Biological test method for toxicity tests using early life stages of rainbow trout: front matter

Front Matter

• Readers' Comments
• Review Notice
• Abstract
• Foreword
• List of Abbreviations and Chemical Formulae
• Terminology
• Acknowledgements

Readers' Comments

Comments regarding the content of this report should be addressed to:

Richard Scroggins
Method Development and Applications Section
Environmental Technology Centre
Environment Canada
335 River Road
Ottawa, Ontario K1A 0H3

Review Notice

This report has been reviewed by the staff of the Environmental Technology Advancement Directorate, Environment Canada, and approved for publication. Mention of trade names or commercial products does not constitute endorsement by Environment Canada or recommendation for use.

Abstract

Revised procedures for toxicity tests using salmonid (i.e., rainbow trout) embryos, alevins, and swim-up fry, are recommended by Environment Canada in this report. Three test options are described: an embryo (E) test for frequent or routine monitoring; an embryo/alevin (EA) test for measuring effects on multiple phases of development; and an embryo/alevin/fry (EAF) test for more definitive investigations. All three test options start at the onset of embryo development, and measure the development and survival of early life stages. The embryo test ends seven days after fertilization. The embryo/alevin test is terminated seven days after half of the alevins are seen to have hatched in the control. The embryo/alevin/fry test ends after 30 days of feeding swim-up fry. Selection of the most suitable test option will depend on the objectives of the test and on the physicochemical characteristics of the substance being tested. Because such early life stages are usually a sensitive part of the life cycle of a fish, the tests should be considered as powerful and meaningful assays.

Rainbow trout (Oncorhynchus mykiss) must be used when performing each of these three test options. Procedures are given for spawning broodstock, handling gametes, and fertilizing eggs of rainbow trout before starting the test, as well as for incubating embryos and alevins and feeding swim-up fry during the tests which include these life stages. General or universal conditions and procedures are outlined for testing a variety of substances for their effects on the early life stages of rainbow trout. 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, and validation.

Foreword

This is the second edition of Environment Canada's three-option biological test method for performing toxicity tests using early life stages of salmonid fish (i.e., EC, 1992) which was published in December 1992. Since that time, investigators within Canadian government and private laboratories as well as scientists in the United States have proposed a number of changes to the test procedures and conditions which simplify and improve the performance of each of the three options (i.e., E, EA, and EAF options) that comprise this biological test method. Unlike the first edition (EC, 1992), which recommended three species of salmonid fish (i.e., rainbow trout, coho salmon, or Atlantic salmon) as candidate test organisms, this second edition specifies that Oncorhynchus mykiss (rainbow trout or steelhead trout) must be used as the species of test organisms when applying any of the three test options using the revised procedures and conditions described herein.

This is one of a series of recommended methods for measuring and assessing the aquatic biological effects of toxic substances or materials. Recommended methods are those that have been evaluated by Environment Canada (EC), and are favoured:

• for use in EC aquatic 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 life of specific test substances or materials within the aquatic environment or destined for it. Depending on the biological test method chosen, substances or materials to be tested for toxicity could include samples of chemical, effluent, elutriate, leachate, receiving water or, where appropriate, sediment or similar particulate material.

List of Abbreviations and Chemical Formulae

CaCO₃

calcium carbonate

cm

centimetre(s)

CV

coefficient of variation

d

day(s)

DO

dissolved oxygen (concentration)

E

embryo

EA

embryo/alevin

EAF

embryo/alevin/fry

EC50

median effective concentration

g

gram(s)

g/kg

grams per kilogram (equivalent to ‰)

h

hour(s)

HCl

hydrochloric acid

H₂O

water

ICp

inhibiting concentration for a (specified) percent effect

L

litre(s)

LC50

median lethal concentration

LOEC

lowest-observed-effect concentration

m

metre(s)

mg

milligram(s)

min

minute(s)

mL

millilitre(s)

mm

millimetre(s)

mS

millisiemen(s)

MSD

minimum significant difference

N

Normal

NaOH

sodium hydroxide

NOEC

no-observed-effect concentration

P

probability

SD

standard deviation

SI

Système internationale d'unités

sp.

species

TOEC

threshold-observed-effect concentration

TIE

toxicity identification evaluation

TM (™)

Trade Mark

°C

degree(s) Celsius

µg

microgram(s)

µm

micrometre(s)

>

greater than

<

less than

≥

greater than or equal to

≤

less than or equal to

±

plus or minus

%

percentage or percent

‰

parts per thousand

~

approximately

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 mean "could".

General Technical Terms

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

Alevin is a recently-hatched, non-feeding salmonid fish with an evident yolk sac (for nutritive requirements). Often referred to as a yolk-sac fry, sometimes referred to as a larva. See also nonviable alevin.

Broodstock are the adult fish which are undergoing physiological changes to produce either eggs or sperm.

Compliance means in accordance with governmental permitting or regulatory requirements.

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 the solution’s temperature. Conductivity is reported as micromhos per centimetre (µmhos/cm) or as millisiemens per metre (mS/m); 1 mS/m = 10 µmhos/cm.

Culture, as a noun, is a stock of animals or plants raised under defined and controlled conditions to produce healthy test organisms. As a verb, it means to carry out this procedure of raising organisms.

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

Egg is an encapsulated, spherical ovum, unfertilized or fertilized, obtained from a sexually mature female fish.

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

Embryo is an undeveloped young fish, before it hatches from the egg.

Exogenous feeding means oral intake and consumption of food available in the water, by free-swimming fry.

Eyed egg is an encapsulated embryo that has reached a stage of development in which its pigmented eyes are clearly evident during routine observations.

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

Gametes are the eggs or sperm obtained from mature adult fish.

Growth is the increase in size or weight as the result of proliferation of new tissues. In this test, it is limited to increase in dry weight.

Hardness is the concentration of cations in water that will react with a sodium soap to precipitate an insoluble residue. In general, hardness is a measure of the concentration of calcium and magnesium ions in water, expressed as mg/L calcium carbonate.

Impermeable means, in reference to the egg membrane, the extent to which the membrane prevents the passage of molecules (e.g., water, ions, proteins, fats, toxicants).

Incubation means the rearing of embryos or alevins under defined conditions compatible with normal development.

Larva is a recently hatched fish or other organism that has physical characteristics other than those seen in the adult. For salmonid fish, the term larva is synonymous with alevin.

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.

Milt is the mixture of sperm and seminal fluid extracted from a sexually mature male fish.

Monitoring means the routine (e.g., daily, weekly, monthly, or 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 for toxicity of samples of effluent, elutriate, leachate, or receiving water.

Nonviable alevin means an egg that has failed to hatch into a normal living, alevin, seven days after 50% hatch is seen to be achieved in the control groups of this toxicity test. The category encompasses failure of egg fertilization, dead embryos, embryos for which development is delayed, and alevins which are obviously deformed or atypical (e.g., two-headed alevins or those with bifurcated tails).

Nonviable at swim-up refers to a test organism which has failed to survive or develop normally, at the time when 50% swim-up is seen to be achieved in the control group of this toxicity test. The category includes failure of egg fertilization, failure to hatch, and mortality, delayed development, or abnormal development among embryos, alevins, and early swim-up fry. Any swim-up fry that is alive and apparently normal at the above- mentioned time would be counted as viable.

Nonviable embryo means an egg which has failed to survive or develop normally, when observed. The category includes failure of egg fertilization, mortality, delayed development, or abnormal development among embryos. Any embryo that is alive and apparently normal when observed is counted as viable.

Percentage (%) is a concentration expressed in parts per hundred parts. One percent represents one unit or part of substance (e.g., effluent, elutriate, leachate, or receiving 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 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 describes the durations of illumination and darkness within a 24-h day.

Precipitation is 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 fish.

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 also be measured directly using a salinity/conductivity meter or other means (see APHA et al., 1995). It is usually reported in grams per kilogram or parts per thousand (‰).

Spawning means the release of eggs or sperm from mature adult fish, or refers to behaviour related to the readiness of fish to release gametes.

Stripping means human handling of mature adult fish in order to extract eggs or sperm from them.

Swim-up fry is a young, post-alevin fish which has commenced active feeding.

Thinning refers to the random removal of a number of individual test organisms from one or more replicates, to reduce crowding, maintain an acceptable loading density, and/or minimize the volumes of test solution required during each renewal. Thinning must not be done at any time during an E or EA test, nor during the embryo or alevin stages of an EAF test. Thinning may only be done, if desired, at the time that the final phase of an EAF test is started.

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.

Water hardening is the process occurring within the first two hours after fertilization, when the egg swells due to uptake of water into the perivitelline space, and before the egg membrane becomes relatively impermeable.

Terms for Test 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 that is being studied. In an aquatic toxicity test, the control must duplicate all the conditions of the exposure treatment(s), but must contain no added test substance. The control is used to determine the absence of measurable toxicity due to basic test conditions (e.g., quality of the dilution water, health of test organisms, or effects due to handling of organisms).

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

Dechlorinated water is 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 fresh water used to dilute a test substance to prepare different concentrations for a toxicity test.

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.

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 just "upstream" or up-current from the discharge point). Further descriptive information must be provided to indicate the intended meaning.

Reconstituted water is deionized or glass-distilled water to which reagent grade chemicals have been added. The resultant synthetic fresh water is free from contaminants and has the desired characteristics of pH, alkalinity, and hardness.

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

Substance is a particular kind of material having more or less uniform properties.

Upstream water is surface water (e.g., in a stream, river or lake) that is not influenced by the effluent (or other test 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 in relation to the life span of the organism, for example a four-day test with fish. An acute toxic effect would be induced and observable within the short period.

Chronic means occurring during a relatively long period, usually a significant portion of the life span of the organism (e.g., 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 refers to long-term effects that are usually related to changes in such things as metabolism, growth, reproduction, or ability to survive. Because of the long life span of salmonids, early life-stage tests do not measure chronic toxicity, although the intent of this test is to estimate approximately, what such sublethal chronic toxicity might be.

Chronic value is a synonym for TOEC (q.v.). TOEC is the recommended term because it can be applied accurately to all sublethal effects whether acute or chronic.

Continuous-flow describes tests in which solutions in test vessels are renewed by the continuous inflow of a fresh solution, or by a frequent intermittent inflow. The meaning is the same as flow-through.

EC50 is the median effective concentration. That is, the concentration of substance in water (e.g., mg/L) that is estimated to cause a discernible sublethal or lethal toxic effect to 50% of the test organisms. In most instances, the EC50 (including its 95% confidence limits) is statistically derived by analysis of an observed biological response (e.g., incidence of nonviable embryos or reduced hatching success) for various test concentrations, after a fixed period of exposure. The duration of exposure must be specified.

Endpoint means the reaction of the organisms to show the effect which is intended to mark completion of the test, and also means the measurement(s) or value(s) derived, that characterize the results of the test (e.g., ICp).

Flow-through describes tests in which solutions in test vessels are renewed continuously by the constant inflow of a fresh solution, or by a frequent intermittent inflow (same as continuous-flow).

ICp is the inhibiting concentration for a (specified) percent effect. It represents a point estimate of the concentration of test substance that is estimated to cause a designated percent impairment in a quantitative biological function such as the size attained by fish during a growth period. For example, an IC25 could be the concentration estimated to cause fish to attain dry weight that is 25% lower than that attained by control fish. This term should be used for any toxicological test which measures a quantitative effect or change in rate, such as growth, reproduction, or respiration. (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.)

LC50 is the median lethal concentration, i.e., the concentration of substance in water estimated to be lethal to 50% of the test organisms. The LC50 and its 95% confidence limits are usually derived by statistical analysis of mortalities in several test concentrations, after a fixed period of exposure. The duration of exposure must be specified (e.g., 96-h LC50).

Lethal means causing death by direct action. Death of fish is defined 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 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 growth of fish was decreased significantly from that of the control groups. LOEC is generally reserved for adverse sublethal effects but can also be used for mortality, which might sometimes be the most sensitive effect observed.

Minimum Significant Difference (MSD) means the difference between values for individual concentrations (e.g., mean percent nonviable embryos; mean percent nonviable alevins) that would have to exist before it could be concluded that there was a significant difference between the groups. The MSD is provided by certain statistical tests including Dunnett's multiple- range test, a standard statistical procedure.

NOEC is the no-observed-effect concentration. This is the highest concentration of a test 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 growth was not decreased significantly from that of the control groups. NOEC customarily refers to adverse sublethal effects, and to the most sensitive effect unless otherwise specified.

Replicate is a single test chamber containing a prescribed number of organisms in either one concentration of test solution or in dilution water as a control. In a toxicity test comprising five test concentrations and a control, and using three replicates, 18 test chambers would be used. For each concentration or control, there would be three test chambers or replicates. A replicate is an independent test unit; therefore, any transfer of organisms or solutions from one replicate to another would invalidate the statistical analysis.

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

Static renewal describes a toxicity test in which test solutions are renewed (replaced) periodically, usually at the beginning of each 24-h period. Synonymous terms are "batch replacement", "renewed static", "renewal", "static replacement" and "semi-static".

Sublethal means detrimental to the fish, but below the level that directly causes death within the test period.

TOEC is the threshold-observed-effect concentration. It is calculated as the geometric mean of NOEC and LOEC. A term variously defined in some other countries is the MATC (maximum acceptable toxicant concentration). Chronic value or subchronic value are alternative terms that might be appropriate depending on the duration of exposure in the test.

Toxicity is the inherent potential or capacity of a substance to cause adverse effect(s) on fish or other organisms. The effect(s) could be lethal or sublethal.

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

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

Acknowledgements

The first edition of this report, printed in December 1992, was co-authored by M.R. Gordon (M.R. Gordon & Associates, Ltd., Sechelt, BC), D.J. McLeay (McLeay Associates Ltd., West Vancouver, BC) and J.B. Sprague (Sprague Associates Ltd., Guelph, ON). Key reviewers who assisted in the development of the first edition included: W.J. Birge (University of Kentucky, Lexington,KY); K.G. Doe (Environment Canada, Dartmouth, NS); P.V. Hodson (Queens University, Kingston, ON); T. Kovacs (Pulp and Paper Research Institute of Canada, Pointe Claire, PQ; D.D. Monteith (BC Research Corporation, Vancouver, BC); C. M. Neville (Ontario Ministry of the Environment, Rexdale,ON); M.D. Paine (EVS Consultants Limited, North Vancouver, BC); R.P. Scroggins (Environment Canada, Ottawa, ON); G.A. Sergy (Environment Canada, Edmonton, AB); J.A. Servizi (Fisheries and Oceans Canada, Cultus Lake, BC): J.D. Somers (Alberta Environmental Centre, Vegreville, AB); G. van Aggelen (BC Ministry of Environment, Lands and Parks, North Vancouver, BC): and R. Watts (Environment Canada, North Vancouver, BC.

This second edition was prepared by D.J. McLeay (McLeay Environmental Ltd., Victoria, BC) and J.B. Sprague (Sprague Associates Ltd., Salt Spring Island, BC). The following persons contributed to the modified test procedures and conditions in this edition of the report, and are thanked accordingly: H.C. Bailey (EVS Environment Consultants Ltd., North Vancouver, BC); W.J. Birge (University of Kentucky, Lexington, KY); J. Black (EA Engineering, Science & Technology Inc., Sparks, MD); E.C. Canaria (EVS Environment Consultants Ltd., North Vancouver, BC); P.M. Chapman (EVS Environment Consultants Ltd., North Vancouver, BC); M. Fennell (Environment Canada, North Vancouver, BC); S. Gibbons (PAPRICAN, Pointe-Claire, PQ); K. Holtze (B.A.R. Environmental Inc., Guelph, ON); E. Jonczyk (Beak Consultants Ltd., Brampton, ON); T. Kovacs (PAPRICAN, Pointe-Claire, PQ); J. Larocque (PAPRICAN, Pointe Claire, PQ); L. Novak (B.A.R. Environmental Inc., Guelph, ON); J. Pickard (B.C. Research Inc., Vancouver, BC); R. Scroggins (Environment Canada, Gloucester, ON);G. van Aggelen (Environment Canada, North Vancouver, BC), R. Watts (Environment Canada, North Vancouver, BC); and S. Yee (Environment Canada, North Vancouver, BC).

Special acknowledgement is made of the technical contributions by S. Yee, M. Fennell, and J. Bruno (Environment Canada, North Vancouver, BC) with respect to laboratory tests undertaken to redesign the test apparatus and improve the test procedures and conditions (Yee et al., 1996; Fennell et al., 1998). Researchers at EVS Environment Consultants Ltd. (North Vancouver, BC) are sincerely thanked for several useful procedural improvements (see Canaria et al., 1996) that were incorporated in this revised biological test method. The past experience of J. Pickard and co-workers at B.C. Research Inc. (Vancouver, BC) in performing an EAF test with rainbow trout and providing related guidance herein is acknowledged. Drs. W.J. Birge (University of Kentucky, Lexington, KY) and J. Black (EA Engineering, Science & Technology, Inc., Sparks, MD) also provided many useful technical comments and suggestions for change following publication of the first edition, which influenced the changes evident in this edition. The assistance of R. Watts (Environment Canada, North Vancouver, BC) with respect to redesigning and illustrating the embryo/alevin incubation unit is acknowledged; as is that of S. Yee (Environment Canada, North Vancouver, BC) for taking and supplying the photographs displayed on the cover of this biological test method. Finally, members of IGATG (see Appendix A) are thanked for their continuing technical support.