Guidelines SOP

E201: Euthanasia of Fish and Amphibians

SOP # / Title
E201 / Euthanasia of Fish and Amphibians
  1. Purpose

The SOP describes acceptable methods of euthanasia of fish and amphibians used in research, teaching, and testing.

  1. General Information/Responsibility

This SOP describes a two-step process of euthanasia as recommended by the Canadian Council on Animal Care and approved by the AREB. Principal investigators may reference this SOP when euthanasia is performedexactly as indicated in the SOP. Any deviation from the SOP must be identified in the Animal Use Protocol and the principal investigator must provide justification for the deviation from standard procedure.

Responsibility: Individuals performing euthanasia must be adequately trained by taking the UACC Practical Skills: Aquatics workshop and be trained by the experienced and trained individuals in the lab group or by the UACC staff.

  1. Equipment/Materials
  • Chemical euthanasia agent: tricaine methane sulfonate (MS-222), benzocaine hydrochloride, metomidate hydrochloride (aquacalm).
  • Physical/mechanical euthanasia tool (guillotine, pithing tool, hammer)
  • Appropriately sized container/bucket
  1. Procedure

NOTE: The procedure requires a two-step process where lethal levels of a central nervous system depressant is first employed to induce anaesthesia, followed by a physical method to cause brain death. In rare instances, a species may not be amenable to a physical method. In such cases prolonged immersion (i.e. 3-4 hours) of the animal in a suitable central nervous system depressant is acceptable.

  1. Step One – Induction of Anaesthesia
  1. Tricaine methane sulfonate (MS-222) Immersion Method
  2. Use pharmaceutical grade MS-222. (NOTE: pharmaceutical grade MS-222 is required, do not use non-pharmaceutical grade.)
  3. MS-222 is acidic and requires buffering to neutral pH before use. Buffer MS-222 with sodium bicarbonate to achieve a pH of 7.0-7.5. Dilute powder using tank water prior to introducing the animal to the container.
  4. For fish: Completely immerse fish in a tank/tub containing a buffered solution at a minimum concentration of 150 mg MS-222/L water (this is a guidance; see note below)*. Immersion may result in excitatory movement – cover container to avoid splash and to prevent the animal from leaving the container. *(Note: Different species have different tolerances to MS-222. Rainbow trout, for example, can be euthanized with only 150 mg/L, while some species require much higher concentrations. To develop a species-specific euthanasia procedure for each species, contact the UACC veterinarians or the UACC Senior Aquatics Advisor.).
  5. For amphibian: Completely immerse in a tank/tub containing a buffered solution at a minimum concentration of 2-3 g MS-222/L water. Immersion may result in excitatory movement – cover container to avoid splash and to prevent the animal from leaving the container.
  6. Allow a minimum of 10 minutes and watch for loss of equilibrium and finally a complete lack of opercular movement. Fish will not respond to external stimuli, such as touch. Once the fish is unresponsive to touch and opercular movement has ceased, apply a physical method of euthanasia to cause brain death as identified in section 4B below.
  1. Benzocaine hydrochloride Immersion Method
  2. Benzocaine HCl is acidic and requires buffering to neutral pH before use. Buffer with sodium bicarbonate to achieve a pH of 7.0-7.5.
  3. Immerse fish or amphibian completely into a tank/tub of buffered solution at a minimum concentration of 250 mg/L.
  4. Once unconscious as described in section 1.a.v. above, apply a physical method of euthanasia to cause brain death as identified in section 4B below.
  1. Step Two – Physical Method to Cause Brain Death
  1. Decapitation (fish or amphibian)
  2. Separate the head from the body swiftly using appropriate sharp equipment and follow with pithing via the open spinal canal (if animal is large enough).
  3. Cervical transection without decapitation (fish)
  4. For larger species of fish and for ease of subsequent dissection
  5. Swifly cut through the musculature at the base of the skull and continue through the spinal canal
  1. Pithing without decapitation (amphibians)
  2. Flex the neck and identify the foramen magnum.
  3. Insert a rigid metal rod into the cranium and pivot and rotate the rod in a manner that assures destruction of the brain tissue and proximal spinal cord.
  1. Crushing blow to head
  2. Use of a heavy blunt object (e.g. hammer) to deliver a crushing blow to the cranium is acceptable. Monitor fish for 10 minutes to observe for opercular movement.
  3. Exsanguination
  4. Complete blood collection under anaesthesia appropriate for the species is an acceptable physical method. Refer to SOPs specific for blood collection in fish and amphibia.
  1. Safety
  1. Wear appropriate protective clothing, gloves, and safety glasses when handling chemicals and animals exposed to chemicals.
  2. Safe practices with MS-222, aquacalm, and benzocaine-HCl:
  3. Use a fume hood, if possible, to prepare concentrated stock solutions by mixing an appropriate amount of chemical with water and stir with a utensil until the powder is fully dissolved.
  4. Dilute the stock solution further as required.
  5. Use fresh solutions but stock solutions can be stored for short periods of time (one hour maximum).
  1. Potential Complications and Troubleshooting

Some species can be rather tolerant to chemical anaesthetic agents. It is recommended that the appropriate concentration be determined from the literature before euthanizing a particular species for the first time. If a less common species is used and no appropriate anaesthetization/euthanization information is available, contact the UACC veterinarians or the UACC Senior Aquatics Advisor.

  1. References
  1. CCAC guidelines: Euthanasia of animals used in science (
  2. CCAC guidelines on: The care and use of fish in research, teaching, and testing (
  1. Revision History

Date Created:February 21, 2014 / Written by:Jane Alcorn
SOP Review and Revision History
Revision Number / Review/Revision Date / Reviewer
1 / May 15, 2018 / Kurtis Swekla/Jason Raine

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