or flow-through methods can be used.
Several standard tests using duckweed are available in the
literature.
11–13
3. References
1. HILLMAN, W.S. 1961. The Lemnaceae, or duckweed, a review of the
descriptive and experimental literature. Bot. Rev. 27:221.
2. WANG, W. 1990. Literature review on duckweed toxicity testing.
Environ. Res. 51:7.
3. WANG, W. 1986. Phytotoxicity tests of aquatic pollutants by using
common duckweed. Environ. Pollut. (Ser. B) 11:1.
4. KING, J.M. & K.S. COLEY. 1985. Toxicity of aqueous extracts of
natural and synthetic oils to three species of Lemna, Spec. Tech.
Publ. 891. American Soc. Testing & Materials, Philadelphia, Pa.
5. HUGHES, J.S., M.M. ALEXANDER &K.BALU. 1988. An evaluation
of appropriate expressions of toxicity in aquatic plant bioassays as
demonstrated by the effects of atrazine on algae and duckweed,
Spec. Tech. Publ. 921. American Soc. Testing & Materials, Phila-
delphia, Pa.
6. ROWE, E.L., R.J. ZIOBRO, C.J.K. WANG & C.W. DENCE. 1982. The
use of an alga Chlorella pyrenoidosa and a duckweed Lemna
perpusilla as test organisms for toxicity bioassays of spent bleach-
ing liquors and their compounds. Environ. Pollut. (Ser. A) 27:289.i
7. WANG,W.&J.WILLIAMS. 1988. Screening and biomonitoring of
industrial effluents using phytotoxicity tests. Environ. Toxicol.
Chem. 7:645.
8. LOCKHART, W.L. & A.P. BLOUW. 1979. Phytotoxicity tests using the
duckweed Lemna minor. In Toxicity Tests Freshwater Organisms.
Canadian Spec. Publ. Fish. Aquat. Sci. 44:112.
* Approved by Standard Methods Committee, 2011.
Joint Task Group: Mathias Eberius (chair), James F. Fairchild, Joe M. King,
Michael A. Lewis, Jerry C. Smrchek, Jane Staveley, James P. Swigert, David E.
Weber.
Figure 8211:1. Common duckweed: Lemna minor. Each plant consists of
frond and root; together, plants form colony.
1

9. TARALDSEN, T.E. & T.J. NORBERG-KING. 1990. New method for
determining toxicity using duckweed (Lemna minor).Environ.
Toxicol. Chem. 9:761.
10. FAIRCHILD, J.R., D.W. RUESSLER, P.S. HAVERLAND & A.R. CARLSON.
1997. Comparative sensitivity of Selenastrum capricornutum and
Lemna minor to sixteen herbicides. Archiv. Contam. Toxicol. 32:
353.
11. AMERICAN SOCIETY for TESTING and MATERIALS. 2008. Standard guide
for conducting static toxicity tests with Lemna gibba G3. E1415-91
(2004). In Annual Book of ASTM Standards, Vol. 11.06. American
Soc. Testing & Materials, W. Conshohocken, Pa.
12. ENVIRONMENT CANADA. 1999. Biological Test Method: Test for
measuring the inhibition of growth using the freshwater macro-
phyte, Lemna minor, EPS 1/RM 37. Environment Canada, Ottawa,
Ont.
13. ORGANIZATION for ENVIRONMENTAL GROWTH and DEVELOPMENT. 2006.
Lemna sp. Growth Inhibition Test. In OECD Guidelines for the
Testing of Chemicals, Section 2, Effects on Biotic Systems, Test
221. Organization for Environmental Growth and Development,
Paris, France.
4. Bibliography
HOLST, R.W. & T.C. ELLWANGER. 1982. Pesticide Assessment Guide-
lines, Subdivision J Hazard Evaluation: Nontarget Plants, EPA
540/9-82-020. U.S. Environmental Protection Agency, Washing-
ton, D.C.
LARSON, J.H., P.C. FROST & G.A. LAMBERTI. 2008. Variable toxicity of
ionic liquid forming chemicals to Lemna minor and the influence of
dissolved organic matter. Environ. Toxicol. Chem. 27:676.
8211 B. Selecting and Preparing Test Organisms
1. Test Species
The procedure is designed for use with Lemna minor. The
organism can be obtained from commercial sources, testing
laboratories, or the field. It must be identified and confirmed
taxonomically before use.
1
If available, use L. minor ST from
culture collections especially for substance testing or for legal
purposes, because this clone is well characterized and widely
used, and behaves very well in permanent lab cultures. Other
duckweed species (e.g., L. gibba, L. perpusilla, L. paucicostata,
and L. polyrrhiza) have been used successfully with modified
procedures.
2,3
2. Culturing Test Organisms
Acclimate a new duckweed culture to the test environment
(e.g., lighting, temperature, and nutrient conditions) for at least 2
weeks before a test—at least up to the stage of demonstrating
growth validation criterion. This culture grows vigorously and
provides a nearly inexhaustible supply for testing under proper
conditions. Grow duckweed in a culture vessel (e.g., an aquar-
ium or stainless steel basin). To prepare a 10-L culture solution,
add 100 mL of each stock nutrient solution A, B, and C (Table
8211:I) to deionized or other suitable water. Do not use tap
water. Use a water depth of at least 40 mm and provide contin-
uous (24 h/24 h) cool-white fluorescent light at 2150 to 4300 lux
at the water surface. Maintain a temperature of 24 ⫾2°C.
Change nutrient solution weekly and avoid overcrowding (more
than one layer of duckweed). Axenic culturing is unnecessary
but covering the vessel loosely helps avoid algal and fungal
contamination.
3. Diseases and Predators
Diseases, phytophagous insects, and other pests normally are
not problems for a duckweed culture. If the culture appears
unhealthy (e.g., chlorotic or necrotic) or contaminated by fungi
or algae, destroy it and start a new one. It is good practice to
maintain several cultures isolated from each other. For long-term
storage or as a backup, use axenic stock cultures on test medium
solidified by 1% agar with 1% glucose added to erlenmeyer
flasks. Autoclave the solidified medium with cotton or foam
plugs. Add 2 to 3 plants axenically after agar has cooled. Cover
the cotton plug with aluminum foil to minimize evaporation, and
store in a cool place with low light conditions for some months.
4. References
1. CORRELL, D.S. & H.B. CORRELL. 1972. Aquatic and Wetland Plants of
Southwestern United States. U.S. Environmental Protection Agency,
Washington, D.C.
TABLE 8211:I. DUCKWEED NUTRIENT SOLUTION
Solution
Stock Solution
Concentration Element
Final
Concentration
A:
NaNO
3
25.5 g/L N 42.0 mg/L
Na 110.0 mg/L
NaHCO
3
15.0 g/L C 21.4 mg/L
K
2
HPO
4
1.04 g/L K 4.69 mg/L
P 1.86 mg/L
B:
CaCl
2
䡠2H
2
O4.41 g/L Ca 12.0 mg/L
MgCl
2
5.7 g/L Mg 29.0 mg/L
FeCl
3
0.096 g/L Fe 0.33 mg/L
Na
2
EDTA䡠2H
2
O0.3 g/L
MnCl
2
0.264 g/L Mn 1.15 mg/L
C:
MgSO
4
䡠7H
2
O14.7 g/L S 19.1 mg/L
H
3
BO
3
0.186 g/L B 325 ␮ g/L
Na
2
MoO
4
䡠2H
2
O7.26 mg/L Mo 28.8
␮ g/L
ZnCl
2
3.27 mg/L Zn 15.7 ␮ g/L
CoCl
2
0.78 mg/L Co 3.54 ␮ g/L
CuCl
2
0.009 mg/L Cu 0.04 ␮ g/L
NOTES:
1. Omit Na
2
EDTA䡠2H
2
O in Solution B if test samples contain toxic metals. In that
case, acidify Solution B to pH 2 to prevent precipitation.
2. To prepare the duckweed nutrient solution, add 1 mL of each stock solution to
100 mL deionized water. Adjust to pH 7.5– 8.0.
DUCKWEED (8211)/Selecting and Preparing Test Organisms
2
DUCKWEED (8211)/Selecting and Preparing Test Organisms

2. KING, J.M. & K.S. COLEY. 1985. Toxicity of aqueous extracts of
natural and synthetic oils to three species of Lemna, Spec. Tech. Publ.
891. American Soc. Testing & Materials, Philadelphia, Pa.
3. HUGHES, J.S., M.M. ALEXANDER &K.BALU. 1988. An evaluation of
appropriate expressions of toxicity in aquatic plant bioassays as
demonstrated by the effects of atrazine on algae and duckweed,
Spec. Tech. Publ. 921. American Soc. Testing & Materials, Phila-
delphia, Pa.
8211 C. Toxicity Test Procedure
1. General Considerations
Use static, or where necessary, renewal methods.
1,2
As a
general rule, if a test solution is stable (e.g., a solution with low
microbial population, high toxicity, or low volatility), use a static
test. If samples are unstable, use repeated renewal methods (see
Section 8010D).
2. Preparing Test Materials
As used herein, dilution water and control water are identical
to duckweed nutrient solution. To prepare this solution, see
Table 8211:I.
To prepare toxicant solutions, see Section 8010F.2b.
3. Test Procedures and Conditions
Screening tests use a predetermined concentration (e.g., 100%
effluent) to determine if a sample is toxic in comparison with the
control water. If the sample is toxic, test it further using range-
finding and definitive tests. In the range-finding test, examine a
series of concentrations, usually at ratios of 10 (10%, 1%, 0.1%,
etc.).
Range-finding-test results are used as a basis for designing
definitive tests. For definitive tests, use at least five concentra-
tions of sample in a constant ratio (geometric series). For exam-
ple, for environmental samples using a recommended ratio of
two, concentrations should be 90% environmental sample (add-
ing tenfold concentrated medium to sample, thus not reaching
100%), 50%, 25%,12.5%, 6.25%, 3.12%, 1.56%, 0.78%, 0.39%,
etc. Ideally, choose a ratio to prepare a series of solutions in
which all effect concentrations (EC values) to be assessed are
bracketed by at least one higher and one lower concentration.
Alternatively, select the highest and lowest concentrations to
produce approximately 80% or higher and 10% or lower inhib-
itory effects.
Use three replicates for each treatment. Include a negative
control: six replicates containing only duckweed nutrient solu-
tion. If any solvents were used for pure chemicals, add an
additional solvent control with six replicates. Prepare a positive
dose–response test with 3,5-dichlorophenol [EC
50
around 2
mg/L test range (0.54 mg/L)]. Preferably use cylindrical vessels
(beakers or petri dishes that are at least 60 mm diam and hold at
least 150 mL). (Although the dish may be shallower than root
length, duckweed growth is not adversely affected.
3
) Vessels
should be inert to expected chemicals and not adsorb them
significantly. This generally leads to a preference for glass ves-
sels. However, cationic substances (e.g., metals) may signifi-
cantly adsorb to borosilicate glass, so specific plastics may be
preferred for such samples. Discard vessels if contamination is
suspected.
Add the same amount of nutrients to all control and test
samples (e.g., 1 to 10 mL of concentrated nutrient solution to
make 100 mL sample). Prepare at least 100 mL portions of test
solution (or control sample). Select duckweed specimens from
stock cultures that have been grown under the same conditions.
Use only unblemished colonies (green and healthy looking; not
chlorotic, necrotic, or irregularly shaped) containing two or three
fronds of approximately equal size per colony. Place 12 duck-
weed fronds (identical numbers of colonies per vessel) in uni-
form glass plates or hourglasses.
Illuminate from above with continuous (24-h) cool-white
fluorescent light (2150 to 4300 lux at water surface). If no
appropriate light meter is available, check light field via a set of
control samples. If all control samples surpass a growth rate of
0.275/d, then there is enough light (this is generally a more
reliable indication of sufficient light than any physical measure-
ment). To avoid dose-dependent shadowing and reflection ef-
fects of colored or turbid solutions, place a black bottom under
all test vessels (very important) and incubate at 24 ⫾2°C.
Test duration is 168 h. Sometimes shorter test durations may
be possible, but this can significantly reduce test sensitivity. If a
shorter test is necessary, do not reduce the number of observa-
tions; instead, eliminate the first 1 or 2 days of growth if this
seems to be an adaptation phase (delayed reaction) in which even
highly contaminated samples will still grow near to control
values. Because results are calculated based on growth rate, such
a reduction is scientifically permitted but should always be noted
in the results [e.g., EC
50
(2–5 d)].
When testing effluent toxicity in a receiving water, renew with
fresh effluent if sample seems to be unstable, and use receiving
water as diluent if necessary for the test goal. Otherwise, use a
standard water as diluent (see Table 8010:III). Complete frond
counts at least every 72 h (at the beginning and end of the test,
and at least two intermediate points) to determine any interme-
diate toxic effects.
4. Test Results
For visual observations, place test vessels on a white back-
ground or apply direct light from the side or bottom into the
vessel. Observe duckweed plants for symptoms, including chlo-
rosis (loss of pigment/yellowing), necrosis (localized dead tis-
sue), colony breakup, root destruction, loss of buoyancy, and
gibbosity (humpback or swelling). Compare affected fronds with
duckweed specimens in the control. Count frond increase daily
to provide a quantal value directly reflecting duckweed growth.
To measure frond increase, count every visible, protruding bud.
DUCKWEED (8211)/Toxicity Test Procedure
3
DUCKWEED (8211)/Toxicity Test Procedure

This nondestructive method allows repeated observations of the
same test vessel.
Using projected lead area measurement based on image pro-
cessing (combining automatic leaf count and area measure-
ment*) provides another nondestructive observational parameter
to help compensate for variability caused by colonies with dif-
ferent start sizes. It also serves as a good estimate for biomass
production. Leaf area measurement is generally at least as sen-
sitive as frond measurement because many toxicants not only
reduce frond number but also average frond size. Use this
additional parameter at least at the beginning and end of the test
(where measurement is destructive); take six additional ran-
domly selected replicates for initial measurement to calculate
mean start value.
Although other scoring methods have been used, including
14
C uptake, chlorophyll content, dry weight, frond area, plant
colony counts, total root number, and root length,
4,5
only dry
weight measurement is recommended as an alternative here to
produce reliable results. Sometimes duckweed may exhibit de-
layed toxic effects (2 to 3 days), when newly developed frond is
affected but not those already developed in the pouches. Lag
phases (delayed start of growth) found in controls after a change
of medium are a clear sign of change-of-growth conditions at test
inception that should be avoided. Stimulatory effects may occur
in some instances due to hormesis. Stimulatory effects due to
extra nutrients are widely minimized by the optimized nutrient
Steinberg medium.
Report this effect if observed, even if you cannot include it in
EC-value calculations.
5. Statistical Analysis
Follow general procedures described in Section 8010G. In
control samples, duckweed growth conforms more to exponen-
tial than linear kinetics, so percent inhibition values must be
calculated from growth rates, not measurement values (which
imply a linear growth pattern).
6
Using growth rate eliminates the
mathematical difference among EC values due to differences in
test durations and the controls’ absolute growth. It also allows
for biologically relevant comparisons of sensitivity between dif-
ferent species (e.g., algae) and the use of segmented growth rates
(e.g., day 3 to 5 or 7, as described above). First, calculate average
growth rate for each parameter X(e.g., frond number, frond area)
separately for each vessel over part or all of the test period using
intermediate values to identify time-dependent changes:
r⫽
ln x
t2
⫺ln x
t1
t
2
⫺t
1
where:
r⫽growth rate per day,
x
t1
⫽value of observation parameter at t
1
days,
x
t2
⫽value of observation parameter at t
2
days, and
t
2
⫺t
1
⫽time period between t
1
and t
2
in days
Differences are measured in days. A graph that plots the log of
the measured parameter versus time (in which slope ⫽growth
rate) also is instructive.
Second, calculate mean values of average growth rate for
controls and treatments to express percent toxicity (or stimula-
tion) relative to the control:
%I⫽100 共C⫺T兲/C
where Cand Tare mean average growth rates of the measured
parameter in control and test samples, respectively.
Inhibition/stimulation values of definitive tests may be
graphed using linear, semi-log, or log–log plots. Typically, the
concentration– effect relationship is sigmoidal. Determine inhi-
bition values (the concentration causing X% inhibitory effect;
X⫽5, 10, 20, 25, 50) via graphical or statistical methods. Each
effective reproduction concentration (ErC) value should be ei-
ther near or bracketed by inhibition values. Whenever possible,
report ErC value with confidence intervals, growth rate, and
parameter used [e.g., ErC
10
(frond area)]. Only such comprehen-
sive reporting in databases allows scientifically reliable compar-
ison between actual and literature values.
In older studies, the calculation method was often not re-
ported; it may be scientifically outdated or have biased final
biomass or yield values [effective biomass concentration (EbC)]
due to exponential growth and different test duration. There is no
retrospective way to recalculate ErC values from such EC values
because the conversion factor depends on the controls’ absolute
growth rate and the slope of the dose–response relationship
(which generally was not reported). As a general estimate, an
EbC
50
value corresponding to ErC
20
values can be used for
duckweed tests. Although they seem less sensitive, growth rate
values are scientifically correct, while EbC values are not. The
slope of dose–response relationship is toxicant-specific and thus
can be valuable information; it should be reported as different
ErC
X
values for each parameter.
6. Quality Control
Negative controls are used to ensure that growth effects are
reasonable based on the test population’s typical growth rates. A
test is unacceptable if the duckweed’s doubling time is less than
2 d, or if the control sample yields less than a sevenfold increase
in fronds in 168 h. If the coefficient of variation for the controls’
growth rate is more than 10%, conditions should be controlled
and optimized, but the test is still considered valid. In most cases,
it is sufficient to avoid placing test vessels at sites with unsuit-
able growth conditions.
Use a reference chemical at one specified concentration as a
positive control. Perform a positive control dose–response test
with 3,5-dichlorophenol regularly [ErC
50
around 2 mg/L test
range (0.5– 4 mg/L)]. Chromate ion can also be used as a
reference toxicant.
6,7
7. References
1. DAVIS, J.A. 1981. Comparison of Static-Replacement and Flow-
Through Bioassays Using Duckweed, Lemna gibba G-3, EPA-560/
6-81-003. U.S. Environmental Protection Agency, Washington, D.C.
2. WALBRIDGE, C.T. 1977. A Flow-Through Testing Procedure with
Duckweed (Lemna minor L.), EPA-600/3-77-108. U.S. Environmen-
tal Protection Agency, Duluth, Minn.
* LemnaTec Scanalyzer or equivalent.
DUCKWEED (8211)/Toxicity Test Procedure
4
DUCKWEED (8211)/Toxicity Test Procedure

3. WANG, W. 1992. Toxicity reduction of photo processing wastewaters.
J. Environ. Sci. Health A27:1313.
4. TARALDSEN, J.E. & T.J. NORBERG-KING. 1990. New method for deter-
mining toxicity using duckweed (Lemna minor). Environ. Toxicol.
Chem. 9:761.
5. WANG,W.&J.WILLIAMS. 1988. Screening and biomonitoring of
industrial effluents using phytotoxicity tests. Environ. Toxicol. Chem.
7:645.
6. WANG, W. 1987. Chromate ion as a reference toxicant in aquatic
phytotoxicity tests. Environ. Toxicol. Chem. 6:953.
7. WANG, W. 1986. The effect of river water on phytotoxicity of barium,
cadmium, and chromium ions. Environ. Poll. (Sec. B) 11:193.
8. Bibliography
U.S. ENVIRONMENTAL PROTECTION AGENCY. 1996. Aquatic plant toxicity
testing using Lemna spp. Tier I and II, Ecological Effects Guide-
lines OPPTS 850.4400 (draft), EPA 712-C-96-156. U.S. Environ-
mental Protection Agency, Washington, D.C.
DUCKWEED (8211)/Toxicity Test Procedure
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DUCKWEED (8211)/Toxicity Test Procedure