Biological test method: fertilization assay using echinoids (sea urchins and sand dollars), chapter 2

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Richard P. Scroggins, Chief
Biological Assessment and Standardization Section
Science and Technology Branch
Environment Canada
335 River Road
Ottawa, ON  K1A 0H3

Lisa Taylor, Manager
Method Development & Applications Unit
Science and Technology Branch
Environment Canada
335 River Road
Ottawa, ON  K1A 0H3

Review Notice

This report has been reviewed by the staff of the Environmental Technology Advancement Directorate, Environment Canada. Mention of trade names or commercial products does not constitute endorsement by Environment Canada for use. Other products of similar value are available.


Methods recommended by Environment Canada for performing a sublethal marine toxicity test using gametes obtained from sea urchins or sand dollars are described in this report. This second edition of EPS 1/RM/27, published in 2011 supersedes the first edition that was published in 1992, and amended in 1997. It includes numerous procedural modifications as well as updated guidance and instructions to assist in performing the biological test method.

In the test, sperm are exposed to the substance or material being tested. Eggs are then added, and the success of fertilization under continued exposure to the same concentration of test substance or material is measured. The endpoint is decreased success of fertilization, described in terms of the concentration estimated to cause a specified percent inhibition (ICp). The test is quick and is among the most sensitive of marine sublethal toxicity tests. Because the gametes and the success of fertilization usually represent a sensitive part of the life cycle, this assay should be considered as a powerful and meaningful sublethal test. The test may be run with a minimum of seven concentrations of test substance or material to determine the threshold of effect, or with one concentration as a regulatory or pass/fail test.

Recommended species for use in this test are the green sea urchin (Strongylocentrotus droebachiensis) found on the Atlantic, Pacific and Arctic coasts of Canada, the Pacific purple sea urchin (Strongylocentrotus purpuratus), the eccentric sand dollar (Dendraster excentricus) found in the Pacific, the Atlantic purple sea urchin commonly called Arbacia (Arbacia punctulata), and the white sea urchin from California (Lytechinus pictus).

Procedures are given for acclimating and holding adult echinoids in the laboratory for extended periods of time, for holding adult echinoids in the laboratory for immediate use (for adults who are spawned within 3 days of arrival at the laboratory), and for obtaining sperm and eggs for a test. General or universal conditions and procedures are outlined for testing a variety of materials or substances for their effect on echinoid fertilization. Additional conditions and procedures are specific for testing sample(s) of chemical, effluent, receiving water, leachate, elutriate, or liquid derived from sediment or similar solid material. Instructions are included for test facilities, handling and storing samples, preparing test solutions and initiating tests, specific test conditions, appropriate observations and measurements, endpoints and methods of calculation, validation of the test, and the use of reference toxicants.


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 program or standard reference method.

The different types of tests included in this series were selected because of their acceptability for the needs of environmental protection and management programs 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 A 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

analysis of variance
degree(s) Celsius
coefficient of variation
dissolved oxygen (concentration)
grams per kilogram
hydrochloric acid
hypersaline brine
inhibiting concentration for a (specific) percent effect
potassium chloride
lowest-observed-effect concentration
molarity (concentration)
sodium hydroxide
no-observed-effect concentration
standard deviation
Système internationale d’unités
toxicity identification evaluation
Trade Mark
× g
relative centrifugal force (times gravity)
greater than
less than
greater than or equal to
less than or equal to
per; alternatively, “or” (e.g., control/dilution water)
plus or minus
percentage or percent
parts per thousand


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

Batch means a single group of adult echinoids received from a supplier at a discrete time, in order to provide all of the gametes intended for use in a discrete toxicity test (including any associated reference toxicity test). It might also refer to the gametes collected from a single male and female or a group of males and females at one time, intended for use in a discrete toxicity test (including any associated reference toxicity test).

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 in fresh waters is measured at 25 °C, and is normally reported in the SI unit of millisiemens/metre, or as micromhos/centimetre (1 mS/m = 10 μmhos/cm). Conductivity is a standard method for measuring salinity, with the result read off as g/kg or “parts per thousand”(‰).

Dispersant is a chemical substance which 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.

Embryo means the undeveloped young animal, before it hatches from the egg.

Euryhaline is the ability to tolerate a wide variation in salinity without stress.

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

Gametes are the sperm or unfertilized eggs obtained from adult echinoids.

Larva (plural, larvae) is a recently hatched organism which has physical characteristics other than those seen in the adult of the species.

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 of 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-candles is highly variable and depends on the light source, the light meter used, the geometrical arrangement, and the possibilities of reflections (see ASTM, 1999). An approximate conversion between quantal flux and lux, for full-spectrum fluorescent light (e.g., Vita-Lite® by Duro-Test®), is as follows: one lux is approximately equal to 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, leachate, elutriate, marine/estuarine receiving water, or pore water for toxicity.

Percentage (%) is a concentration expressed in parts per hundred parts. One percent represents one unit or part of material or substance (e.g., chemical, effluent, leachate, elutriate, receiving water, or pore water) diluted with water to a total of 100 parts. Concentrations can be prepared on a volume- to-volume or weight-to-weight 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 signifying increasingly greater acidic reactions, and numbers greater than 7 indicating increasingly basic or alkaline reactions.

Photoperiod is 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.

Pre-treatment is, in this report, treatment of a sample or dilution thereof, before exposure of gametes.

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.

Salinity is the total amount of solid substance, in grams, dissolved in 1 kg of water. 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 be measured directly using a salinity/conductivity meter or other means (see APHA et al., 1989, 2005). Salinity is reported here as g/kg. The term “parts per thousand” (‰) is synonymous with g/kg.

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 chemical substance that might enter the aquatic environment through spillage, application, or discharge. Examples of chemicals that are applied to the environment are insecticides, herbicides, fungicides, sea lamprey larvicides, and agents for treating oil spills.

Control is a treatment in an investigation or study that duplicates all the conditions and factors that might affect the results of the investigation, except the specific condition that is 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 measurable toxicity due to basic test conditions (e.g., quality of dilution water, health of test organisms, or effects due to their handling).

Control/dilution water is the water used for diluting the test material or substance, or for the control test, or both.

Control sediment means uncontaminated (clean) sediment which does not contain concentrations of one or more contaminants that could affect the fertilization of echinoid eggs. In this report, control sediment is natural, field-collected sediment from an uncontaminated site, with pore water that is known to enable an acceptable egg fertilization rate. This sediment must contain no added test material or substance, and may provide a basis for interpreting data derived from toxicity tests using test sediment(s).

Dechlorinated water means a chlorinated water (usually municipal drinking water) that has been treated to remove chlorine and chlorinated compounds from solution.

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 seawater or other saline water used to dilute a test substance or material in order to prepare different concentrations for the various toxicity test treatments.

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 material (e.g., sediment, tailings, drilling mud, dredge spoil), shaking the mixture, then centrifuging or filtering it or decanting the supernatant.

Estuarine water is brackish seawater, from or within a coastal body of ocean water that is measurably diluted with fresh water derived from land drainage.

Hypersaline brine is a solution of sea salts in water, in stronger concentration than in oceanic water. It can be obtained from high quality filtered seawater by partial freezing and draining off the unfrozen liquid, freezing and partially thawing, or slow heating and evaporation. It can also be prepared by adding commercially available ocean salts or reagent-grade salts to fresh or distilled water. The strength of brine used for this fertilization assay should be 90 ± 1 g/kg.

Interstitial water - see pore water.

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

Marine water is seawater from or within the ocean, sea, or an inshore location where there is no appreciable dilution of water by natural fresh water derived from land drainage.

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.

Pore water (also called interstitial water) is the water occupying space between sediment particles. The amount of pore water is expressed as a percentage of the wet sediment, by weight.

Produced water is mainly salty water brought up along with oil and gas during its production. Produced water originates from water contained in oil and gas reservoirs that is produced along with the oil and gas. Produced water might be problematic in the environment due to its highly saline nature.

Receiving water is a natural seawater (e.g., in a marine or estuarine waterbody) that has received a discharged waste, or else is about to receive such a waste (e.g., it is just “upstream” or up-current from the discharge point). Further descriptive information must be provided to indicate which meaning is intended.

Reconstituted seawater is fresh water (deionized or glass distilled) to which commercially available dry ocean salts, reagent-grade salts, or hypersaline brine has been added, in a quantity that provides the seawater salinity (and pH) desired for holding organisms and for testing purposes (control/dilution water).

Reference sediment is a field-collected sample of presumably clean (uncontaminated) sediment, selected for properties (e.g., particle size, compactness, total organic content) representing sediment conditions that closely match those of the sample(s) of test sediment except for the degree of chemical contaminants. It is often selected from a site that is uninfluenced or minimally influenced by the source(s) of anthropogenic contamination but within the general vicinity of the site(s) where samples of test sediment are collected. One or more samples of reference sediment should be included in each series of toxicity tests with test sediment(s). This sediment might or might not prove to be toxic due to the presence of naturally occurring chemicals such as hydrogen sulphide or ammonia, or the unanticipated presence of contaminants from human influence at harmful-effect concentrations. The use of such (toxic) sediment as reference sediment in future toxicity tests should be avoided, unless the experimental design is cognizant of this and the investigator(s) wish to compare test results for this material with those for one or more samples of test sediment.

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 of results obtained by the laboratory for that chemical.

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

Sediment is a natural particulate material, which has been transported and deposited in water and then deposited on the sea floor. The term can also describe a material that has been experimentally prepared (formulated) using selected particulate material (e.g., sand of a particular grain size, bentonite clay, etc.) for experimental purposes.

Stock solution is a concentrated aqueous solution of the substance or material to be tested. Measured volumes of a stock solution are added to dilution water in order to prepare the required strengths of test solutions.

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.

Upstream water is natural seawater (e.g., in a marine or estuarine waterbody) 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 in relation to the life span of the organism, and would be within a few days for echinoids, which generally have a life span of several years, e.g., four to eight years for sea urchins. An acute toxic effect would be induced and observable within the short period.

Chronic means occurring during a relatively long period of exposure, usually a significant portion of the life span of the organism such as 10% or more.

Chronic toxicity implies long-term effects that are 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).

Endpoint means the measurement(s) or value(s) that characterize the results of the test (e.g., IC25). It also means the response of the test organisms that is measured (e.g., fertilization).

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). At higher concentrations, deleterious effects are seen.

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 a growth rate, or number of young per brood, compared to the control. For example, an IC25 could be the concentration estimated to cause a 25% reduction in growth rate, relative to the control. This term should be used for any toxicological test which measures a quantitative effect or change in rate, such as growth, respiration, or reproductive rate. In the present echinoid test, unmeasured effects on sperm, on eggs, and on the fertilization process are given an overall assessment by the percent inhibition of fertilization. The term effective concentration for a specified percent effect (ECp), such as the median effective concentration (EC50) or an EC25, is not appropriate in tests of this kind because it is limited to quantal measurements, e.g., an estimate that 25% of the individual organisms exposed to that concentration would show a particular effect. The echinoid fertilization assay does not completely conform with the requirements for an ECp (such as the EC50), because the number of quantal observations is large (i.e., ≥100). As such, the change in percent effect caused by one individual reacting would be low enough that the data can be treated as if they represent a continuous distribution. Environment Canada (2005), therefore recommends estimating the ICp, a quantitative endpoint, for the echinoid fertilization test.

Lethal means causing death by direct action. Death is defined here as the cessation of all visible signs of movement or other activity.

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, effects which are detected by the observer and are statistically significant. For example, the LOEC might be the lowest concentration at which fertilization success 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 effect on the organism. For example, the NOEC might be the highest tested concentration at which an observed variable such as fertilization success did not differ significantly from that in the control. NOEC customarily refers to sublethal effects, and to the most sensitive effect unless otherwise specified.

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 might 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 an egg might be fertilized or not fertilized. Quantal effects are typically expressed as numerical counts or percentages thereof. Quantitative estimates are performed on quantal data if the number of quantal observations is large (i.e., counting 100 - 200 eggs per container) (EC, 2005).

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 number of progeny produced, or the weight attained by individual organisms at the end of a test.

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

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

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 chemical or material might cause adverse effects on living organisms, if present in sufficient amount. Toxic is an adjective or adverb, and should not be used as a noun; whereas toxicantis a 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 (TIE) describes a systematic sample pre-treatment (e.g., pH adjustment, filtration, aeration) followed by tests for toxicity. This evaluation is used to identify the agent that is 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, tissues, cells or other living material, under defined conditions. An aquatic toxicity test usually measures either (a) the proportions of organisms affected (quantal), or (b) the degree of effect shown (quantitative or graded), after exposure to specific concentrations of chemical, effluent, receiving water, leachate, elutriate, or interstitial water derived from sediment or similar solid material.

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 one or more substances or materials.

Treatment is, in general, an intervention or procedure whose effect is to be measured. More specifically, in testing for toxicity, it is a condition or procedure applied to the test organisms by an investigator, with the intention of measuring the effect(s) 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 concentration causing an effect 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.


The first edition of this biological test method, printed in December 1992 and amended in November 1997, was co-authored by John B. Sprague (Sprague Associates Ltd., Guelph, Ontario) and Donald J. McLeay (McLeay Associates Ltd., West Vancouver, B.C.). It was based on pre-existing procedures of several authors, laboratories, agencies, and other groups in Europe, Japan, U.S.A., and Canada, and on a supporting review of relevant publications (Sprague and McLeay, 1991). Gary A. Sergy and Richard P. Scroggins (Environmental Protection, C&P, Environment Canada) acted as Scientific Authorities and provided technical input and guidance throughout the preparation of the original Report EPS 1/RM/27. Members of the Inter-Governmental Ecotoxicological Testing Group (IGETG, Appendix B) participated actively in the development and review of the first edition of this document and are thanked accordingly.

Special acknowledgement is made of the technical contributions provided by the IGETG Sub- Committee which was responsible for initial and final reviews of the first edition of this report: Gary A. Chapman (USEPA, Newport, Oregon); Paul A. Dinnel (Univ. of Washington, Seattle, Washington); Timothy J. Hall (NCASI, Anacortes, Washington); John H. Himmelman (Laval University, Quebec); Emilia Jonczyk (Beak Consultants Ltd., Brampton, Ont.); Cathy A. McPherson (EVS Consultants, North Vancouver, B.C.); and IGETG members Kenneth G. Doe, Richard P. Scroggins, Gary A. Sergy, Graham C. van Aggelen, Peter G. Wells, and Stewart G. Yee. Substantial guidance for the first edition of this biological test method came from laboratory tests on methodology by laboratories of Environment Canada (Atlantic, K.G. Doe; Pacific and Yukon, S.G. Yee), Beak Consultants Ltd. (Jennifer Miller, E. Jonczyk), and EVS Consultants (C.A. McPherson).

The following people provided information and many useful comments on final or early drafts of the first edition of this report: Brian S. Anderson (Univ. of California, Santa Cruz); Glenn F. Atkinson (Applied Statistics Div., of Environment Canada, Ottawa); Robert S. Carr (U.S. Fish & Wildlife Service, Corpus Christi, Texas); Guy L. Gilron (Sentar, Surrey, B.C.); Larry R. Goodman (USEPA, Gulf Breeze, Florida); Christopher W. Hickey (Water Quality Centre, Hamilton, N.Z.); Harold H. Lee (Univ. of Toledo, Toledo, Ohio); Richard Lloyd (Chelmsford, Essex, United Kingdom); Marian L. Nipper (Companhia de Technologia de Saneamento Ambiental, São Paulo, Brazil); James M. Osborne (C & P, Ottawa); Jerry F. Payne (Fisheries & Oceans, St. John’s, Newfoundland); and Linda Porebski (C & P, Ottawa).

Photographs for front cover of the first edition of this report were supplied by Stewart Yee, Aquatic Toxicology Laboratory, Pacific and Yukon Region, Environment Canada.

This (second) edition was prepared by Jennifer Miller (Miller Environmental Sciences Inc., King City, ON), with assistance and guidance from Lisa Taylor (Manager, Method Development and Applications Unit) and Rick Scroggins (Chief, Biological Methods Section) of Environment Canada, Ottawa, ON. Statistical guidance on regression analyses was provided by Barry Zajdlik (Zajdlik & Associates, Rockwood, ON), who is sincerely thanked. This second edition incorporates the November 1997 Amendments (with modification as appropriate), and includes numerous updates such as revised guidance on salinityadjustment, revised methodology for porewater testing, and the use of regression analyses for quantitative endpoint data. Numerous comments and suggestions provided in response to a questionnaire circulated among toxicity testing laboratories in Canada and the US performing this sublethal test, formed the basis for many of the changes to the first edition, and were considered when preparing this second edition of Report EPS 1/RM/27.

Grateful acknowledgement is made of the many useful comments and suggestions provided by the following laboratory personnel who responded to the circulated questionnaire: Ken Doe and Paula Jackman (Atlantic Laboratory for Environmental Testing, Environment Canada, Moncton, NB); Emila Jonczyk and Keith Holtze (AquaTox Testing and Consulting Inc., Guelph, ON); Janet Pickard (Cantest Ltd., Burnaby, BC); Scott Carr, Marion Nipper, and Jim Biedenbach (Columbia Environmental Research Centre, TAMU-CC, Centre for Coastal Studies, Corpus Christi, TX); Cathy McPherson (Golder Associates Ltd., North Vancouver, BC); Armando Tank (Nautilus Environmental, Burnaby, BC); Suzanne Agius (Ocean Disposal Program, Environment Canada, Hull, QC); Craig Buday (Pacific and Yukon Laboratory for Environmental Testing, North Vancouver, BC); Steve Bay and Darrin Greenstein (Southern California Coastal Water Research Project, Costa Mesa, CA). Don McLeay (McLeay Environmental Ltd., Victoria BC) is also thanked for his help in addressing the many comments received in response to the questionnaire, and his review of the 2nd edition document.

Revisions to the sediment testing procedures provided in this 2nd edition test method document are predominantly based on the results of an inter-laboratory study carried out in 2008 designed to investigate the improvement of the porewater-testing component of EPS 1/RM/27. Participants of this inter-laboratory study are gratefully acknowledged for their participation and their input into the report, and include: Ken Doe and Paula Jackman (Atlantic Laboratory for Environmental Testing, Environment Canada, Moncton, NB); Craig Buday (Pacific and Yukon Laboratory for Environmental Testing, North Vancouver, BC); and Scott Carr and Jim Biedenbach (Columbia Environmental Research Centre, TAMU-CC, Centre for Coastal Studies, Corpus Christi, TX, USA). Special thanks to Rick Scroggins and Lisa Taylor (Biological Methods Section, Environment Canada, Ottawa, ON) who acted as Scientific Authorities and provided technical input and guidance throughout the study.

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