&EPA
United States
Environmental Protection
Agency
Office of Chemical Safety
and Pollution Prevention
(7101)
EPA712-C-008
January 2012
Ecological Effects
Test Guidelines
OCSPP 850.4400:
Aquatic Plant Toxicity
Test Using Lemna spp
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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 850.4400: Aquatic plant toxicity test using Lemna spp.
(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 materials used in developing this harmonized OCSPP test
guideline include OPPT guideline under 40 CFR 797.1160 Lemna Acute Toxicity Test;
the OPP Non-target Plants: Growth and Reproduction of Aquatic Plants - Tiers 1 and 2
Standard Evaluation Procedure; OPP 122-2 Growth and Reproduction of Aquatic Plants
(Tier 1), OPP 123-2 Growth and Reproduction of Aquatic Plants (Tier 2) (Pesticide
Assessment Guidelines Subdivision J—Hazard Evaluation: Nontarget Plants); OPP
Pesticides Reregi strati on Rejection Rate Analysis: Ecological Effects; and ASTM E
1415-91, Standard guide for conducting static toxicity tests with Lemna gibba G3.
(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. This guideline prescribes test procedures and conditions using the freshwater
vascular aquatic plants Lemna gibba or Lemna minor to develop data on the phytotoxicity of test
substances. The Environmental Protection Agency will use data from these tests in assessing the
hazard and risks a test substance may present in the aquatic 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
the plant test guidelines and OCSPP 850.1000 (Background and special considerations for
conducting ecological effects tests with aquatic and sediment-dwelling fauna and aquatic
microcosms), which provides general information for conducting toxicity tests in an aqueous
matrix.
(c) Definitions. The definitions in OCSPP 850.1000 and OCSPP 850.4000 are applicable to this
guideline. In addition, the more specific definitions in this paragraph also apply:
Colony is an aggregate of mother and daughter fronds attached to each other.
Frond is a single Lemna "leaf-like" structure.
Frond mortality refers to dead fronds which are identified by a total discoloration
(yellow, white, black, or clear) of the entire frond.
(d) General considerations—
(1) Summary of the test. Organisms of a particular species of duckweed (Lemna gibba
or Lemna minor} are maintained in test vessels containing nutrient medium alone and
nutrient medium to which the test substance has been added. Over an exposure period of
7 days, data on population growth are obtained on a regular basis. In addition to
measurements of effects on frond number, effects on frond size (dry weight or frond area)
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are determined. The test is designed to determine the quantity of test substance that
results in a 50 percent inhibition (1C50) in yield and average growth rate based on number
of fronds and yield, and average growth rate based on frond size (dry weight or frond
area), and to determine the no observed effect concentration (NOEC) for these effect
measures. The results are used to establish toxicity levels, evaluate hazards or risks to
aquatic vascular plants, and to indicate if further testing at a higher tier is necessary.
Note historically in OCSPP pesticide and industrial chemical guidelines the term ECX was
used to cover both the current OCSPP 850.4000 definition of ECX (concentration where x
percent (x%) of the population exhibit the effect (e.g., mortality)) and ICX (concentration
resulting in an x% decrease or inhibition effect on an attribute of the population (e.g.,
growth rate)).
(2) General test guidance. The general guidance in OCSPP 850.4000 and OCSPP
850.1000 applies to this guideline except as specifically noted herein.
(3) Range-finding test. A range-finding test is usually conducted to establish the
appropriate test solution concentrations for the definitive test. In the range-finding test,
the test organisms are exposed to a series of widely-spaced concentrations of the test
substance (e.g., 0.1, 1.0, 10, 100 milligrams per liter (mg/L), etc.), usually under static
conditions. The details of the range-finding test do not have to be the same as the
definitive testing in that there are no replicates, and the number of test organisms used,
and duration of exposure may be less than in definitive testing. In addition, the types and
frequency of observations made on test organisms are not as detailed or as frequently
observed as that of a definitive test and results are analyzed using nominal
concentrations. However, the range-finding test will be more useful the greater the
similarity between the range-finding and the definitive test.
(4) Definitive test. The goal of the definitive test is to determine for Lemna sp. the
concentration-response curve for yield and growth rate, and the median inhibition
concentration (ICso) value for each of these responses (with 95% confidence interval and
standard error). If possible the slopes of the concentration-response curves, associated
standard errors, and the 95% confidence intervals of the slopes should also be
determined. However, at a minimum, the full concentration-response curve (curve
between ICos and ICgo) is determined for the most sensitive measure of effect using a
minimum of five concentrations of the test chemical, plus appropriate controls. For a
satisfactory test, the lowest treatment concentration is below the ICso value for all
measures of effect. Analytical confirmation of test concentrations is performed as
described in OCSPP 850.1000. A summary of test conditions is provided in Table 2 in
paragraph (g) of this guideline and validity elements for an acceptable definitive test are
listed in Table 3 in paragraph (h) of this guideline.
(5) Limit test. In some situations, it is only necessary to ascertain that the 7-day ICso
value for yield, area under the growth curve, and growth rate are above a certain limit
concentration, and that at this limit concentration there is no observable adverse effect.
For pesticides a limit test has also been referred to as a Tier I test or Maximum Challenge
Concentration test. In a Lemna sp. limit test, at least four replicate test vessels are
exposed to a single "limit concentration," with the same number of test vessels containing
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the appropriate control solution(s). The multiple-concentration definitive test may be
waived if the following two conditions are met for all measures of effect (yield and
average specific growth rate based on number of fronds and frond size (dry weight or
frond area). First, the "limit" treatment response is both statistically less than a 50%
decrease from the control response (i.e., ICso values > limit concentration), and second,
limit treatment responses are not significantly reduced (or inhibited) as compared to the
control response (i.e., NOEC > limit concentration). For most industrial chemicals 1,000
mg/L or the limits of water solubility or dispersion are considered appropriate as the limit
concentration. For pesticides the limit concentration is equivalent to the maximum label
rate (pounds of active ingredient per acre (Ibs a.i./A)) directly applied to a one acre pool
that is 6 inches deep (21,280 cubic feet (ft3) or 602,581 liters). For example, a 1 Ib a.i./A
(or 453,592 milligrams (mg) a.i. per acre) application rate and assuming a water density
of 1 gram per milliliter would have a limit concentration of 0.75 mg a.i./L. Except for the
number of treatment groups, 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 3 in paragraph (h) of this guideline). Acceptable limit tests like
definitive tests include analytical confirmation of the test exposure concentration.
(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 use of the typical end-use product (TEP) instead of the
technical grade active ingredient (TGAI) is preferred for all aquatic plant phytotoxicity
tests. 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. Adjuvants are not used with TEP or TGAI
testing of algae. OCSPP 850.1000 and OCSPP 850.4000 list the type of information that
should be known about the test substance before testing, and discuss methods for
preparation of test substances.
(2) Test duration. The duration of the test is 7 days.
(3) Test organism—
(i) Species. The test species is L. gibba G3 or L. minor. L. gibba has been widely
used for testing with pesticides and other chemicals in the United States, while L.
minor has been used more frequently for testing of environmental samples and in
Europe. The identity of the organism should be verified using an appropriate
taxonomic key and it is also desirable to identify the clone (see paragraph (j)0) of
this guideline).
(ii) Source. Axenic cultures may be obtained from laboratory cultures or
commercial sources.
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(iii) Age and condition. The plants used in testing should be from stock cultures
that have been actively growing in growth medium under the same conditions as
used in the test for at least eight weeks immediately preceding the start of the test.
Plants should be aseptically transferred on a regular schedule (such as weekly) to
fresh growth medium. Long-term maintenance of cultures on a solid medium
containing 1% agar in sterile Petrie dishes or test tubes may be desirable.
However for a satisfactory test, the plants used to initiate toxicity tests are from a
liquid culture. Plants used in a test should be randomly selected from cultures
which are between 7 and 12 days old.
(iv) Culturing procedures. Lemna spp. cultures should be maintained in growth
medium using the culturing procedures described in the reference in paragraph
(j)(l) of this guideline. The cultures should be maintained under the same
conditions as used for testing.
(4) Administration of test substance—
(i) Preparation of test solutions—
(A) Stock solutions or direct addition. Test solutions are prepared by
adding the test substance directly to the nutrient medium or by addition of
a stock solution to the nutrient medium. Typically, a stock solution of the
test substance is prepared and aliquots of the stock solution added to
nutrient medium to obtain the desired test concentrations. Guidance for
preparation of test solutions, especially for difficult or low solubility test
substances, is provided in OCSPP 850.1000.
(B) Solvents. The recommended solvent for Lemna spp. toxicity tests is
N,N-dimethyl-formamide, as solvents such as acetone can cause
stimulation of bacterial growth. The concentration of solvent should
preferably be the same in all test treatments and should not exceed 0.1
milliliter per liter (mL/L).
(C) Exposure technique.
(1) For pesticides, this test should be conducted using the static
renewal exposure technique. For industrial chemicals, this test
may be conducted using a static, static renewal, or flow-through
with guidance on the selection of the appropriate exposure
technique based on the stability of the test substance as provided in
OCSPP 850.1000. To conduct tests with duckweed using the flow-
through exposure technique see the references in paragraphs (j)(2),
(j)(4) and (j)(13) of this guideline, which may be necessary when
testing volatile industrial test substances.
(2) Static renewal of test solutions. For static renewal tests, the
plants should be transferred to fresh test solutions of initial test
concentration levels at intervals necessary to maintain the test
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concentrations (for example, on days 3 and 5 or on days 2 and 4).
Transfer should be done using aseptic technique in a clean, draft-
free area as quickly as possible to minimize contamination of the
colonies.
(3) When a substance is know to have a tendency to vaporize,
closed test flasks with increased head-space may be used.
Attempts should be made to determine the amount of the substance
which remains dissolved in solution, and extreme caution is
advised when interpreting results of test with volatile chemicals
using closed systems.
(ii) Treatment levels. At a minimum five test solution concentrations are tested
for multi-concentration definitive testing, plus the appropriate control(s). A
range-finding test can be used to establish the appropriate test solution
concentrations for the definitive test (see paragraph (d)(3) of this guideline). For
scientifically sound estimates of a given point estimate (e.g., ICso), test substance
concentrations should immediately bracket the point estimate(s) of concern. The
concentrations should be a twofold geometric progression (e.g., 0.1, 0.2, 0.4, 0.8,
and 1.6 milligrams per liter (mg/L)). While a twofold progression is preferred,
threefold and fourfold progressions are acceptable. If a fourfold or higher series
progression is used, the rationale for using this large an interval between
concentrations and the effect on the accuracy and reproducibility of the point
estimate and NOEC should be provided. For an acceptable study, the lowest test
treatment level should be lower than the ICso values for yield and average specific
growth rate based on number of fronds and frond size. The lack of a NOEC for
an effect measure is not critical as long as the response-curve for the effect
measure is acceptable for calculation of the 5% inhibition concentration (ICos). It
is recommended that one or two additional test concentrations in the lower tail of
the concentration-response curve of the most sensitive endpoint be added to
insure bracketing of both the most sensitive ICso value and the most sensitive
NOEC (or ICos) value. For a limit test, there is single treatment concentration,
plus the appropriate control(s). Guidance on the limit concentration is provided in
paragraph (d)(5) of this guideline.
(iii) Introduction of test organisms.
(A) In preparation for the test, containers are filled with appropriate
volumes of nutrient medium and/or the test solutions. The test is initiated
by introducing Lemna sp. fronds into each of the test vessels within 30
minutes of addition of test substance to test solutions. Plants should be
placed in the test vessels using a sterile inoculating loop or hook, Nitex
screen, or other aseptic technique. The plants should be impartially or
randomly distributed among the test vessels in such a manner that test
results show no significant bias from the distributions. The test vessels are
then immediately placed in a growth chamber or the controlled laboratory
environment.
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(B) Test vessels within the testing area are positioned in a random manner
or in a way in which appropriate statistical analyses can be used to
determine the variation due to placement.
(5) Controls.
(i) Every test includes a negative control treatment consisting of the same nutrient
medium, number of test organisms, environmental conditions, and procedures as
the treated test vessels except that none of the test substance is added. In addition,
vehicle (solvent) controls are also included if a solvent is used to dissolve or
suspend the test substance.
(ii) At test termination the control coefficient of variation for yield should
generally be less than 20% and substantially less than 20% for growth rate, which
is a logarithmically-transformed variable.
(iii) For a satisfactory test the doubling time of number of fronds in the control
should be less than 2.5 days (60 hours), corresponding to approximately a seven-
fold increase in seven days and an average specific growth rate of 0.275 per day
(d-1).
(6) Number of test organisms and replicates.
(i) The minimum number of replicates per treatment and control(s) is four, each
containing three to five plants (consisting of three to four fronds per plant). Plants
of similar size and appearance should be selected, and the number of plants and
number of fronds should be identical in each test vessel. A total of at least 12, but
no more than 16 fronds, per test vessel are recommended (e.g. three 4-frond plants
and one 3-frond plant could be used, for a total of 15 fronds).
(ii) For determination of initial dry weight of fronds at test initiation, an additional
control set of replicates are created at test initiation using plants of similar size
and appearance as those selected for the test. The number of replicates should be
equivalent in number to that used for the test.
(7) Facilities, apparatus and supplies—
(i) Containers for culturing and testing. Glass beakers (250 - 1000 milliliters
(mL)), 250 mL flat-bottomed test tubes, and Erlenmeyer flasks (250 - 500 mL)
have been used successfully. For a satisfactory test, test vessels should be large
enough to hold the Lemna colonies without crowding (i.e., space and nutrient
availability should not be growth limiting factors) for the duration of the test.
Test vessels should be covered to keep out extraneous contaminants and to reduce
evaporation of test solutions. Containers and covers that may contact the test
solution should be chosen to minimize sorption of test substances, and not contain
substances that can be leached or dissolved into aqueous solutions in quantities
that can affect the test results. Beakers may be covered with a clear watch glass
while Erlenmeyer flasks may be covered with foam plugs, stainless steel caps,
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Shimadzu enclosures, glass caps or screw caps. (The acceptability of foam plugs
should be investigated prior to use because some brands have been found to be
toxic). For a satisfactory test, all test vessels and covers in a test are identical.
(ii) Growth chamber or laboratory environment.
(A) A controlled environment growth chamber or an enclosed laboratory
area capable of maintaining the specified number of test vessels and able
to maintain the air temperature, light intensity rate, and photoperiod
specified in this guideline.
(B) Facilities should be well ventilated and free of fumes that may affect
the test organisms. Construction materials and equipment that may
contact the stock solution, test solution, or nutrient medium should be
chosen to minimize sorption of test substances and not contain substances
that can be leached or dissolved into aqueous solutions in quantities that
can affect the test results. Refer to OCSPP 850.1000 for additional
information on appropriate construction materials.
(iii) Environmental monitoring equipment. Equipment for determination of
test environmental conditions (e.g., pH meter, photosynthetically active radiation
(PAR) light sensor, etc.)
(iv) Cleaning and sterilization. Apparatus for sterilizing glassware, preparing
sterile nutrient media, and maintaining aseptic technique during culturing and
testing. All glassware and equipment used in Lemna spp. culturing or testing is to
be cleaned and sterilized prior to use. The Nitex screen or inoculating loops used
for transferring the Lemna sp. should be discarded after use or thoroughly cleaned
and sterilized before reuse.
(v) Nutrient media and diluent. Different media are recommended for culturing
and testing L. gibba and L. minor.
(A) Medium for L. gibba. 20X-AAP medium is recommended for
maintaining L. gibba cultures and for use as the diluent in the preparation
of the various test solution concentrations. Medium is sterilized by
autoclaving or filtering (0.22 micrometer (jim) filter). Preparation of this
medium is described under paragraph (j)(l) of this guideline and in Table
1. Water used for preparation of nutrient medium should be of reagent
quality (e.g., ASTM Type I water). If prepared in advance, medium
should be stored under refrigeration in the dark at 4 degrees Celsius (°C).
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Table 1.—Preparation of 20X-AAP Medium
Add 20 ml of each of the macronutrient stock solutions and 20 ml of the micronutrient stock solution
listed in this table to approximately 800 ml reagent grade water and then dilute to 1 liter (L).
Macronutrient stock solutions are made by dissolving the following chemicals into 500 ml of reagent
grade water:
Sodium nitrate (NaNO3) -12.750 grams (g)
Magnesium chloride hexahydrate (MgCI2 • 6H2O) - 6.082 g
Calcium chloride dihydrate (CaCI2 • 2H2O) - 2.205 g
Magnesium sulfate heptahydrate (MgSO4 • 7H2O) - 7.350 g
Potassium phosphate (K2HPO4) - 0.522 g
Sodium bicarbonate (NaHCO3) - 7.500 g
The micronutrient stock solution is made by dissolving the following chemicals into 500 ml of reagent
grade water:
Boric acid (H3BO3) - 92.760 milligrams (mg)
Manganese chloride tetrathydrate (MnCI2 • 4H2O) - 207.690 mg
Zinc chloride (ZnCI2) -1.635 mg
Ferric chloride hexahydrate (FeCI3 • 6H2O) - 79.880 mg
Cobalt chloride hexahydrate (CoCI2 • 6H2O) - 0.714 mg
Sodium molybdate dihydrate (Na2MoO4 • 2H2O) - 3.630 mg
Copper chloride dihydrate (CuCI2 • 2H2O) - 0.006 mg [Typically must be prepared by serial dilution].
Ethylenediaminetetraacetic acid disodium salt dihydrate (Na2EDTA • 2H2O) -150.000 mg
Adjust pH to 7.5 ± 0.1 with 0.1 Normal (A/) or 1.0 N sodium hydroxide (NaOH) or hydrochloric acid (HCI).
Filter the media into a sterile container through a 0.22 urn membrane filter. Store medium in the dark at
approximately 4 degrees Celsius (°C) until use.
(B) Medium for L. minor. A modification of the Swedish standard
(Standardiseringen I Sverige (SIS)) Lemna spp. medium is recommended
for culturing and testing with L. minor. Preparation of this medium is
described in references in paragraphs (j)(5) and (j)(8) of this guideline.
(vi) Equipment to observe plants. A lighted magnifying lens, dissecting
microscope, or other device may be used to facilitate observations of the fronds.
(8) Environmental conditions. Environmental conditions during the test should be
maintained as follows:
(i) Temperature. Test solution temperature should be 25 ± 2 °C throughout the
duration of the test.
(ii) Lighting and photoperiod. Continuous lighting should be used to provide a
light intensity in the range of 57 - 90 micromoles per meter square per second
(|imol/m2/s). For cool-white fluorescent lighting, this is approximately equivalent
to 4200 to 6700 lux. Warm-white or cool-white fluorescent lighting has been
used for testing with Lemna spp. Additional information on the use of lighting in
plant toxicity tests can be found in the references given in OCSPP 850.4000.
(iii) Nutrient medium pH. Prior to use, the pH of the nutrient medium should be
adjusted to 7.5 ±0.1. 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
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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. Chelating agents, such as
ethylenediamine-tetraacetic acid, are present in the 20X-AAP medium to ensure
that trace nutrients will be available to the L. gibba fronds.
(9) Observations—
(i) Measurement of test substance. Analytical confirmation of dissolved test
concentrations is performed at test initiation and at test termination and as
described for renewal tests in OCSPP 850.1000. Samples for analysis should be
collected as described in OCSPP 850.1000, with the following exception: if there
is insufficient volume at test termination to perform the analysis, the contents of
the replicate test vessels may be pooled after the plants have been removed for
observations. The analytical methods used to measure the amount of dissolved
test substance in a sample are validated before beginning the test, as described in
OCSPP 850.1000.
(ii) Test solution appearance. Observations are made daily on test substance
solubility (e.g., surface slicks, clarity, precipitates, or material adhering to the
sides of the test vessels) and recorded.
(iii) Dilution water quality. The dilution water source used to prepare media
should be periodically analyzed to document and characterize the hardness,
alkalinity, pH, conductivity, total organic carbon (TOC) or chemical oxygen
demand magnitude and variability, and to ensure that pesticides, PCBs and toxic
metals are not present at concentrations that are considered toxic. See OCSPP
850.1000 for guidance on dilution water.
(iv) Environmental conditions—
(A) Temperature. It is impractical to measure the temperature of the
solutions in the test vessels while maintaining axenic conditions.
Therefore, one or two extra test vessels may be prepared for the purpose of
measuring the solution temperature during the test. Alternatively, hourly
measurements of the air temperature (or daily measurements of the
maximum and minimum) are acceptable. Because the vessels are placed
in an environmental chamber or incubator, the air temperature is more
likely to fluctuate than the water temperature.
(B) Light intensity. Light intensity should be monitored at test initiation
at the approximate level of the test solution at each test vessel position in
the growth chamber or laboratory. If it is suspected that light intensity has
changed by 15% or more, monitoring of light intensity should be
conducted daily. A photosynthetically active radiation (PAR) sensor
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should be used to measure light quality. The light intensity at each
position should not differ by more than 15% from the selected light
intensity. Because illumination may vary at different positions, and since
this environmental parameter has an important influence on duckweed
growth, it is recommended that the test vessels be randomly re-positioned
on a regular schedule (e.g., daily) to minimize spatial differences.
(C) pH. The pH in the control(s) and test solutions should be measured.
This measurement is made on the bulk test solutions at the beginning of
each renewal period and at the end of each renewal period on samples of
pooled replicates of each test treatment provided none of the replicates
appear to be "outliers" with respect to growth. In the case of outlier
growth response individual pH measurements should be made in each test
vessel. For a flow-through test for industrial chemicals see OCSPP
850.1000 for guidance on frequency and sampling scheme for pH.
(v) Measures of effect—
(A) Frond number. Using a dissecting microscope or lighted magnifying
lens the number of fronds in each test vessel is determined and recorded at
least every three days during the test and at test termination (e.g. days 3, 5,
and 7 or days 2, 4, and 7). A frond is counted regardless of size as long as
it is visible adjacent to the parent frond. Any bud which is visible when
viewed under a hand lens or dissecting microscope should be counted as a
frond.
(B) Frond size. Acceptable indicators of frond size include determination
of dry weight and/or measurement of frond area. Frond size determined
using dry weight, is made at test initiation and test termination. Frond size
determined using frond area is determined at a minimum at test initiation
and termination.
(1) Dry weight. Dry weight is a destructive procedure. In order to
evaluate the increase in dry weight over the course of the test, a
representative sample of fronds at test initiation should be
processed (see paragraph (e)(6)(ii) of this guideline), using the
same drying method as used at test termination, to establish the
treatment and control mean dry weight of fronds at test initiation.
To determine dry weight, the plants (including roots and root
fragments) for a test vessel (replicate) are removed from the test
solution, rinsed with distilled or deionized water, blotted to remove
excess water, and placed in previously dried, tared weighing pan
for that replicate. The plants should be dried at approximately 60
°C, cooled in a dessicator, and weighed to the nearest 0.1 milligram
(mg). The drying process is repeated until constant weight is
obtained.
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(2) Frond area. Frond area for a test vessel (replicate) is
determined at a minimum at test initiation and test termination but
may also be determined at various intervals during the test. Frond
area can be determined using image analysis, in which an image of
each plant in a test vessel (replicate) is captured using a video
camera and then digitized. An alternative procedure is to
photograph the test vessel (replicate) from the top, cut out the
silhouette of the plants, and determine the area using a leaf area
analyzer or graph paper.
(C) Appearance or condition. The appearance or condition (e.g.,
decrease in size, necrosis, chlorosis, sinking of fronds, or other
abnormalities) of plants in each test vessel (replicate) is determined at
least every three days during the test and at test termination (e.g. days 3, 5,
and 7 or days 2, 4, and 7).
(D) Phytostatic and phytocidal effects. At test termination, it may be
desirable to determine phytostatic and phytocidal effects, e.g. whether or
not plants that were inhibited during the exposure period are able to
resume growth when transferred to test substance-free medium. This
optional procedure may be conducted as described in the reference in
paragraph (j)(6) of this guideline.
(f) Treatment of results—
(1) Determination of crowding. For each treatment and control vessel (replicate)
calculate the dry weight per frond (wfrond) at test termination using Equation 1. If nutrient
availability or space was limiting to growth, the dry weight per frond may be observed to
increase as the number of fronds decreases.
w frond = Wtotal/l Equation 1
where:
wtotal = total dry weight biomass in a test vessel at test termination; and
n = the number of fronds in the test vessel at test termination.
(2) Response variable calculations. There are two response variables calculated from
number of fronds and frond size: yield and average specific growth rate.
(i) Yield calculations. For each treatment and control test vessel (replicate)
calculate the yield by subtracting the initial number of fronds, frond area, or dry
weight from the number of fronds, frond area, or dry weight, respectively,
obtained at test termination (Equation 2).
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Y = bl-b0 Equation 2
where:
7= yield of observed biomass (number of fronds or dry weight or frond
area)
bo = biomass (number of fronds or dry weight or frond area, respectively)
at test initiation
b\ = biomass (number of fronds or dry weight or frond area, respectively)
at test termination
(ii) Average specific growth rate calculations. Average specific growth rate
(sometimes called relative growth rate) is the rate of growth over a given time
interval. The growth rate for number of fronds, frond area, and dry weight for
each test vessel (replicate) over a given time interval is calculated as given in
Equation 3. At a minimum, the average specific growth rate is calculated for the
time interval between test initiation and termination.
Info,)-Info.)
r,-j = —^ — Equation 3
where:
ri-j= average specific growth rate per day (day"1) of observed biomass
(number of fronds or dry weight or frond area) from time / toy.
b[ = observed biomass (number of fronds or dry weight or frond area,
respectively) at beginning of the observation interval, time /'
&j = observed biomass (number of fronds or frond area or dry weight,
respectively) at end of the observation interval, timey
t = time period from /' toj in days
(3) Summary statistics—
(i) Environmental conditions. Calculate descriptive statistics (mean, standard
deviation, coefficient of variation, minimum, maximum) by treatment level for
temperature and pH. Calculate descriptive statistics (mean, standard deviation,
coefficient of variation, minimum, maximum) by test vessel position for light
intensity.
(ii) Test substance concentration. For each treatment level compare the test
substance concentration at the start and end of each renewal period. For a stable
test substance calculate the time-weighted average concentration. For industrial
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chemicals if the test substance was unstable calculate a rate of decline and the
associated time-weighted mean concentration (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. For pesticides under unstable test substance
conditions, use the mean test substance concentration in solution measured at test
initiation and in bulk renewal solutions for calculating concentration-response and
NOEC-LOEC values. Such an approach is used rather than the exponential time-
weighted averaged because of the exposure estimate currently used by OPP for
calculating risk estimates.
(iii) Number of fronds. For each treatment level and observation time calculate
the mean, standard deviation, and coefficient of variation for number of fronds.
Calculate the mean treatment yield and average specific growth rate based on
changes in number of fronds from test initiation to test termination.
(iv) Frond size —
(A) Dry weight. Calculate the mean treatment yield and average specific
growth rate based on changes in dry weight from test initiation to test
termination.
(B) Frond area. For each treatment level and observation time calculate
the mean, standard deviation, and coefficient of variation for frond area.
Calculate the mean treatment yield and average specific growth rate based
on changes in frond area from test initiation to test termination.
(v) Appearance and condition. 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.
(4) Percent inhibition. For yield and average specific growth rate for each response
variable calculate the percent inhibition (%I) at each treatment level using Equation 4.
Equation4
where:
C = the control mean response value (yield or average specific growth rate); and
X = the treatment mean response value (yield or average specific growth rate,
respectively). Stimulation is reported as negative %I.
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(5) Doubling time of control. The doubling time (Td) of the number of fronds in the
control is calculated using Equation 5.
Td = ln(2)/ Equation 5
/ T 'Control
where:
r control = mean of the control average specific growth rate test vessel values from
paragraph (f)(l)(ii) of this guideline.
(6) Limit test—
(i) ICso values. To ascertain that the yield and average specific growth rate ICso
values, based on number of fronds and frond size, 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 measures. For a comparison of sample means, the difference defining
the ICso compared to controls is operationally defined as a 50% reduction or
inhibition from the control sample mean (Equation 6). The null hypothesis (Ho)
stated in terms of true population parameters is that the difference of the "limit"
treatment response (|iiimit) from the control mean response (^control) is greater than
or equal to a 50% inhibition or 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: Hcont-oi - Hiimit < So). An example of a
parametric two-sample comparison test of this is the Student's t-test. If the null
hypothesis is rejected, the inhibition level for the given response measure (i.e.
yield and average specific growth rate based on frond number and size) in the
limit treatment as compared to the control is declared to be less than 50% (i.e.,
ICso > limit concentration). If the null hypothesis is not rejected, the limit
treatment as compared to the control response is declared to be 50% or greater
(i.e., ICso < limit concentration).
Equations
where:
So = difference between two parameters, defined in this case as a p%
reduction from the control sample mean;
^control = control sample mean response (e.g., yield and average mean
biomass); and
p = percent reduction from the control sample mean, which is 50 in the
case of the ICso.
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(ii) NOEC. To ascertain that there is no observable effect at the limit treatment
(i.e., NOEC > limit dose) for a given response measure (yield and average
specific growth rate based on frond number and size), the limit treatment response
is compared to the control treatment response using a one-sided two-sample
parametric or nonparametric test, as appropriate (see OCSPP 850.4000). The
minimum significant difference detectable by the test or a similar estimate of the
sensitivity of the test should be determined and reported.
(iii) Multiple-concentration definitive testing.
(A) A multiple-concentration definitive test is performed if either the
effect or inhibition level for one or more response measures (i.e., yield and
average specific growth rate based on frond number and size) in the limit
treatment as compared to the control response at test termination are
declared to be 50% or greater effect (i.e., the null hypothesis is not
rejected) or the NOEC is less than the limit concentration.
(B) Multiple-concentration definitive testing may be waived if at test
termination the "limit" treatment response is both statistically less than a
50% decrease from the control response and there is no observable
adverse effect from the control response for all measures of effect (yield
and average specific growth rate based on frond number and size).
(7) 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 yield and growth rate based on number of fronds and
frond size (dry weight or frond area) at test initiation and test termination. If a
concentration-response curve model was fit to the data to determine an ICso value,
the model parameters (e.g., slope) and their uncertainty estimates (e.g., standard
error) should be recorded. The response values for each test vessel, not the mean
response for each treatment level, should be used in fitting the model. Where the
concentration-response range tested does not result in the determination of a
definitive ICso value for a given response measure, test and document that the ICso
value is above the highest treatment level tested (see the statistical guidance in
paragraph (f)(6) of this guideline). Such an event may arise if one of the other
response measures is much more sensitive, and while the full response curve for
that response measure is captured too many additional treatments would be
needed to capture the full response relationship for the other less sensitive
response measure(s).
(ii) NOEC. The 7-day NOEC for yield and growth rate based on number of
fronds and frond size (dry weight or frond area) are determined (see OCSPP
850.4000). If a 7-day NOEC value can not be determined for a given response
measure, the concentration at which there is a 5% inhibition (i.e., an ICos value for
yield or growth rate) is estimated and used in place of the given NOEC. The
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standard error and 95% confidence interval should also be calculated for the ICos
value. For industrial chemicals, the specific ICX used in place of a NOEC that can
not be determined will vary, consult with the Agency. Methods, assumptions, and
results of the statistical approaches used should be recorded.
(iii) Statistical methods. Statistical procedures for modeling continuous toxicity data are
available and should be used (see references in paragraphs (j)(3), G)(7) and (j)(12) of this
guideline). Additional discussion about endpoints and statistical procedures is found in
OCSPP 850.1000 and OCSPP 850.4000.
(g) Tabular summary of test conditions. Table 2 lists the important conditions that should
prevail during the definitive test. Except for the number of treatment levels, 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.
Table 2.—Summary of Test Conditions for the Lemna sp. Toxicity Test
Test type
Test duration
Test matrix
Temperature
Light quality
Light intensity
Photoperiod
Test vessel size
Age of inoculum
Size of inoculum
Number of replicate test vessels per concentration
Test concentrations
Test concentration preparation
Measures of effect (measurement endpoints)
Static renewal (pesticides)
Static, static renewal, or flow through (industrial
chemicals)
7 days
Synthetic growth medium
25 ± 2 °C
Warm-white or cool-white fluorescent
57 - 90 umol/m2/s
Continuous
Sufficient to prevent crowding (e.g., 250 - 1000 mL
beakers or flasks)
From healthy stock cultures 7-12 days old
12-16 fronds total, with the same number of plants
and fronds in each test vessel
Four (minimum)
Unless performing limit test, minimum of 5 test
concentrations plus appropriate controls
Aqueous solutions prepared by adding test
substance to synthetic nutrient medium, directly or
via vehicle
IC50 and NOEC (or IC05) values for yield, and
average specific growth rate based on frond
number
IC50 and NOEC (or IC05) values for yield and
average specific growth rate based on frond size
(dry weight or frond area)
(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 and OCSPP 850.1000, it is unlikely a study will be rejected when there are
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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.
Table 3.—Test Validity Elements for the Lemna Toxicity Test
1. All test vessels and closures were not identical.
2. The duckweed plants were not impartially or randomly assigned to the test vessels.
3. A medium (untreated) control [and solvent (vehicle) control, when a solvent was used] was not
included in the test.
4. For testing with industrial chemicals, a surfactant or dispersant was used in the preparation of a stock
or test solution.
5. The concentration of solvent in the range used affected growth of the test species.
6. A minimum of five test concentrations were not used in the definitive test.
7. Controls were contaminated with the test substance.
8. Temperature and light intensity were not measured as specified during the test.
9. The doubling time of number of fronds in the control exceed 2.5 days.
10. The lowest test concentration level was not less than the 7-day yield and average specific growth
rate IC50 values based on number of fronds and frond area (dry weight or frond area).
(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
OCSPP 850.4000.
(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, the reasons for these changes, and any
resulting effects on test endpoints noted and discussed.
(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)).
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(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 test species.
(i) Scientific and common name, plant family, and strain.
(ii) Source and method of species and strain verification.
(iii) Culture practices, including culturing media used, and conditions.
(iv) Acclimation period, if applicable.
(v) Age (stage) of inoculum at test initiation.
(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) Description of the incubator, growth chamber, or laboratory location, type of
lights and aeration or agitation of test vessels.
(ii) Description of the test container used: size, type, material, fill volume.
(iii) Number of fronds per plant and number of plants and fronds added to each
test vessel at test initiation.
(iv) Number of test vessels (replicates) per treatment level and control(s).
(v) Description of the preparation of the synthetic growth media used including
the preparation date, concentration of all constituents, the initial pH, and storage
conditions and duration prior to use in test.
(vi) Description of the dilution water and any water pretreatment: source/type;
pH; total organic carbon content; particulate matter content; metals, pesticides,
and chlorine concentration. Describe the frequency and sample date(s) for
documenting dilution water quality.
(vii) Methods used for treatment randomization and assignment of plants to test
vessels.
(viii) Date of introduction of test organisms to test solutions and test duration.
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(ix) Exposure technique and frequency of renewal if static renewal and flow rate
if flow-through.
(x) The photoperiod and light source.
(xi) Methods and frequency of environmental monitoring performed during the
definitive or limit study for temperature, light intensity, pH.
(xii) Methods and frequency of measuring test substance to confirm exposure
concentrations.
(xiii) Methods and frequency of measuring number of fronds, frond size (dry
weight or frond area), and any other symptoms.
(xiv) For the definitive and limit test, all analytical procedures should be
described. The accuracy of the method, method detection limit, and limit of
quantification should be given.
(6) Results.
(i) Tabulation of test substance analytical results by test vessel and treatment
(provide raw data) and descriptive statistics (time-weighted mean, standard
deviation, minimum, maximum, coefficient of variation).
(ii) Environmental monitoring data results (test solution temperature, light
intensity, and pH) in tabular form (provide raw data for measurements not made
on a continuous basis), and descriptive statistics (mean, standard deviation,
minimum, maximum).
(iii) For preliminary range-finding tests, if conducted, the number of fronds and
frond size (dry weight or frond area) at each treatment level and in the control(s).
A description and count of visual phytotoxic effects, if recorded, at each treatment
level and in the control(s).
(iv) For a limit test, tabulate for the limit concentration and the control(s) by
replicate, the number of fronds and frond size (dry weight in representative
samples or frond area) at test initiation and termination, the number of fronds and,
if measured, frond area in each test vessel at each observation time during the test
(provide the raw data).
(v) For the definitive test, tabulation by test vessel and treatment of the number of
fronds and frond size (dry weight or frond area) at test initiation and termination,
and the number of fronds and, if measured, frond area in each test vessel at each
observation time during the test (provide the raw data).
(vi) For the limit and definitive tests, tabulation by test vessel and treatment of
yield and average specific growth rate for number of fronds and frond area (dry
weight or frond area).
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(vii) For the limit and definitive tests, tabulation of the mean treatment yield and
average specific growth rate values, treatment standard deviations for these
variables, and the treatment %I (or stimulation) in yield and average specific
growth rate relative to the control values.
(viii) For the limit and definitive test, tabulation of observed morphologic signs of
toxicity (chlorosis, necrosis, mortality, pigmentation, abnormal shape).
(ix) Graphs of the concentration-response data for yield and average specific
growth rate based on number of fronds and frond size (dry weight or frond area).
(x) For a limit test, provide the results of hypothesis tests.
(xi) For the limit test, provide a description of the statistical methods used
including software package, and the basis for the choice of method.
(xii) For the definitive study and for those effect measures (yield and average
specific growth rate for number of fronds and frond size (dry weight or frond
area)) with data sufficient to fit a concentration-response relationship, tabulation
of the slope of the concentration-response curve and its standard error and 95%
confidence limits and any goodness of fit results.
(xiii) For the definitive test, tabulation of ICso values for yield and average
specific growth rate for number of fronds and frond size (dry weight or frond
area).
(xiv) For the definitive test, a tabulation of the NOAEC and LOAEC for each
response variable (yield and average specific growth rate based on number of
fronds and frond size (dry weight or frond area)). The ICos and 95% confidence
interval should be reported for response data where an NOAEC could not be
determined.
(xv) Description of statistical method(s) used for point estimates, including
software package, for determining ICso values, fitting the dose-response model,
and the basis for the choice of method. Provide results of any goodness-of-fit
tests.
(xvi) Description of statistical method(s) used for NOAEC and LOAEC
determination, including software package, and the basis for the choice of
method. If an ICos value is used in place of a NOAEC provide a description of
statistical method(s) used for point estimates, including software package, for
determining ICos values, fitting the dose-response model, and the basis for the
choice of method. Provide results of any goodness-of-fit tests.
(xvii) If determined, report the phytostatic and phytocidal concentrations.
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(j) References. The following references should be consulted for additional background
material on this test guideline.
(1) American Society for Testing and Materials. ASTM E 1415-91(2004). Standard
guide for conducting static toxicity tests with Lemna gibba G3. In Annual Book of
ASTM_Standards, Vol. 11.06, West Conshohocken, PA. Current edition reapproved
2004.
(2) Bishop, W.E., and R.L. Perry, 1981. The development and evaluation of a flow-
through growth inhibition test with duckweed (Lemna minor), In Aquatic Toxicology and
Hazard Assessment, ASTM STP 737, ASTM, Philadelphia, PA, pp. 421 - 435.
(3) Bruce, R.D. and DJ. Versteeg, 1992. A statistical procedure for modeling continuous
toxicity data. Environmental Toxicology and Chemistry 11:1485-1494.
(4) Davis, J.A., 1981. Comparison of static-replacement and flow-through bioassays
using duckweed, Lemna gibba G3. EPA-560/6-81-003, U.S. Environmental Protection
Agency, Washington, DC.
(5) Environment Canada, 1999. Biological Test Method: Test for Measuring the
Inhibition of Growth using the Freshwater Macrophyte, Lemna minor. Report EPA
l/RM/37, Method Development and Application Section, Environmental Technology
Centre, Environment Canada, Ottawa, Ontario.
(6) Hughes, J.S., Alexander, M.M., and Balu, K, 1988. An evaluation of appropriate
expressions of toxicity in aquatic plant bioassays as demonstrated by the effects of
atrazine on algae and duckweed, Aquatic Toxicology and Hazard Assessment: 10*
Volume, ASTM STP 971, W.J. Adams, GA. Chapman and W.A. Landis, eds., ASTM,
Philadelphia, PA, pp. 531-547.
(7) Nyholm, N., P.S. Sorenson, K.O. Kusk, and E.R. Christensen, 1992. Statistical
treatment of data from microbial toxicity tests, Environmental Toxicology and Chemistry
11:157-167.
(8) Organization for Economic Co-operation and Development, 2006. OECD Guidelines
for Testing of Chemicals, Test No. 221, Lemna sp. Growth Inhibition Test, Adopted 23
March, 2006.
(9) U.S. Environmental Protection Agency, 1994. Pesticides Reregi strati on Rejection
Rate Analysis: Ecological Effects, Office of Prevention, Pesticides and Toxic Substances,
Washington, D.C. EPA 738-R-94-035
(10) U.S. Environmental Protection Agency, 1986. Hazard Evaluation Division Standard
Evaluation Procedure, Non-target Plants: Growth and Reproduction of Aquatic Plants
Tiers 1 and 2. Office of Pesticides Programs, Washington, D.C. EPA 540/9-86-134.
Page 21 of 22
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(11) U.S. Environmental Protection Agency, 1982. Pesticide Assessment Guidelines,
Subdivision J Hazard Evaluation: Non-target plants. Office of Pesticides and Toxic
Substances, Washington, D.C. EPA 540/9-82-020, October 1982.
(12) VanEwijk, P.H. and J.A. Hoekstra, 1993. Calculation of the EC50 and its confidence
interval when subtoxic stimulus is present, Ecotoxicology and Environmental Safety
25:25-32.
(13) Walbridge, C.T., 1977. A flow-through testing procedure with duckweed (Lemna
minor L.), EPA -600/3-77-108, U.S. Environmental Protection Agency, Duluth, MN.
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