Biological test method for toxicity tests using early life stages of rainbow trout: chapter 2

Section 2: Test Organism

• 2.1 Species and Life Stages
• 2.2 Source

2.1 Species and Life Stages

Rainbow trout (Oncorhynchus mykiss) is the test organism recommended as the source of gametes for use with any of the three test options (i.e., E, EA, or EAF options) described herein (see Sections 1.3 and 4.3.1). Steelhead trout (an anadromous subspecies of O. mykiss) may also be used if available.

The generalized appearance of salmonid early life stages is shown in Figure 2. The test is designed to determine effects on rainbow trout from the onset of embryo development through to a particular stage of development, depending on the test option selected (see Section 4.3.1). Since toxicant exposure is initiated immediately after the eggs are fertilized, the test requires that the eggs be fertilized with milt in the laboratory (see Appendix D for recommended procedures). The time taken for embryo/larval development varies with water temperature (see Appendix D).

Three major transitions of the different developmental stages in the early life of rainbow trout are important and integral to the test option to be chosen. The first transition is when recently fertilized eggs change to an embryo, and develop within an egg membrane that quickly becomes relatively impermeable as the result of "water hardening" after fertilization. Rapid cell division takes place in the developing embryo, and this phase is used in the shortest test option (i.e., the embryo or E test option). The second transition is hatching, and the former embryo becomes an alevin or yolk-sac fry. This stage is included in the embryo/alevin (EA) test option. The third transition is from alevin to swim-up fry, when the young fish changes from using its yolk as food, to feeding on outside sources. The embryo/alevin/fry (EAF) test option includes this change and 30 days of exposure for the feeding fry. Further details on the stages and the timing of stages with respect to these three test options are given in Section 4.3.6. Appendix D should be consulted for additional information on the ranges of size, temperature tolerance, and temperature-dependent developmental rates of these life stages.

2.2 Source

Gametes or broodstock should be obtained from a single population and source of rainbow trout. The best sources are likely to be government hatcheries, government research stations, and private culture facilities that are known to have disease-free fish. To simplify procedures, it is preferable to obtain gametes, since handling, transport, holding and stripping of broodstock require additional holding facilities and experienced personnel. Egg fertility is influenced markedly by the ripeness of female gonads, and timing of stripping for egg procurement should be optimal (Appendix D) to enable good fertilization success.

The pool of eggs to be used in a toxicity test must be obtained from a minimum of four females (see Appendix D, including footnote 46). Additionally, the milt must be obtained from a minimum of three males.

Figure 2 General Appearance of Salmonid Early Life Stages (from Sedgewick, 1982)

Pre-fertilization screening of milt for sperm mobility must be carried out to improve the likelihood of good fertilization success. The following procedure (Novak, 1996) has proven effective, and is recommended herein. Since experience has shown that inactive milt can be obtained from 25 to 50% of males, samples from three or more sexually mature individuals must be collected and held in separate vials (Novak, 1996; Fennell et al., 1998). At the testing facility, a thin film of milt from each vial must be placed on a dry glass slide, and examined immediately under a compound microscope at ~100 × magnification. The sperm should appear inactive. A small amount of fresh water (or ovarian fluid) is then added, and mixed quickly with the milt on the slide. Sperm should become vigorously active for approximately 20 to 30 seconds, and will be inactive after 60 seconds. Vials that contain inactive sperm must not be used for fertilization. Samples of fresh milt must be obtained if all vials contain inactive sperm.

If broodstock are obtained, screening of fish for bacterial diseases is recommended before stripping (see Appendix D). Procurement, shipment, and transfer of gametes or broodstock should be approved, if required, by provincial or regional authorities. Provincial governments might require a permit to import fish or their gametes whether or not the species is native to the area, or movements of fish stocks might be controlled by a Federal-Provincial Introductions and Transplant Committee. Advice on contacting the committee or provincial authorities and on sources of fish, can be obtained from the regional Environmental Protection office (Appendix B). In areas where O. mykiss is not native, such as in the northern regions of some provinces or the Yukon and Northwest Territories (see species distribution in Appendix D), application for a permit must be made to the above-mentioned committee, to the appropriate provincial agency, or the Regional Director General of the Department of Fisheries and Oceans (DFO), depending on procedures in place locally.