United States

Environmental Protection 

Agency

Office of Chemical Safety

and Pollution Prevention

(7101)

EPA 712-C-008

January 2012

Ecological Effects

Test Guidelines

OCSPP 850.4400:

Aquatic Plant Toxicity 

Test Using Lemna spp.
i

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.gov/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.
Page 1 of 22

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 Reregistration 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 (IC50) 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 (IC50) 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 IC05 and IC90) 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 IC50 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 IC50

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 
Page 3 of 22

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., IC50 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 (lbs 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 lb 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 enduse 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)(1) of 

this guideline).

(ii) Source. Axenic cultures may be obtained from laboratory cultures or 

commercial sources.
Page 4 of 22

(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)(1) 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 flowthrough 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 
Page 5 of 22

concentrations (for example, on days 3 and 5 or on days 2 and 4). 

Transfer should be done using aseptic technique in a clean, draftfree 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., IC50), 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 IC50 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 (IC05). 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 IC50 value and the most sensitive 

NOEC (or IC05) 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.
Page 6 of 22

(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 sevenfold 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 than16 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, 
Page 7 of 22

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 (μm) filter). Preparation of this 

medium is described under paragraph (j)(1) 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 (oC).
Page 8 of 22

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 (MgCl2 • 6H2O) - 6.082 g

 Calcium chloride dihydrate (CaCl2 • 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 (MnCl2 • 4H2O) - 207.690 mg

 Zinc chloride (ZnCl2) - 1.635 mg

 Ferric chloride hexahydrate (FeCl3 • 6H2O) - 79.880 mg

 Cobalt chloride hexahydrate (CoCl2 • 6H2O) - 0.714 mg

 Sodium molybdate dihydrate (Na2MoO4 • 2H2O) - 3.630 mg

 Copper chloride dihydrate (CuCl2 • 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 (N) or 1.0 N sodium hydroxide (NaOH) or hydrochloric acid (HCl).

Filter the media into a sterile container through a 0.22 μm membrane filter. Store medium in the dark at 

approximately 4 degrees Celsius (oC) 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 oC 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 

(μmol/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 
Page 9 of 22

acid (HCl) 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 
Page 10 of 22

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 

oC, cooled in a dessicator, and weighed to the nearest 0.1 milligram 

(mg). The drying process is repeated until constant weight is 

obtained.
Page 11 of 22

(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.

n

w

w total

frond  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).
Page 12 of 22

Y  b1  b0 Equation 2

where:

Y = yield of observed biomass (number of fronds or dry weight or frond 

area)

b0 = biomass (number of fronds or dry weight or frond area, respectively) 

at test initiation

b1 = 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.

   

t

b b

r

j i

i j

ln  ln

  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 i to j.

bi = observed biomass (number of fronds or dry weight or frond area, 

respectively) at beginning of the observation interval, time i

bj = observed biomass (number of fronds or frond area or dry weight, 

respectively) at end of the observation interval, time j

t = time period from i to j 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 
Page 13 of 22

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 timeweighted 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.

  

C

C X

I

100 %

 Equation 4

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.
Page 14 of 22

(5) Doubling time of control. The doubling time (Td) of the number of fronds in the 

control is calculated using Equation 5.

Control

d

r

T

ln(2)

 Equation 5

where:

rControl = mean of the control average specific growth rate test vessel values from 

paragraph (f)(1)(ii) of this guideline.

(6) Limit test―

(i) IC50 values. To ascertain that the yield and average specific growth rate IC50

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 IC50 compared to controls is operationally defined as a 50% reduction or 

inhibition from the control sample mean (Equation 6). The null hypothesis (H0) 

stated in terms of true population parameters is that the difference of the “limit” 

treatment response (μlimit) from the control mean response (μcontrol) is greater than 

or equal to a 50% inhibition or reduction, compared to the control (i.e., H0: μcontrol

- μlimit > δ0). The alternative hypothesis (HA) is that this difference is less than a 

50% reduction, compared to the control (HA: μcontrol - μlimit < δ0). 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., 

IC50 > 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., IC50 < limit concentration).

   100  0  xcontrol  p Equation 6

where:

δ0 = difference between two parameters, defined in this case as a p% 

reduction from the control sample mean;

control x = 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 IC50.
Page 15 of 22

(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 IC50. For dose-response tests 

statistical procedures are employed to calculate the IC50 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 IC50 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 IC50 value for a given response measure, test and document that the IC50

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 IC05 value for 

yield or growth rate) is estimated and used in place of the given NOEC. The 
Page 16 of 22

standard error and 95% confidence interval should also be calculated for the IC05

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), (j)(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 Static renewal (pesticides)

Static, static renewal, or flow through (industrial 

chemicals)

Test duration 7 days 

Test matrix Synthetic growth medium

Temperature 25 ± 2 oC

Light quality Warm-white or cool-white fluorescent

Light intensity 57 - 90 μmol/m2/s

Photoperiod Continuous

Test vessel size Sufficient to prevent crowding (e.g., 250 - 1000 mL 

beakers or flasks)

Age of inoculum From healthy stock cultures 7 -12 days old

Size of inoculum 12 -16 fronds total, with the same number of plants 

and fronds in each test vessel

Number of replicate test vessels per concentration Four (minimum)

Test concentrations Unless performing limit test, minimum of 5 test 

concentrations plus appropriate controls

Test concentration preparation Aqueous solutions prepared by adding test 

substance to synthetic nutrient medium, directly or 

via vehicle

Measures of effect (measurement endpoints) 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 
Page 17 of 22

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)(1) 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)).
Page 18 of 22

(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.
Page 19 of 22

(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).
Page 20 of 22

(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 IC50 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 IC05 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 IC50 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 IC05 value is used in place of a NOAEC provide a description of 

statistical method(s) used for point estimates, including software package, for 

determining IC05 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.
Page 21 of 22

(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 flowthrough 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 D.J. 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 

1/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: 10th

Volume, ASTM STP 971, W.J. Adams, G.A. 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 Reregistration 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 22 of 22

(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.