UW Policy for the Euthanasia of Fish Species - 2008

Background: Small fish species (2-6cm in length) typically utilized in laboratory research include zebrafish, medaka, fathead minnow, goldfish, swordtail, and platyfish among others. Large fish species (>6cm in length) typically used include salmon, trout, tilapia, catfish, hybrid-striped bass, bass, bluegill, sturgeon and others.

Policy: Chemical agents such as tricaine methanesulfonate (MS-222 or TMS) or benzocaine hydrochloride (ethyl aminobenzoate), should be used as the method of choice for the euthanasia all fish species. The dosage required for euthanasia with either of these compounds is higher than the anesthetic dose, but varies greatly with species, size, and water temperature. In addition, a longer time of exposure to the chemical agent (relative to anesthesia) is required to ensure that death occurs. Fish should be removed only after ten minutes have passed since their last observed opercular movements (respiration) have occurred.

A. Primary euthanasia methods for fish (methods that can be used alone) include:

1. MS-222/TMS- dosage: 50 to 250 mg/L
   [buffered to a neutral pH with sodium bicarbonate (NaHCO₃)]
   Note: Fish euthanized with MS-222 cannot be used for human consumption.
   
   
2. Benzocaine-dosage: 50 to 500 mg/L
   [buffered to a neutral pH with sodium bicarbonate (NaHCO₃)]
   Note: Fish euthanized with Benzocaine cannot be used for human consumption.
   
   
3. Exposure to a solution saturated with carbon dioxide (CO₂):
   When possible, carbon dioxide should be used from a compressed gas cylinder source. However, the AVMA Guidelines on Euthanasia (2) does not address the issue of the source/production of carbon dioxide as it relates to aquatic animal species. The use of chemical methods for the production of carbon dioxide, such as a saturated solution of sodium bicarbonate or Alka-Seltzer ®, has been proven quite effective with fish as noted in the literature. When dosed adequately sodium bicarbonate renders rapid loss of consciousness and death (3,5). As a result, the use of carbon dioxide liberated by chemical means will also be acceptable for the euthanasia of fish when compressed carbon dioxide gas cylinders cannot be used. Individuals who have demonstrated competency with this technique to qualified personnel (i.e. aquatic animal veterinarian or his/her designate) should only perform the euthanasia of fish by carbon dioxide.
   
   Sodium bicarbonate dosage: (30g/L or 120g/gallon)
   
   Alka-Seltzer ® dosage: (1 tablet/ 20L or 2 tablets/ 10gallons)
   Note: Fish euthanized with sodium bicarbonate or Alka-Seltzer® should not be used for human consumption.
   
   Protocol: Fish should be placed into a container with an appropriate volume of water that will provide free movement of fish to be euthanized. Water should then be saturated with carbon dioxide from either a compressed gas cylinder (via the use of an air stone with a moderate flow rate for ~5-10 minutes) or by dissolving enough sodium bicarbonate or Alka-Seltzer® into the water (via the dosages previously provided). Fish should be removed only after ten minutes have passed since their last observed opercular movements (respiration) have occurred.
   
   
4. Chilling of Tropical species- The AVMA Guidelines on Euthanasia (2) concludes that cooling to 4°C will decrease metabolism and facilitate handling of poikilothermic species. This method by itself should only be used to facilitate handling, and it does not constitute an acceptable method of euthanasia since there is no evidence that it reduces pain or is clinically efficacious. However, the guidelines do not discuss chilling to 4°C as a method for euthanasia for tropical species. Because tropical fish species, (i.e. zebrafish, medaka, and platyfish), have minimal to no physiologic adaptation mechanism for adjusting to cold (4°C) water, cooling to 4°C should be considered an acceptable method of euthanasia since the rapid decrease in temperature from 26°C (or higher) to 4°C induces rapid loss of consciousness and is lethal to these species. Fish euthanized by this method should not come in direct contact with the ice, because this may cause thermal burns and induce pain, but rather the ice should be added to a lowered amount of water for a contact time of 20 minutes.
   
   Protocol: In a container of an appropriate size for the number and size of fish to be euthanized, place a small volume of water that will still allow the fish to freely move about. To this volume of water add four times the volume of ice such that there is a 1:4 ratio of water to ice. Allow the fish to remain in ice-water bath for 20 minutes prior to removing. Afterwards, transfer the fish to the freezer for storage prior to disposal.

B. Secondary euthanasia methods for fish (methods that cannot be used alone and must be followed by an adjunct method to ensure death):

1. Sharp blow to the head- This method must be followed by exsanguination (via severing the large branchial vessels or large caudal tail vessels) or decapitation (via the use of a sharp blade).
   
   
2. Alternating or Direct electrical current- This method must be followed by exsanguination (via severing the large branchial vessels or large caudal tail vessels) or decapitation (via the use of a sharp blade). [Note: the use of electrical current (electrofishing) must follow the protocol/guidelines provided by the Marine Resources Division of the Washington State Department of Fish and Wildlife.]
   
   
3. Chilling of Non-tropical species in salt water - Since non-tropical species have adaptive mechanisms to handle decreased temperature ranges, the use of chilling to 4°C (without direct contact of the fish to the ice) must be followed by decapitation. However, if non-tropical species are exposed to chilled salt water (-10°C) or fresh water at a temperature significantly less than 4°C (i.e. 0°C) decapitation is not necessary 10 minutes after the observed cessation of opercular movements.

Euthanasia Policy References:

1. Canadian Council on Animal Care, Guide Vol. 1 (2^nd Ed.). 1993. Chapter XII – Euthanasia.
2. AVMA Guidelines on Euthanasia, June 2007.
   http://www.avma.org/issues/animal_welfare/euthanasia.pdf.
3. Stoskopf, M.K. 1993. Fish Medicine. W.B. Saunders Co. Philadelphia.
4. The Guide for the Care and Use of Laboratory Animals. 1996. National Academy Press, Washington, D.C.
5. Noga, E.J. 1996. Fish Diseases: Diagnosis and Treatment. Mosby-YearBook, Inc, St. Louis.
6. Casebolt, D.B., D.J. Speare, and B.S. Horney. 1998 Care and Use of Fish as Laboratory Animals: Current State of Knowledge. Lab Animal Science 48(2): 124-136.
7. Burns R, McMahan B. Euthanasia methods for ectothermic vertebrates. In: Bonagura JD, ed.
   Continuing veterinary therapy XII. Philadelphia: WB Saunders Co. 1995:1379-1381.
8. Guidelines for the Use of Fishes in Research. 2004. American Fisheries Society
   http://www.fisheries.org/afs/docs/policy_guidelines2004.pdf
9. Guidelines for Use of Fishes in Field Research. In: Fisheries, Vol. 13, No. 2, p. 16-23, 1988 or http://199.245.200.110/pubs/fishguide.html
10. Electrofishing Boat Operation and Maintenance Policy. 1996. Marine Resources Division of the Washington State Department of Fish and Wildlife.
11. Electrofishing Guidelines for Stream Typing. 1996. Marine Resources Division of the Washington State Department of Fish and Wildlife.
12. USFW 241 FW 6, Electrofishing Guidelines. 1992. http://policy.fws.gov/241fw6.html
13. Kolz, A.L., A. Temple, and D. Lam. 1998. Principles and Techniques of Electrofishing. U.S. Fish and Wildlife Service National Conservation Training Session.