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

Appendices

1. Members of the Inter-Governmental Aquatic Toxicity Group (as of October, 1998)
2. Environment Canada, Environmental Protection Service, Regional and Headquarters Offices
3. Review of Procedural Variations for Undertaking Early Life-stage Tests Using Salmonid Fish
4. Distribution, Life History, and Husbandry of Rainbow Trout
5. Logarithmic Series of Concentrations Suitable for Toxicity Tests

Appendix A: Members of the Inter-Governmental Aquatic Toxicity Group (as of October, 1998)

Federal, Environment Canada

C. Blaise
Centre St. Laurent
Montreal, PQ

S. Blenkinsopp
Environmental Technology Advancement Directorate
Edmonton, AB

C. Boutin
National Wildlife Research Centre
Hull, PQ

C. Buday
Pacific Environmental Science Centre
North Vancouver, BC

A. Chevrier
Marine Environment Division
Hull, PQ

K. Day
National Water Research Institute
Burlington, ON

K. Doe
Environmental Conservation Branch
Moncton, NB

G. Elliott
Ecotoxicology Laboratory
Edmonton, AB

M. Fennell
Pacific Environmental Science Centre
North Vancouver, BC

M. Harwood
Centre St. Laurent
Montreal, PQ

P. Jackman
Environmental Conservation Branch
Moncton, NB

R. Kent
Evaluation and Interpretation Branch
Hull, PQ

N. Kruper
Ecotoxicology Laboratory
Edmonton, AB

D. MacGregor
Environmental Technology Centre
Gloucester, ON

D. Moul
Pacific Environmental Science Centre
North Vancouver, BC

W.R. Parker
Atlantic Region
Dartmouth, NS

L. Porebski
Marine Environment Division
Hull, PQ

D. Rodrigue
Environmental Technology Centre
Gloucester, ON

R. Scroggins
Environmental Technology Centre
Gloucester, ON

A. Steenkamer
Environmental Technology Centre
Gloucester, ON

D. St.-Laurent
Quebec Region
Montreal, PQ

G. van Aggelen
Pacific Environmental Science Centre
North Vancouver, BC

R. Watts
Pacific Environmental Science Centre
North Vancouver, BC

P. Wells
Atlantic Region
Dartmouth, NS

W. Windle
Commercial Chemicals and Evaluation Branch
Hull, PQ

S. Yee
Pacific Environmental Science Centre
North Vancouver, BC

Federal, Atomic Energy Control Board

P. Thompson
Radiation and Protection Division
Federal Natural Resources
Ottawa, ON

Provincial

S. Abernethy
Ministry of Environment and Energy
Etobicoke, ON

C. Bastien
Ministre de l'environnement et de la faune
Ste-Foy, PQ

D. Bedard
Ministry of Environment and Energy
Etobicoke, ON

M. Mueller
Ministry of Environment and Energy
Etobicoke, ON

C. Neville
Ministry of Environment and Energy
Etobicoke, ON

D. Poirier
Ministry of Environment and Energy
Etobicoke, ON

G. Westlake
Ministry of Environment and Energy
Etobicoke, ON

Appendix B: Environment Canada, Environmental Protection Service, Regional and Headquarters Offices

Headquarters
351 St. Joseph Boulevard
Place Vincent Massey
Hull, Quebec
K1A 0H3

Atlantic Region
15th Floor, Queen Square
45 Alderney Drive
Dartmouth, Nova Scotia
B2Y 2N6

Quebec Region
105 McGill Street
14th Floor
Montreal, Quebec
H2Y 2E7

Ontario Region
4905 Dufferin St., 2nd Floor
Downsview, Ontario
M3H 5T4

Western and Northern Region
Room 210, Twin Atria No. 2
4999 - 98th Avenue
Edmonton, Alberta
T6B 2X3

Pacific and Yukon Region^Footnote 45
224 Esplanade Street
North Vancouver, British Columbia
V7M 3H7

Appendix C: Review of Procedural Variations for Undertaking Early Life-stage Tests Using Salmonid Fish^Footnote 46

Appendix D: Distribution, Life History, and Husbandry of Rainbow Trout

Distribution

Rainbow trout are native to western North America, and are found from Baja California to Alaska. However, the largest numbers of fish are found from northern California into northern British Columbia, particularly in larger rivers and their tributaries, as well as lakes and streams. In central British Columbia, these fish are sometimes referred to as Kamloops trout. Rainbow have been introduced successfully around the world, and now frequent waters of all Canadian provinces as a result of intentional or unintentional releases. Populations spend their entire life in fresh water, although they can also frequent estuarine waters as juveniles or adults, and subspecies (i.e., steelhead) on both coasts of Canada run to sea and return to streams for spawning. In Canada and elsewhere, these trout are widely reared in hatcheries for stocking natural waters to support sports fishing. O. mykiss is among the most common species used in commercial aquaculture and is one of the standard species used worldwide for aquatic toxicity tests, particularly in Canada.

Life History

Rainbow spawn from late winter through the spring. Spawning fish are usually three to four years of age and weigh 1.5 to 4 kg, but repeat spawners can be considerably older and larger in size. Fecundity is approximately 1000 to 1400 eggs/kg spawning female. Eggs 3.0 to 5.0 mm in diameter are laid in gravel redds. After hatching, alevins ranging from 80 to 175 mg (wet weight) remain in the redds until their yolk is absorbed, and emerge as 0.1 to 0.2 g swim-up fry in late May or June. Young hatched in streams commonly remain there for the first winter, after which they migrate to lakes. Fry and juvenile fish usually feed on insect larvae and zooplankton (e.g., daphids). Adults are known to feed on insects, crustaceans, and other fish (Carl et al., 1973); Gordon et al., 1987).

Young (to ~12 cm) have 9 to 13 dark oval parr marks along the lateral line, which are overlain by fine black spots on back and sides. A series of 5 to 10 median parr marks lie along the mid-dorsal line ahead of the dorsal fin. The dorsal fin has a dark leading edge in small fish (fry) and a series of distinct black bars or spots in older fish. Distinct white or pale orange tips appear on the dorsal and anal fins. One or two black spots are common on the adipose fin; with few or no spots on the tail. No red dashes are found on the underside of the lower jaw (Carl et al., 1973).

Husbandry

Stripping of Broodstock

Practical considerations might dictate that gametes for the toxicity test should be obtained from broodstock held and spawned at the test facility. If this approach is being considered, there are several important factors to take into account. Some of the more fundamental aspects of stripping broodstock fish are described here, but more detailed information on specific procedures should be thoroughly studied before undertaking stripping.

The seasonal availability of mature gametes depends on local situations and populations, and on hatchery practices (i.e., seasonal water temperatures and photoperiods). Certain hatcheries have successfully selected and manipulated different populations of broodstock to provide gametes year round.

Broodstock are normally sorted to separate males from females and ripe individuals from sexually immature ones. It is straightforward to separate the sexes and determine ripe males, but selecting ripe females for stripping takes experience and practice. If female ripeness is not checked at frequent intervals, there is a high risk of acquiring infertile eggs. Maximum fertility of eggs is achieved within a three- to four-day period, between 4 to 8 days post-ovulation. For optimal fertilization success, eggs taken for toxicity tests should be stripped during this 4- to 8-day period following ovulation. Allowing the eggs to over-ripen affects survival adversely, not only at fertilization, but also at the eyed stage through to swim-up fry.

Careful handling of the fish while checking for ripeness is essential; they are easily damaged internally, and broken eggs result in infertility. External signs of ripeness include a soft, enlarged abdomen, swollen and red urogenital papilla protruding from the vent, and spontaneous flow of eggs from the vent. Extruded eggs can be checked for ripeness by clearing them and examining the position of the germinal vesicle and lipid droplets in the yolk. Proper handling of broodstock and checking ripeness requires experienced personnel.

Stripping can be carried out with one or two people, depending on their experience. Typically, one holds the fish while the other performs the stripping. Eggs can be removed from the females by various methods, depending on whether the female is to be killed or anaesthetized. Excess force during stripping of eggs should be avoided. A mature male can be stripped more than once. If he is to be stripped again, a period of one week should lapse between stripping sessions, otherwise milt quality might be compromised.

Handling of Gametes

The procedure detailed in this report requires the fertilization of eggs just before the start of the test. This necessitates the coordinated and timely procurement and handling of unfertilized eggs and milt. Although gametes can be obtained from sexually mature broodstock held at the laboratory, it is frequently easier and less costly to obtain them from a hatchery, and transport the milt and unfertilized eggs to the test facility (see Section 2.2). Provided that care is taken and conditions are optimal, both milt and unfertilized eggs can be transported and stored for a few days before fertilization. Minimizing the storage period to as brief as possible (ideally, <24 h) is desirable, though, to enhance the likelihood of good fertilization success.

Milt, if handled and stored properly, normally maintains 70 to 90% fertility for at least five days. The sperm in freshly collected milt remains immotile in the seminal fluid, due to the fluid's potassium content. Subsequent quality of sperm is affected during transportation and storage by temperature, depth of milt in container, sterility of the container, and humidity. Lower temperatures (ideally, 0 to 4°C) allow longer storage of sperm. Even if shipped and stored cold, however, more stored sperm are required to fertilize a batch of eggs than if there were no delay in fertilization. Keeping the depth of milt in the container at a minimum (<6 mm) is important to ensure that the sperm receive adequate oxygen. Flushing the milt with oxygen is also desirable. The use of moisture-saturated oxygen or air can significantly increase storage time, since it helps to prevent drying of the gametes.

Unfertilized eggs can be transported and stored in much the same way as milt. Eggs should be collected as soon after ovulation as possible, since a decreased storage ability occurs as eggs ripen. Eggs should be shipped and stored chilled (0 to 4°C), no more than four layers thick, in insulated containers designed to minimize breakage. Unfertilized eggs, if handled in this manner, should retain normal fertilization rates for about three days. To maximize fertilization, stored eggs should be fertilized with fresh milt.

Fertilization

Although fertilization can take place with water, this technique must be avoided since it triggers closure of the micropyle before the freshly fertilized eggs are exposed to test solutions. Accordingly, the dry method of fertilization must be used. Using this method, it is recommended that the eggs from four or more females^Footnote 47 (see Section 2.2) be spawned into a dry, clean bucket or plastic tray. The milt, from one or more vials containing motile sperm when activated (see Section 2.2), is then added. It is preferable to fertilize eggs with milt from more than one male, to improve fertilization success. However, sperm used to fertilize the eggs must be taken from only those vials (one to four, depending on sperm motility) for which sperm have been demonstrated to be active when mixed with fresh water or ovarian fluid (Section 2.2). Upon addition of milt, the gametes are gently mixed (e.g., by hand using clean surgical gloves; or using a goose-wing feather). A period of five minutes for mixing and fertilization is recommended (Fennell et al., 1998); although a period of up to 20 minutes for mixing and fertilization may be used (Birge et al., 1985). Fertilization should take place under low lighting intensity. Immediately after fertilization, groups of fertilized eggs should be transferred as quickly as possible to the test solutions. The transfer of all groups of freshly-fertilized embryos to the test solutions is normally achieved within 10 minutes or less per test (Yee et al., 1996), and must be completed within 30 minutes per test.

Various techniques have been used for transferring groups of freshly fertilized eggs to test solutions. Choice of technique is left to the discretion of the investigator, provided that it limits the pre-test exposure of newly fertilized eggs to water (for the purpose of washing off any unwanted debris or excess milt) to no more than 10 seconds, and enables all groups of eggs to be transferred quickly (i.e., as soon as possible, and within 30 minutes) to test solutions after fertilization (see Section 4.2). A proven and recommended technique (Canaria et al., 1996; Yee et al., 1996; Fennell et al., 1998) is to partially fill labelled weighing boats with each test solution, and then transfer groups of freshly fertilized eggs to the boats (one group per boat). A plastic scoop (e.g., for measuring coffee) or spoon is useful for this purpose, to enable the quick transfer of the approximate number of eggs required per replicate to each weighing boat. An initial count of eggs is made following each transfer. Once all groups of eggs have been distributed to the weighing boats, the number of eggs per boat is recounted and adjusted as required. Following an additional period of no more than 30 minutes (to ensure water hardening and minimize stress during that period; Fennell et al., 1998), the contents of each boat must then be transferred gently to its assigned test chamber.

Another recommended technique (Birge, 1996) is to transfer each group of freshly fertilized eggs from the fertilization chamber directly to the test chamber, using a suitably sized container (e.g., plastic scoop or graduated beaker with small holes drilled to enable draining or brief rinsing with water). To remove debris or excess milt, the fertilized eggs can be washed by dipping the scoop in control water which has been previously adjusted to the test temperature. Upon completion of all transfers, the number of eggs placed in each incubation unit can be counted and adjustments for numbers or appearance (e.g., size uniformity) of eggs made as necessary (see Section 4.2).

Incubation and Development of Embryos

Table D.1 provides guidance on the optimal and lethal temperatures for rainbow trout embryos. Water temperature is the major variable governing development of the embryos, and can be used to predict the time when the various stages of development are reached. Values vary between races of the same species. Table D.2 gives predicted incubation periods to achieve 50% hatch.

Embryos are especially sensitive to mechanical shock (physical agitation) at certain developmental stages. Embryos cannot be handled, stirred, poured, or transported without significant mortality during these sensitive stages. Sensitivity to mechanical shock has been found to occur at three stages of embryonic development, each successive stage being more sensitive. The first occurs 10 to 45 minutes after the immersion of embryos in water following fertilization. During this time, fusion of the male and female chromosomes takes place. The second stage occurs 2 to 72 hours after the embryos are immersed, at which time the cells are undergoing rapid division. The third and most sensitive stage occurs 4 to 14 days post-fertilization, when the embryo is undergoing rapid cellular differentiation. Sensitivity to mechanical shock decreases thereafter and is no longer detectable at and after the eyed stage is reached.

Since the embryonic developmental rate depends on temperature, the changes in sensitivity will vary for different incubation conditions. However, to minimize losses, any handling of embryos should be completed within 24 hours of immersing the embryos in the test solutions. Although the embryos are sensitive during this time, they are not overly so. Embryos should not be handled at all throughout the period from 24 hours post-fertilization until the eyed stage is reached.

Appendix E: Logarithmic Series of Concentrations Suitable for Toxicity Tests^Footnote 48