Biological test method for toxicity tests using early life stages of rainbow trout: chapter 1


1.1 Background

Aquatic toxicity tests are used within Canada and elsewhere to measure, predict, and control the discharge of discrete substances or complex mixtures that could adversely affect aquatic life. Recognizing that no single test method or test organism can be expected to satisfy a comprehensive approach to environmental conservation and protection, the Inter-Governmental Aquatic Toxicity Group (IGATG) (Appendix A) proposed the development and standardization of a set of aquatic toxicity tests that would be broadly acceptable, and would measure different toxic effects using organisms representing different trophic levels and taxonomic groups (Sergy, 1987). A test based on the development, growth, and mortality of early life stages of salmonid fish is one of several "core" aquatic toxicity tests that was selected to be standardized sufficiently for use in Environment Canada's regional laboratories (Appendix B), as well as in provincial and private laboratories, to help meet Environment Canada's testing requirements (e.g., EC, 1991).

Universal procedures and conditions for conducting early life-stage tests using rainbow trout (Oncorhynchus mykiss), are described in this report. Also presented are specific sets of conditions and procedures required or recommended when using the test for evaluating different types of substances (namely, samples of chemical, effluent, elutriate, leachate, or receiving water). For guidance on the implementation of this and other Environment Canada biological test methods, and on the interpretation and application of the endpoint data, the reader should consult Environment Canada (1998a).

Figure 1 gives a general picture of topics covered in this report. Some details of methodology are discussed in explanatory footnotes.

The biological test method presented in this report is based largely on other embryo- larval and early life-stage methods developed in North America and Europe (USEPA, 1985a; Birge et al., 1985; Rexrode and Armitage, 1987; van Aggelen, 1988; Birge and Black, 1990; ASTM, 1991a; Hodson et al., 1991; Paine et al., 1991; OECD, 1992a;b;c; Neville, 1995a;b; OECD, 1996; 1997). It has been developed following a review of specific procedural variations indicated in existing "methodology" documents (Appendix C) and in other related reports and publications.

This report replaces the biological test method "Toxicity Tests Using Early Life Stages of Salmonid Fish (Rainbow Trout, Coho Salmon, or Atlantic Salmon", which was published by Environment Canada in 1992 (EC, 1992a). Since that time, certain pretest and test conditions and procedures have been simplified, clarified, or otherwise improved, based on (a) the experience of a number of Canadian laboratories performing the "E" or “EA” test options to meet regulatory requirements for monitoring environmental effects (EC, 1991), and (b) controlled studies with the objectives of simplifying or improving methods (Canaria et al., 1996; Yee et al., 1996; Fennell et al., 1998). Improvements in procedures, system design, statistical treatments of data, and more explicit or more detailed guidance for the performance of tests, are provided herein.

Three test options are described: an embryo (E) test suitable for frequent or routine monitoring; an embryo/alevin (EA) test for measuring the effects of toxicants on multiple phases of development; and an embryo/alevin/fry (EAF) test for definitive investigations. All three test options start with the onset of embryo development, and measure the development and survival of early life stages. The E test ends seven days after fertilization. The EA test is terminated during the alevin stage, when 50% hatching success is seen in the control, with no feeding of fish being required. The EAF test ends after 30 days of exposing swim-up fry, with feeding. Any of these three test options may be used to evaluate samples of chemical or chemical product, effluent, elutriate, leachate, or receiving water. Selection of the most suitable test options will depend on the objectives of the test and the nature of the substance being tested (see Sections 4.3.1, 5.1, 6.1, and 7.1).

When formulating these procedures, an attempt was made to balance scientific, practical, and financial considerations, and to ensure that the results would be accurate and precise enough for most situations in which they would be applied. It is assumed that the user has a certain degree of familiarity with aquatic toxicity tests. The explicit instructions that might be required in a regulatory test are not provided, although this report is intended as a guidance document for this and other uses.

Figure 1 Considerations for Preparing and Performing Toxicity Tests Using Early Life Stages of Rainbow Trout and Various Types of Test Substances

Universal Procedures

  • Obtaining and handling gametes
  • Culturing of embryos and alevins
  • Preparing test solutions
  • Test conditions (temp., DO, etc.)
  • Beginning the test
  • Water quality measurements
  • Reference toxicants for "E" test
  • Observations during test
  • Endpoints
  • Calculations
  • Validity of results
  • Legal considerations

Items Covered in Specific Sections

Chemicals

  • Choosing control/dilution water
  • Preparation of solutions
  • Observations during tests
  • Measurements during tests
  • Endpoints
  • Chemical properties
  • Labelling and storage
  • Chemical measurements

Effluents, Elutriates, and Leachates

  • Choosing control/dilution water
  • Preparation of solutions
  • Observations during tests
  • Measurements during tests
  • Endpoints
  • Containers and labelling
  • Sample transit and storage

Receiving Waters

  • Choosing control/dilution water
  • Preparation of solutions
  • Observations during test
  • Measurements during test
  • Endpoints
  • Containers and labelling
  • Sample transit and storage

1.2 Historical Use of Test

Chronic toxic effects on fish have been studied with either life-cycle tests (egg-to-egg), or partial life-cycle tests (egg-to-juvenile), depending on the nature of the studies and the fish species used. For salmonids, complete life-cycle studies are largely impractical because it takes two to five years for these fish to reach maturity. However, over the past thirty years, results from full and partial life-cycle tests with several fish species and a variety of chemicals indicate that the early developmental stages (i.e., embryo, larval, and early juvenile) can be equally or more sensitive to aquatic contaminants than the adults (McKim, 1977, 1985; Hodson and Blunt, 1981; Woltering, 1984). Based on this experience, a number of procedures have been developed to measure toxic effects on early life stages of salmonid fish (Birge et al., 1985; van Aggelen, 1988; Birge and Black, 1990; Hodson et al., 1991; Paine et al., 1991; Neville, 1995a;b). These procedures are based on the assumption that the highest concentrations which are without sublethal effects in early life-stage tests, will approximate the chronically safe concentrations for the salmonid species tested.Footnote 1

Early life-stage tests using rainbow trout or other species of salmonids for regulatory and research purposes have initiated toxicant exposures at the onset of embryological development, and ended them during the alevin stage, when the fish begin to exhibit swim-up behaviour, or after the fry have been feeding for several weeks (Rexrode and Armitage, 1987; ASTM, 1991a; Hodson et al., 1991; OECD, 1992a;b; OECD, 1996). Different early life stages can vary in their sensitivity to different toxicants (Mayer et al., 1986; Kristensen, 1990); therefore, it is preferable to monitor effects of continuous toxicant exposure on several early life stages, and during the transition from one stage to the next, to obtain a good estimate of a sublethally safe concentration. Depending on the species and temperature, and on the number of days that alevins are observed before ending the test, the duration of an EA test with salmonid fish might be from as little as ~30 days (Fennell et al., 1998) to as much as ~80 days. The duration of an EAF test might also vary from as little as 70 days to as much as 120 days or longer, depending on species and temperature and on the number of days that the survival and growth of fry are monitored. In any case, such early life-stage tests can be conducted in much less time and at much less expense than full life-cycle tests using salmonid fish.

Various short-term tests of 7 to 28 days have been developed using rainbow trout embryos and/or alevins (Birge et al., 1985; Birge and Black, 1990; Paine et al., 1991; OECD, 1992a; 1996; Neville, 1995a;b), or fry (OECD, 1992c; 1997). These tests focus on one or more sensitive transitional periods of development (e.g., early embryo development, alevin development and yolk conversion, or feeding and growth of young fry) and have been standardized to use only rainbow trout. These relatively new methods are promising,Footnote 2 but in some cases their endpoints might be difficult to define or measure with confidence, or they might require special technical skills to obtain reproducible results. When using these tests, it is advisable to undertake preliminary tests to determine the reproducibility of results, and to compare the sensitivity of results with more conventional early life- stage tests using salmonids (Rexrode and Armitage, 1987; ASTM, 1991a; OECD, 1992b).

The purpose of this report is to provide a "standardized" Canadian methodology for estimating the toxicity of various substances to rainbow trout in fresh water, by exposing one or more of the early life stages of this species using one of the three test options herein (i.e., E, EA, or EAF test options). The procedures for salmonid early life-stage toxicity tests in existing Canadian, U.S., and international methodology documents vary in terms of recommended test species, duration of exposure, temperature regime(s), substance examined, test conditions and systems, biological observations and endpoints, statistical design, and criteria for validity (Appendix C). This report gives guidance for evaluating sublethal toxicity of samples of chemical, effluent, leachate, elutriate, or receiving water, and the rationale for selecting certain approaches. The three test options (i.e., E, EA, or EAF tests) herein are for use with rainbow trout acclimated to fresh water, with fresh water as the dilution and control water, and with test substances that include wastewaters that are essentially fresh water (i.e., salinity ≤10 g/kg) or are saline but are destined for discharge to fresh water. The application of one or more of these three test options may be varied but includes instances in which the impact or potential impact of substances on the freshwater environment is under investigation. Other tests, using other species acclimated to seawater, may be used to assess the impact or potential impact of substances in estuarine or marine environments, or to evaluate wastewaters having a salinity >10 g/kg that are destined for estuarine or marine discharge.

1.3 Salmonid Species Studied and Recommended

In Canada and the United States, the culture and handling of many species of salmonid fish are well established and understood by fisheries scientists, hatchery specialists, fish biologists, and researchers. The performance and sensitivity of several salmonid species have also been examined in a wide variety of toxicity studies in the laboratory. Toxicity to early life stages of salmonid fish has been studied primarily for rainbow trout, brook trout, Pacific salmon, and Atlantic salmon (McKim and Benoit, 1971; Benoit, 1976; Benoit et al., 1976; Davies et al., 1976; Burkhalter and Kaya, 1977; Brenner and Cooper, 1978; Servizi and Martens, 1978; Daye and Garside, 1979; McLeay and Gordon, 1980; Martens et al., 1980; Helder, 1981; Hodson and Blunt, 1981; Birge et al., 1985; NCASI, 1985; Peterson et al., 1988; Hodson et al., 1991; Neville, 1995a;b).

Rainbow trout is the test organism recommended for use in each of the three test options (i.e., E, EA, or EAF tests) described in this report. Relevant information on the distribution, life history, and husbandry of rainbow trout is given in Appendix D. The test options described herein might also be applied to steelhead trout (an anadromous subspecies of Oncorhynchus mykiss); likely without a need for procedural change. Other species of salmonid fish (e.g., cutthroat trout, brook trout, brown trout, chinook salmon, chum salmon, coho salmon, Atlantic salmon, arctic grayling, or whitefish) might also be used with any of these three test options, although modifications to certain procedures and/or conditions (e.g., temperature and test duration) might be necessary. Investigators wishing to use a species other than O. mykiss are advised to carefully consider a temperature regime that is known or likely to be compatible with the early development of the desired species, and to determine in one or more preliminary tests (using control/dilution water) the appropriate temperature regime and related test duration for any E, EA, or EAF test option to be applied to that species using a test substance. In particular, the criteria for test validity described herein for rainbow trout (see Section 4.6) should be demonstrated to be achievable with an alternate test species before it is used for measuring the toxicity of a test substance according to any of the test options herein (with or without modification). Additionally, any test- specific report which identifies one of the test options herein as the biological test method used must identify the species and common name of the test organism as well as any modifications to definitive pre-test or test conditions and procedures described herein (see Section 8).

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