Biological test method for measuring the inhibition of growth using freshwater macrophyte: front matter


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Lisa Taylor, Manager
Method Development & Applications Section
Environmental Technology Centre
Environment Canada
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Abstract

A biological test method recommended by Environment Canada for performing toxicity tests that measure the inhibition of growth using the aquatic macrophyte, Lemna minor, is described in this report. This second edition of EPS 1/RM/37, published in 2007 supersedes the first edition that was published in 1999. It includes numerous procedural modifications as well as updated guidance and instructions to assist in performing the biological test method.

The test is conducted at 25 ± 2°C in test vessels containing a minimum of 100 mL of test solution and two, 3-frond plants. The test may be run as a multi-concentration assay to determine the threshold of effect, or with only one concentration as a regulatory or pass/fail test. This test uses ≥3 replicated test vessels/treatment for a single-concentration test, and ≥4 replicated test vessels/treatment for a multi-concentration test. A second option for test design in a multi-concentration test includes unequal replicates per treatment (i.e., six per treatment for control(s); four replicates for each of the lowest 3-5 test concentrations; and three replicates for each of the highest 4-5 test concentrations).

The test may be performed either as a static (i.e., no renewal) assay or as a static-renewal toxicity test. The static option is recommended as the standard procedure, whereas the static-renewal option is recommended for test solutions where the concentration of the test substance (or a biologically active component) can be expected to decrease significantly (i.e., >20%) during the test period. If the static-renewal option is chosen, test solutions are replaced at least every three days during the test. The endpoints for the test are frond number and frond dry weight at the end of a 7-day toxicity test.

Procedures are given for culturing L. minor in the laboratory. General or universal conditions and procedures are outlined for testing a variety of materials or substances for their effects on Lemna growth. Additional conditions and procedures are stipulated, which are specific for testing samples of chemical, effluent, elutriate, leachate, or receiving water. Instructions and requirements are included on apparatus, facilities, handling and storing samples, preparing test solutions and initiating tests, specific test conditions, appropriate observations and measurements, endpoints, methods of calculation, validation, and the use of reference toxicants.

Foreword

This is one of a series of recommended methods for measuring and assessing the toxic effect(s) on single species of aquatic or terrestrial organisms, caused by their exposure to samples of toxic or potentially toxic substances or materials under controlled and defined laboratory conditions. Recommended methods are those that have been evaluated by Environment Canada (EC), and are favoured:

  • for use in EC environmental toxicity laboratories;
  • for testing that is contracted out by Environment Canada or requested from outside agencies or industry;
  • in the absence of more specific instructions, such as are contained in regulations; and
  • as a foundation for the provision of very explicit instructions as might be required in a regulatory protocol or standard reference method.

The different types of tests included in this series were selected because of their acceptability for the needs of programs for environmental protection and management carried out by Environment Canada. These reports are intended to provide guidance and to facilitate the use of consistent, appropriate, and comprehensive procedures for obtaining data on the toxicity to aquatic or terrestrial life of samples of specific test substances or materials destined for or within the environment. Depending on the biological test method(s) chosen and the environmental compartment of concern, substances or materials to be tested for toxicity could include samples of chemical or chemical product, effluent, elutriate, leachate, receiving water, sediment or similar particulate material or soil or similar particulate material. Appendix H provides a listing of the biological test methods and supporting guidance documents published to date by Environment Canada as part of this series.

Words defined in the Terminology section of this document are italicized when first used in the body of the report according to the definition. Italics are also used as emphasis for these and other words, throughout the report.

List of Abbreviations and Chemical Formulae

ANOVA
analysis of variance
°C
degree(s) Celsius
CaCl2
calcium chloride
Ca(NO3)2
calcium nitrate
CoCl2
cobalt chloride
Co(NO3)2
cobalt nitrate
cm
centimetre(s)
CuCl2
copper chloride
CuSO4
copper sulphate
CV
coefficient of variation
d
day(s)
EC50
median effective concentration
EDTA
ethylenediamine tetraacetic acid (C 10H 16O 8N 2)
FeCl3
ferric chloride
g
gram(s)
g/kg
gram(s) per kilogram
g/L
gram(s) per litre
h
hour(s)
H3BO3
boric acid
HCl
hydrochloric acid
H2O
water
%I
percent growth inhibition
ICp
inhibiting concentration for a (specified) percent effect
ID
inside diameter
KCl
potassium chloride
kg
kilogram(s)
KH2PO4
potassium dihydrogen phosphate anhydride
K2HPO4
potassium phosphate
KNO3
potassium nitrate
KOH
potassium hydroxide
kPa
kilopascal
L
litre(s)
LOEC
lowest-observed-effect concentration
m
metre(s)
mg
milligram(s)
MgCl2
magnesium chloride
MgSO4
magnesium sulphate
min
minute(s)
mL
millilitre(s)
mm
millimetre(s)
mS
millisiemens
MnCl2
manganese chloride
MOPS
4-morpholinepropane sulphonic acid
N
Normal
NaCl
sodium chloride
Na2CO3
sodium carbonate
Na2EDTA
disodium ethylenediamine tetraacetic acid (C 10H 14N 2O 8 · 2H 2O)
Na4EDTA
tetrasodium ethylenediamine tetraacetic acid (C 10H 12N 2O 8 · 2H 2O)
NaHCO3
sodium bicarbonate
Na2MoO4
sodium molybdate
NaNO3
sodium nitrate
NaOH
sodium hydroxide
nm
nanometer
NOEC
no-observed-effect concentration
SD
standard deviation
s
second
spp.
species (plural)
SRC
Saskatchewan Research Council
TIE
Toxicity Identification Evaluation
TM (TM)
Trade Mark
µg
microgram(s)
µm
micrometre(s)
µmhos/cm
micromhos per centimetre
µmol/(m2 · s)
micromole per metre squared per second
UTCC
University of Toronto Culture Collection
v:v
volume-to-volume
ZnCl2
zinc chloride
ZnSO4
zinc sulphate
>
greater than
<
less than
greater than or equal to
less than or equal to
±
plus or minus
/
per, alternatively, “or” (e.g., control/dilution water)
~
approximately
approximately equal to
%
percentage or percent
parts per thousand

Terminology

Note: all definitions are given in the context of the procedures in this report, and might not be appropriate in another context.

Grammatical Terms

Must is used to express an absolute requirement.

Should is used to state that the specified condition or procedure is recommended and ought to be met if possible.

May is used to mean “is (are) allowed to”.

Can is used to mean “is (are) able to”.

Might is used to express the possibility that something could exist or happen.

Technical Terms

Acclimation is physiological adjustment to a particular level of one or more environmental factors such as temperature. The term usually refers to the adjustment to controlled laboratory conditions.

Axenic cultures contain organisms of a single species, in the absence of cells or living organisms of any other species.

Biomass is the total dry weight (mass) of a group of plants or animals.

Chlorosis is the loss of chlorophyll (yellowing) in frond tissue.

Clone is a group of individuals reproducing vegetatively (by mitosis) from a single ancestor (i.e., frond).

Colony means an aggregate of mother and daughter fronds (usually 2 to 4) attached to each other. Sometimes referred to as a plant.

Compliance means in accordance with governmental regulations or requirements for issuing a permit.

Conductivity is a numerical expression of the ability of an aqueous solution to carry an electric current. This ability depends on the concentrations of ions in solution, their valence and mobility, and on the solution’s temperature. Conductivity is measured at 25°C, and is reported as millisiemens/metre (mS/m), or as micromhos/centimetre (µmhos/cm); 1 mS/m = 10 µmhos/cm.

Culture, as a noun, is the stock of organisms raised in the laboratory under defined and controlled conditions through one or more generations, to produce healthy test organisms. As a verb, it means to carry out the procedure of raising healthy test organisms from one or more generations, under defined and controlled conditions.

Dispersant is a chemical substance that reduces the surface tension between water and a hydrophobic substance (e.g., oil), thereby facilitating the dispersal of the hydrophobic substance or material throughout the water as an emulsion.

Emulsifier is a chemical substance that aids the fine mixing (in the form of small droplets) within water of an otherwise hydrophobic substance or material.

Flocculation is the formation of a light, loose precipitate (i.e., a floc) from a solution.

Frond is the individual leaf-like structure of a duckweed plant. It is the smallest unit (i.e., individual) capable of reproducing.

Gibbosity means fronds exhibiting a humped or swollen appearance.

Growth is the increase in size or weight as the result of proliferation of new tissues. In this test, it refers to an increase in frond number over the test period as well as the dry weight of fronds at the end of the test.

Growth rate is the rate at which the biomass increases.

Lux is a unit of illumination based on units per square metre. One lux = 0.0929 foot-candles and one foot-candle = 10.76 lux. For conversion of lux to quantal flux [µmol/(m2 · s)], the spectral quality for the light source must be known. Light conditions or irradiance are properly described in terms of quantal flux (photon fluence rate) in the photosynthetically effective wavelength range of approximately 400 to 700 nm. The relationship between quantal flux and lux or foot-candle is highly variable and depends on the light source, the light meter used, the geometrical arrangement, and the possibilities of reflections (see American Society for Testing and Materials (ASTM), 1995). Approximate conversion between quantal flux and lux, however, for full spectrum fluorescent light, is 1 lux ≅ 0.016 µmol/(m2 · s) (Deitzer, 1994; Sager and McFarlane, 1997).

Monitoring is the routine (e.g., daily, weekly, monthly, quarterly) checking of quality, or collection and reporting of information. In the context of this report, it means either the periodic (routine) checking and measurement of certain biological or water-quality variables, or the collection and testing of samples of effluent, elutriate, leachate, or receiving water for toxicity.

Necrosis indicates dead (i.e., with brown or white spots) frond tissue.

Percentage (%) is a concentration expressed in parts per hundred parts. One percentage represents one unit or part of material or substance (e.g., chemical, effluent, elutriate, leachate, or receiving water) diluted with water or medium to a total of 100 parts. Concentrations can be prepared on a volume-to-volume or weight-to-weight basis, or less accurately on a weight-to-volume basis, and are expressed as the percentage of test substance or material in the final solution.

pH is the negative logarithm of the activity of hydrogen ions in gram equivalents per litre. The pH value expresses the degree or intensity of both acidic and alkaline reactions on a scale from 0 to 14, with 7 representing neutrality, numbers less than 7 indicating increasingly greater acidic reactions, and numbers greater than 7 indicating increasingly basic or alkaline reactions.

Photoperiod describes the duration of illumination and darkness within a 24-h day.

Precipitation means the formation of a solid (i.e., precipitate) from some or all of the dissolved components of a solution.

Pretreatment means treatment of a sample, or dilution thereof, before exposure of test organisms.

Protocol is an explicit set of procedures for a test, formally agreed upon by the parties involved, and described precisely in a written document.

Reference method refers to a specific protocol for performing a toxicity test, i.e., a biological test method with an explicit set of test procedures and conditions, formally agreed upon by the parties involved and described precisely in a written document. Unlike other multi-purpose (generic) biological test methods published by Environment Canada, the use of a reference method is frequently restricted to testing requirements associated with specific regulations.

Root is that part of the Lemna plant that assumes a root-like structure.

Salinity is the total amount of solid material, in grams, dissolved in 1 kg of seawater. It is determined after all carbonates have been converted to oxides, all bromide and iodide have been replaced by chloride, and all organic matter has been oxidized. Salinity can also be measured directly using a salinity/conductivity meter or other means (see American Public Health Association (APHA) et al., 1989). It is usually reported in grams per kilogram (g/kg) or parts per thousand (‰).

Stock culture is an ongoing laboratory culture of a specific test organism from which individuals are selected and used to set up separate test cultures.

Strain is a variant group within a species maintained in culture, with more or less distinct morphological, physiological, or cultural characteristics.

Subculture is a laboratory culture of a specific test organism that has been prepared from a pre-existing culture, such as the stock culture. As a verb, it means to conduct the procedure of preparing a subculture.

Surfactant is a surface-active chemical substance (e.g., detergent) that, when added to a nonaqueous liquid, decreases surface tension and facilitates dispersion of substances in water.

Test culture means the culture established from organisms isolated from the stock culture to provide plants for use in a toxicity test. Here, it refers to the 7- to 10-day old Lemna cultures maintained in modified Hoagland’s medium that are then transferred to control/dilution water for an 18- to 24-h acclimation period.

Turbidity is the extent to which the clarity of water has been reduced by the presence of suspended or other matter that causes light to be scattered and absorbed rather than transmitted in straight lines through the sample. It is generally expressed in terms of Nephelometric Turbidity Units.

Terms for Test Materials or Substances

Chemical is, in this report, any element, compound, formulation, or mixture of a substance that might be mixed with, deposited in, or found in association with water.

Control is a treatment in an investigation or study that duplicates all the conditions and factors that might affect the results, except the specific condition being studied. In toxicity tests, the control must duplicate all the conditions of the exposure treatment(s), but must contain no contaminated test material or substance. The control is used as a check for the absence of toxicity due to basic test conditions (e.g., quality of the dilution water, health of test organisms, or effects due to their handling).

Control/dilution water is the water, or in this report, the test medium used for the control treatment, for diluting the test material or substance, or for both.

Deionized water is water that has been purified to remove ions from solution by passing it through resin columns or a reverse osmosis system.

Dilution water is the water, or in this report, the test medium used to dilute a test substance or material to prepare different concentrations for a toxicity test.

Dilution factor is the quotient between two adjacent concentration levels (e.g., 0.32 mg/L ÷ 0.1 mg/L = 3.2 dilution factor).

Distilled water is water that has been passed through a distillation apparatus of borosilicate glass or other material, to remove impurities.

Effluent is any liquid waste (e.g., industrial, municipal) discharged to the aquatic environment.

Elutriate is an aqueous solution obtained after adding water to a solid substance or material (e.g., contaminated soil or sediment, tailings, drilling mud, dredge spoil), shaking the mixture, then centrifuging it, filtering it, or decanting the supernatant.

Leachate is water or wastewater that has percolated through a column of soil or solid waste within the environment.

Material is the substance or substances from which something is made. A material would have more or less uniform characteristics. Effluent, leachate, elutriate, or surface water are materials. Usually, the material would contain several or many substances.

Medium is deionized or glass-distilled water (ASTM Type-1 water) to which reagent-grade chemicals have been added. The resultant synthetic fresh water is free from contaminants.

Nutrient-spiked wastewater is a wastewater sample to which the same nutrients that are used to make up the test medium have been added at the same concentrations (e.g., effluent is spiked with the modified APHA nutrient stock solutions A, B, and C, at a ratio of 10 mL of each per 1000 mL of effluent) before test solutions are prepared.

Nutrient-spiked receiving water is a sample of receiving water to which the same nutrients that are used to make up the test medium have been added at the same concentrations (e.g., receiving water that is to be used as control/dilution water for effluent testing is spiked with the modified APHA nutrient stock solutions A, B, and C, at a ratio of 10 mL of each per 1000 mL of receiving water) before test solutions are prepared.

Receiving water is surface water (e.g., in a stream, river, or lake) that has received a discharged waste, or else is about to receive such a waste (e.g., it is immediately “upstream” or up-current from the discharge point). Further descriptive information must be provided to indicate which meaning is intended.

Reference toxicant is a standard chemical used to measure the sensitivity of the test organisms in order to establish confidence in the toxicity data obtained for a test material or substance. In most instances, a toxicity test with a reference toxicant is performed to assess the sensitivity of the organisms at the time the test material or substance is evaluated, and the precision and reliability of results for that chemical obtained by the laboratory.

Reference toxicity test is a test conducted using a reference toxicant in conjunction with a toxicity test, to appraise the sensitivity of the organisms and/or the precision and reliability of results obtained by the laboratory for that chemical at the time the test material or substance is evaluated. Deviations outside an established normal range indicate that the sensitivity of the test organisms, and the performance and precision of the test, are suspect.

Stock solution is a concentrated aqueous solution of the substance or material to be tested or the chemicals used to prepare growth/test media. Measured volumes of a stock solution are added to dilution water to prepare the required strengths of test solutions or media.

Substance is a particular kind of material having more or less uniform properties. The word substance has a narrower scope than material, and might refer to a particular chemical (e.g., an element) or chemical product.

Test medium is the complete synthetic culture medium (in this case modified American Public Health Association (APHA), Swedish Standards Institute (SIS), or modified Steinberg medium) that enables the growth of test plants during exposure to the test substance. The test substance will normally be mixed with, or dissolved in, the test medium.

Test sample refers to the aqueous sample that is to be tested. It might be derived from chemical stock solutions or collected from effluents, elutriates, leachates, or receiving waters.

Test solution refers to an aqueous solution that consists of a particular concentration of prepared test sample. In the case of this test, the test substance/wastewater is dissolved in test medium or spiked upstream receiving water, which is then subjected to testing.

Upstream water is surface water (e.g., in a stream, river, or lake) that is not influenced by the effluent (or other test material or substance), by virtue of being removed from it in a direction against the current or sufficiently far across the current.

Wastewater is a general term that includes effluents, leachates, and elutriates.

Statistical and Toxicological Terms

Acute means within a short period of exposure (seconds, minutes, hours, or a few days) in relation to the life span of the test organism. An acute toxic effect would be induced and observable within a short period of time.

Chronic means occurring within a relatively long period of exposure (weeks, months, or years), usually a significant portion of the life span of the organism such as 10% or more. A chronic toxic effect might take a significant portion of the life span to become observable, although it could be induced by an exposure to a toxic substance that was either acute or chronic.

Chronic toxicity implies long-term effects that are usually related to changes in such things as metabolism, growth, reproduction, or ability to survive.

Coefficient of Variation (CV) is the standard deviation (SD) of a set of data divided by the mean of the data-set, expressed as a percentage. It is calculated according to the following formula:

  • CV (%) = 100 (SD ÷ mean).

EC50 is the median effective concentration. It is the concentration of material in water (e.g., mg/L), soil or sediment (e.g., mg/kg) that is estimated to cause a specified toxic effect to 50% of the test organisms. In most instances the EC50 and its 95% confidence limits are statistically derived by analyzing the percentages of organisms showing the specified effect at various test concentrations, after a fixed period of exposure. The duration of exposure must be specified (e.g., 72-h EC50). The EC50 describes quantal effects, lethal or sublethal, and is not applicable to quantitative effects (see ICp). Other percentages could be used, see ECp.

ECp has the same meaning as EC50, except that “p” can represent any percentage, and is to be specified for any particular test or circumstance. Some investigators and agencies, particularly European and international, have mistakenly used ECp to mean ICp, but the distinction is important and should be maintained.

Endpoint means the measurement(s) or value(s) that characterize the results of the test (e.g., ICp). It also means the response of the test organisms that is being measured (e.g., number of fronds or frond dry weight).

Geometric mean is the mean of repeated measurements, calculated on a logarithmic basis. It has the advantage that extreme values do not have as great an influence on the mean as is the case for an arithmetic mean. The geometric mean can be calculated as the nth root of the product of the “n” values, and it can also be calculated as the antilogarithm of the mean of the logarithms of the “n” values.

Homoscedasticity refers herein to data showing homogeneity of the residuals within a scatter plot. This term applies when the variability of the residuals does not change significantly with that of the independent variable (i.e., the test concentrations or treatment levels). When performing statistical analyses and assessing residuals (e.g., using Levene’s test), for test data demonstrating homoscedasticity (i.e., homogeneity of residuals), there is no significant difference in the variance of residuals across concentrations or treatment levels.

Hormesis is an effect in which low concentrations of the test material or substance act as a stimulant for performance of the test organisms compared to that for the control organisms (i.e., performance in one or more low concentrations is enhanced and “better” than that in the control treatment). This stimulation must be accompanied by inhibition at higher test concentrations to be defined as hormesis. Hormesis is a specific subset of a stimulatory effect. (See also stimulatory effect).

ICp is the inhibiting concentration for a (specified) percent effect. It represents a point estimate of the concentration of test substance or material that causes a designated percent impairment in a quantitative biological function such as growth. For example, an IC25 could be the concentration estimated to cause fronds to attain a dry weight that is 25% lower than that attained by control fronds at the end of the test. This term should be used for any toxicological test which measures a quantitative effect or change in rate, such as dry weight at test end. (The term EC50 or median effective concentration is not appropriate in tests of this kind since it is limited to quantal measurements, i.e., number of exposed individuals which show a particular effect.)

LOEC is the lowest-observed-effect concentration. This is the lowest concentration of a test material or substance to which organisms are exposed, that causes adverse effects on the organism which are detected by the observer and are statistically significant. For example, the LOEC might be the lowest test concentration at which the dry weight of exposed organisms at test end differed significantly from that in the control.

NOEC is the no-observed-effect concentration. This is the highest concentration of a test material or substance to which organisms are exposed, that does not cause any observed and statistically significant adverse effects on the organism. For example, the NOEC might be the highest test concentration at which an observed variable such as dry weight or frond number at test end does not differ significantly from that in the control.

Normality (or normal distribution) refers to a symmetric, bell-shaped array of observations. The array relates frequency of occurrence to the magnitude of the item being measured. In a normal distribution, most observations will cluster near the mean value, with progressively fewer observations toward the extremes of the range of values. The normal distribution plays a central role in statistical theory because of its mathematical properties. It is also central in biological sciences because many biological phenomena follow the same pattern. Many statistical tests assume that data are normally distributed, and therefore it can be necessary to test whether that is true for a given set of data.

Precision refers to the closeness of repeated measurements of the same quantity to each other, i.e., the degree to which data generated from repeated measurements are the same. It describes the degree of certainty around a result, or the tightness of a statistically derived endpoint such as an ICp.

Quantal is an adjective, as in quantal data, quantal test, etc. A quantal effect is one for which each test organism either shows the effect of interest or does not show it. For example, an animal might either live or die, or it might develop normally or abnormally. Quantal effects are typically expressed as numerical counts or percentages thereof.

Quantitative is an adjective, as in quantitative data, quantitative test, etc. A quantitative effect is one in which the measured effect can take any whole or fractional value on a numerical scale. An example would be the weight attained by individual organisms, or the number of progeny produced at the end of a test.

Replicate (treatment, test vessels) refers to a single test chamber containing a prescribed number of organisms in either one concentration of test material or substance, or in the control or reference treatment(s). A replicate of a treatment must be an independent test unit; therefore any transfer of organisms or test material from one test vessel to another would invalidate a statistical analysis based on replication.

Static describes toxicity tests in which test solutions are not renewed during the test period.

Static-renewal describes a toxicity test in which test solutions are renewed (replaced) periodically (e.g., at specific intervals) during the test period. Synonymous terms are batch replacement, renewed static, renewal, intermittent renewal, static replacement, and semi-static.

Stimulatory effect refers to enhanced performance (i.e., “stimulation”) that is observed in one or more test concentrations relative to that for the control treatment. In this document, stimulatory effect refers specifically to enhanced performance (i) at one or more of the higher concentrations tested or (ii) across all concentrations tested. Hormesis is a specific subset of a stimulatory effect. (See also hormesis).

Sublethal (toxicity) means detrimental to the organism, but below the concentration or level of contamination that directly causes death within the test period.

Toxic means poisonous. A toxic substance or material can cause adverse effects on living organisms, if present in sufficient amounts at the right location. Toxic is an adjective or adverb, and should not be used as a noun; whereas toxicant is legitimate noun.

Toxicant is a toxic substance or material.

Toxicity is the inherent potential or capacity of a substance or material to cause adverse effects on living organisms. These effects could be lethal or sublethal.

Toxicity Identification Evaluation describes a systematic sample pretreatment (e.g., pH adjustment, filtration, aeration), followed by tests for toxicity. This evaluation is used to identify the agent(s) that are primarily responsible for toxicity in a complex mixture. The toxicity test can be lethal or sublethal.

Toxicity test is a determination of the effect of a substance or material on a group of selected organisms (e.g., Lemna minor), under defined conditions. An aquatic toxicity test usually measures: (a) the proportions of organisms affected (quantal); and/or (b) the degree of effect shown (quantitative), after exposure to specific concentrations of chemical, effluent, elutriate, leachate, or receiving water.

Toxicology is a branch of science that studies the toxicity of substances, materials, or conditions. There is no limitation on the use of various scientific disciplines, field or laboratory tools, or studies at various levels of organization, whether molecular, single species, populations, or communities. Applied toxicology would normally have a goal of defining the limits of safety of chemical or other agents.

Treatment is, in general, an intervention or procedure whose effect is to be measured. More specifically, in toxicity testing, it is a condition or procedure applied to the test organisms by an investigator, with the intention of measuring the effects on those organisms. The treatment could be a specific concentration of a potentially toxic material or substance. Alternatively, a treatment might be a particular test material (e.g., a particular sample of effluent, elutriate, leachate, receiving water, or control water).

Warning chart is a graph used to follow changes over time in the endpoints for a reference toxicant. The date of the test is on the horizontal axis and the effect-concentration is plotted on the vertical logarithmic scale.

Warning limit is plus or minus two standard deviations, calculated on a logarithmic basis, from the historic geometric mean of the endpoints from toxicity tests with a reference toxicant.

Acknowledgements

The first edition of this biological test method, printed in March 1999 was written by Jennifer A. Miller (Miller Environmental Sciences Inc., Stroud, Ontario (ON)). It was based on a method developed by Ms. Mary Moody and Dr. Hans Peterson at the Saskatchewan Research Council (1997), which was a modification of the “8211 Duckweed (Proposed)” toxicity test (APHA et al., 1995). The report also incorporated test modifications from existing guidance documents and reports (published or otherwise) that describe procedures and conditions used in the United States, Canada, and Europe for culturing Lemna minor, and for conducting toxicity tests using this species of freshwater aquatic macrophyte.

Mr. R. P. Scroggins (Method Development and Applications Section, Environmental Technology Centre, Environment Canada, Ottawa, ON) acted as Scientific Authority and provided technical input and guidance throughout the work. Special acknowledgement is made of the many useful comments and suggestions provided by each member of the Environment Canada (EC) committee of scientific experts responsible for the initial and final reviews of the first edition of this report: G. Gilron (ESG International Inc., Guelph, ON); E. Jonczyk, Beak International Inc., Brampton, ON); D.J. McLeay (McLeay Environmental Ltd., Victoria, British Columbia (BC)); M. Moody (Saskatchewan Research Council, Saskatoon, Saskatchewan (SK)); J. Rathbun (AScI Corporation, Livonia, Michigan (MI)); J. Staveley (ARCADIS Geraghty & Miller, Inc., Raleigh, North Carolina (NC)); L. Taylor (McMaster University, Hamilton, ON); and P. Whitehouse (WRc, Marlow, Bucks, U.K.).

In addition to the members of the Environment Canada scientific advisory committee who contributed to the development of the first edition of this biological test method, many useful comments and suggestions were provided by the following persons, who reviewed the final draft of the first edition of this document: J. Acreman (UTCC, University of Toronto, Toronto, ON); J. Amato (AScI Corporation, Duluth, Minnesota (MN)); R. Baudo (CNR Istituto Italiano di Idrobiologia, Verbania Pallanza [NO], Italy); C. Boutin (Environment Canada, Gatineau, Quebec (PQ)); D. Forrow (National Centre for Ecotoxicology & Hazardous Substances, Waterlooville, Hants, U.K.); P. Gnemi (Laboratories and Clinics Group Bioscience, Calleretto Giacosa [TO], Italy); B. Greenberg (University of Waterloo, Waterloo, ON); J. Hoberg (Springborn Laboratories, Inc., Wareham, MA); J. Kranzfelder (ABC Laboratories, Columbia, Missouri (MO)); J.D. Madsen (CEWES-ES-P, Vicksburg, MS); R. Morcock (USEPA, Washington, DC); E. Öhlén (Kemi, Solna, Sweden); B. Pénzes (Laboratory of Hydrobiology, Százhalombatta, Hungary); R. Roshon, (Pesticide Management Regulatory Agency, Ottawa, ON); J. Schroeder (Beak International Inc., Brampton, ON); and E. Schultz (Finnish Environment Institute, Helsinki, Finland).

Grateful acknowledgement is made of the numerous technical contributions and support provided by M. Moody (SRC, Saskatoon, SK). The experience and input of J. Acreman (UTCC, Toronto, ON) with respect to Lemna culturing herein is gratefully acknowledged. Dr. D. McLeay (McLeay Environmental Ltd., Victoria, BC) is sincerely thanked for his technical and editorial expertise and input. The assistance of Dr. A. Stomp and her technical staff (N.C. State University, Raleigh, NC) and Dr. E. Landolt (Geobotanisches Institut ETH, Zuerich, Switzerland) for their invaluable input into the taxonomy and characteristics of Lemna minor and its clones is acknowledged with thanks. Finally, members of Inter-Governmental Environmental Toxicity Group (IGETG) (see Appendix A) are thanked for their continuing technical support.

This (second) edition was prepared by J. A. Miller (Miller Environmental Sciences Inc., King, ON), with assistance and guidance from L. Taylor (Manager, Method Development and Applications Section), R. Scroggins (Chief, Biological Methods Division) and L. Van der Vliet (Method Development and Applications Section) of the Environmental Technology Centre, Environment Canada, Ottawa, Ontario, as well as M. Moody (Saskatchewan Research Council, Saskatoon, SK). The second edition includes numerous updates such as modified culture and test media and the use of regression analyses for quantitative endpoint data. Numerous comments and suggestions for change to the first edition, which were forwarded to Environment Canada’s Method Development and Applications Section by Canadian laboratory personnel performing this toxicity test method were considered when preparing this second edition of Report EPS 1/RM/37. Procedures outlined in the 2005 International Organization for Standardization (ISO) draft Lemna minor growth inhibition test have also been incorporated. Many procedural modifications were provided through research carried out by M. Moody.

Photographs for the method were supplied by M. Moody (SRC, Saskatoon, SK).

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