STANDARD OPERATING PROCEDURE for FISH

USED IN TEACHING AND RESEARCH(updated October 13, 2011)

5.1 Intent

The intent of this Standard Operating Procedure (SOP) is to describe acceptable methods of transport, housing, care, and euthanasia of fish. This SOP is intended for personnel that use fish in research or teaching. This procedure is approved by the UWSP Institutional Animal Care and Use Committee (IACUC; October 15, 2007). All investigators using live fish in teaching or research must submit a protocol to the IACUC for approval prior to animal use. This SOP is intended to expedite the preparation of a protocol for review; it does not replace the investigator-prepared protocol. Reference to this SOP is sufficient to answer many questions on the IACUC protocol submission form. If fish use deviates from this SOP, the changes, additions, or omissions must be clearly outlined in the animal use protocol and must be submitted for approval to the IACUC prior to implementation. A protocol is required for each project where live fish will be used.

This SOP follows the Guidelines for the Use of Fishes in Research (American Fisheries Society, 2004) and the Report of the AVMA Panel on Euthanasia (AVMA 2007). Please see the last page for full references and contact information.

5.2 Requirements of personnel implementing this SOP

The principal investigator or lead instructor must ensure that all individuals responsible for euthanasia:

a. Receive appropriate training to perform the procedure.

b. Adhere to IACUC-approved protocols and institutional policies.

c. Are knowledgeable in species differences in metabolism, respiration, and tolerance to cerebral hypoxia.

All student workers must complete the UWSP, Department of Biology, or Northern Aquaculture Facility (NADF) laboratory safety training, read and sign the safety rule document and have it on file in the Biology Department Stockroom, Room 193 CNR or NADF office.

5.3 Safety

Personnel should use rubber gloves and safety glasses when working with noxious chemicals used for treating fish, cleaning, and disinfecting fish tanks and equipment (e.g. bleach, formalin, Sanaqua®). Lab coats, safety glasses, and gloves if deemed necessary, should be worn when performing water chemistry analysis and when working with chemical reagents. A facemask with appropriate filter or fume hood should be worn when working with noxious chemicals such as formalin and hydrogen peroxide. All MSDS forms are located in the Biology Stockroom, Room 193 CNR, or the water quality lab and facility manager’s office, at NADF, and should be reviewed prior to using any chemicals. Personnel should be aware of the location of the first aid kit, rubber gloves, safety glasses, facemasks, and lab coats.

5.3a Security & Visitation policy

All fish laboratories and facilities should have restricted locked access. Visitors are not normally allowed in the laboratory, unless with the permission of those individuals listed in section 5.5. Visitors must be supervised at all times. NADF has an additional SCADA security system to prevent intrusion by unauthorized visitors into the facility. Because fish are attuned to their environment, it is important that tranquility be maintained by minimizing physical disturbances.

5.4 Approved sources of animals

Approved sources of fish included licensed or registered commercial fish suppliers and tribal, state or federal hatcheries. Animals from approved commercial suppliers can be ordered by the purchasing agent for the CNR or facility manager for NADF.

Approved suppliers currently used by UWSP faculty and staff include:

  • Members of the Wisconsin Aquaculture Association:
  • Registered fish farms:
  • Licensed or permitted commercial fishing operations:
  • Carolina Life Science:
  • Nasco:

If fish are not be obtained from an approved source as listed above, the protocol must describe the procedure for obtaining the animals and for ensuring that the health, husbandry, and transport of the animals is in accordance with Public Health Service (PHS) policy.

Investigators capturing animals from the field require permits from the Wisconsin Department of Natural Resources (WI DNR), and from federal wildlife agencies for threatened or endangered species. Health assessment and certification may be required when purchasing fish or holding fish in laboratories or facilities. The Wisconsin Department of Agriculture, Trade, and Consumer Protection – Division of Animal Health should be contacted regarding fish health inspection prior to obtaining fish. Evidence of proper permitting may be required by the IACUC for protocol approval. Investigators should be familiar with fish capture techniques as outlined in Guidelines for the Use of Fishes in Research (American Fisheries Society, 2004). Copies of all relevant permits

5.5 Emergency

In the event of an animal health emergency, facility failure, or environmental disaster, immediately contact the UWSP faculty member in charge of the projects located in the fish laboratory or facility. If they are unavailable, please contact the next person on the contact list posted in each laboratory or facility.

On-campus, the contact list is generally:

  • Faculty investigator
  • Attending veterinarian
  • Ellen Jo Holguin, Biology stockroom manager: 346-3794
  • Jerry Kummer, CNR building manager: 346-4238
  • Campus security: 911

For NADF the contact list is generally:

  • Facility manager
  • Facility technician
  • Project investigator
  • Red Cliff Police Department: 715-779-3733

5.6 Transportation

Animals collected in the field or shipped from a commercial distributor should be confined and transported in a manner that does not expose them to temperature extremes, low dissolved oxygen, or overcrowding. Supplemental oxygen, aeration, salt, ice and “no foam” may be used while hauling fish to reduce stress. Caution must be employed when requesting shipment during the dangerously cold Wisconsin winters. The Biology Stockroom manager, the CNR purchasing agent, or the NADF facility manager can arrange for expedient and safe shipment and arrival of animals. Fish should be moved using nets that have been sterilized before use. NaCl may be added to the transport water at 0.5-1.0%, to reduce the effects of transport stress. The transportation container should be well insulated to minimize temperature changes during transport. Fish should be taken off food for 2-3 days prior to transport, so they will void their digestive tracts and not foul their shipping water. Depending on the species and length of time in transit, this fasting period may last from 1-5 days.

5.7 Fish Inventory

Fish may be housed on a short-term basis (< 12 hours) in teaching or research labs. Tanks should be constructed of non-porous materials that are easily cleaned and should be escape-proof. The appropriate size of the tank will vary by species but should generally be no smaller than 4 body-lengths long and 2 body-lengths wide with a height appropriate to allow normal locomotion. The tank arrangement should provide appropriate thermal, oxygen, lighting, and filtration conditions. Good water quality, food, and nutrition adequate to maintain good health should be provided.

For long-term, fish should be housed in laboratories and/or facilities with wall coverings, floor treatments, door thresholds, and to a lesser extent ceilings constructed of material impervious to water. Plumbing: Lead-based solders should be avoided, and adequate drainage or floor drains should be used. Lighting: Photoperiod is critical in maintaining normal physiological/environmental health in aquatic organisms. Fish laboratories/facilities should be equipped with natural or artificial lighting controlled by timers. Ventilation: All laboratories/facilities should provide a source of clean air and be equipped with compressed (low or medium pressure) air or liquid oxygen for proper tank aeration. Electrical: All electrical circuits should be ground fault (GFI) and of appropriate amperage. Appropriate backup systems should be in place for main water supply, and other critical components. Emergency power should be provided to critical life support systems and stand-pipes should be used on tanks where complete drainage is possible during a power outage.

Fish inventory sheets should include source of fish and date of arrival, species and sex (if identifiable), estimated age and weight, name of principal investigator, transfer history, number of fish in tank, daily records of husbandry (feeding schedules, water temperature, water quality parameters, photoperiod, unique experimental procedures, maintenance, and morbidity/mortality) for each tank and should be posted on a clip board in the vicinity of each fish tank or in a notebook in the fish holding room. If several tanks are included in the same study, all sheets should be posted near the tanks or be included in the same notebook. Short-term records should be summarized at least annually, or at the end of the study.

5.8 Husbandry

All personnel should have working knowledge of systems that provide clean, tempered water, filtration, lighting and aeration to fish tanks. No one except trained workers should alter or adjust these systems. Plans should be in place to ensure that fish cannot escape from the laboratory/facility, especially those not native to the watershed. Specific barriers should be in place to prevent the escape of fish and diseases. Risk management procedures and emergency management plans should be developed for each laboratory/facility and specific guidelines should be followed if non-indigenous, transgenic, or other genetically modified fishes are housed at each laboratory/facility. Effluents should be properly discharged into wastewater drains, treatment systems, or collected for proper disposal. Regulatory authority and determination of acceptable effluent contributions rests with the USEPA or and EPA-designated state or local authority. Discharge of wastes or pollutants entering water of the U.S. may require a National Pollution Discharge Elimination System (NPDES) permit.

5.8a Observation & monitoring

Each tank should be observed daily for condition and cleanliness. Log sheets must be completed daily. If system failure seems apparent, proceed as indicated in section 5.5.

Tanks should be monitored for:

  • Proper flow rate or filtration rate
  • Water source or treatment (e.g. dechlorination)
  • Temperature
  • Dissolved oxygen levels/Gas supersaturation (TDG)
  • Water pH
  • Nitrogen levels (ammonia and nitrite)
  • Photoperiod
  • Mortalities
  • Stocking/tank density

5.8b Feeding

All fish should generally be fed daily to satiation (except when part of research protocol) a diet of commercially prepared fish feed or a similar diet. Feed should be stored in dedicated areas that are dark or covered, temperature and humidity controlled, and pest-free to insure nutritional quality. Feed used for daily feeding should be kept in sealed-top containers to protect it from humidity and light, and frequently replaced with feed from storage. Recommendation from the feed manufacturer should be followed regarding shelf life and storage.

5.8c Mortality & removal

Mortalities should be removed daily from each tank, recorded with the daily log, placed in sealable bags and frozen. The Biology stockroom manager or the NADF facility manager should be notified and arrangements made for pick up by the contracted cremation service or refuse disposal. Tanks showing mortalities >10% over a 3-day period should be suspected of having disease or environmental problems. Recheck all monitored parameters (section 5.8a) and, if all environmental parameters are good, contact the principal investigator and/or facility manager, then proceed to section 5.8f Disease control.

5.8d Tank cleaning

Tanks containing actively feeding, rapidly growing fish should be cleaned regularly. Tank cleaning should consist of the removal of feces, uneaten feed, and algal and bacterial growth. Care should be taken when cleaning tanks to minimize or avoid excessive disturbance and stresses to the fish.

5.8e Room and facility cleaning

Aquatic facilities are complex systems that must be well designed to minimize stress to the fishes, promote efficient operation of the facility, and ensure a safe working environment for personnel. The most basic principle in the design of fish holding facilities is that a healthy fish population is dependent on a stress-free environment. Each fish laboratory or facility should be cleaned as frequently as weekly, or as infrequently as quarterly, depending on use and the status of the fish. Cleaning should not be done too frequently, because bright lights and activity disturb and stress the fish. Regular cleaning should include wet-brushing of the benches, outside of the tanks & floors, and hosing the debris down floor drains. If fish are present in the room, no chemical should be used. When all fish are removed from a room, and before fish are re-introduced, the room should be cleaned using a 1% bleach solution or appropriate approved disinfectant.

5.8f Disease control

All nets and other equipment that are used between tanks should be disinfected with a solution of household bleach after each use (1 cup of bleach {5% sodium hypochlorite} to 15 gallons water) or other approved disinfectant. Automatic feeders should be cleaned as needed, and disinfected with bleach solution between uses. All personnel should disinfect boots/shoes upon entering and leaving the laboratory and/or facility. Only a few theraputants for fish diseases are legally available: reference current Food and Drug Administration policy ( Disease outbreaks at university laboratories or facilities are rare. External parasites can be detected by visual/microscopic inspection of fish. Fish infected with external parasites should be treated with a 2% NaCl dip for 30 minutes. For any other suspected diseases, contact the attending veterinarian. Zoonotic diseases associated with fish are rare and infrequent, but can occur. Table 1 lists select zoonotic agents transmissible from fish to humans. If a zoonotic disease is suspected or detected, contact the facility manger, principal investigator and the attending veterinarian. The major exposure routes for zoonotic diseases in fish to humans include ingestion and introduction of organisms through open wounds or abrasions. More specifically, ingestion includes consumption of raw or under-cooked infected fish tissue, ingestion of fish tissue contaminated with feces from infected fish, and ingestion of water harboring infectious organisms. Dermal exposure includes introduction of infectious agents into open wounds or abrasions through handling infected fish or infected water.

5.8g Quarantine

When new fish are brought into fish laboratories or facilities, they should be kept in solitary, isolated tanks for at least 2 weeks. Fish should be acclimated to the temperature, salinity, pH and hardness of the water in the holding facility. Quarantine areas should be managed according to rigorous infectious agent control practices.

5.9 Euthanasia

Acceptable methods of euthanasia of fish include submersion in tricaine methane sulfonate, benzocaine hydrocloride, or liquid nitrogen, application of cold-shock or electrical-shock, followed by physical methods of pithing, spinal cord dislocation, decapitation and blunt trauma. Fishes destined for food and/or human consumption must not be exposed to chemicals, or must be held for the required number of days post-chemical treatment, and alternate euthanasia methods should be employed.

Unacceptable methods of euthanasia of fishes include oxygen deprivation, administration of clove oil, or use of physical methods (decapitation and/or pithing) without anesthesia.

5.9a Tricaine methane sulfonate

Immerse fish in a solution of tricaine methanesulfonate (TMS, MS-222, Finquel®, or Tricaine-S®) at 500 mgl-1 buffered with sodium bicarbonate to pH 7.0 – 7.5. Verify that an animal is dead before disposing of the carcass by monitoring for respiratory or opercular movement. Time to effect may vary, with death occurring in up to 30 minutes. Follow immersion with a physical method of euthanasia to ensure death. Examples of physical methods include pithing and decapitation.

5.9b Benzocaine hydrochloride

Immerse fish in a solution of benzocaine hydrochloride solution at 250 mgl-1. Verify that an animal is dead by monitoring for respiratory or opercular movement. Time to effect may vary, with death occurring in up to 30 minutes. Follow immersion with a physical method of euthanasia to ensure death. Examples of physical methods include pithing and decapitation.

5.9c Liquid nitrogen

Submersion in liquid nitrogen should only be performed on small fish (< 10 cm). Anesthesia or sedation must be applied prior to the use of liquid nitrogen unless scientifically justified by user and approved by the IACUC.

5.9d Cold-shock or electrical-shock

Both cold and electrical shock should be applied at a level that quickly euthanizes the fish. The use of hypothermia/freezing and electrocution are less preferable means and should be used in combination with anesthesia or sedation because of concerns about the induction of pain during ice crystal formation and spinal fractures, respectively.

5.9e Pithing, spinal cord dislocation, decapitation, or blunt trauma

Anesthesia or sedation should be applied prior to the use of physical techniques unless scientifically justified by user or if the fish are designated as food fish (i.e. for human consumption).Use sharp and/or suitable equipment of appropriate size for the species to be euthanized to ensure that the head is quickly separated from the body rapidly and completely or death is immediate. Ensure that both the brain and the proximal end of the spinal cord are destroyed.

5.10 Disposal of carcasses

Animals euthanized by injection, immersion, or application of drugs must be disposed of in a manner that they may not be consumed by humans or other animals, or contaminate water sources.

5.11 Resources and references

Contacts / Title / Phone / Room / Responsibility
Emily Starke / Manager of animal care facility / x 4823 / 222TNR / Live animals in TNR
Tammy Naczek / CNR purchasing agent / x 4685 / 194 TNR / ordering fish
Ellen Jo Holguin / Biology stockroom manager / x 3794 / 193 TNR / ordering chemicals, chemical safety & disposal
Chris Hartleb / Professor: Fisheries / x 3228 / 442 TNR / advising re: fish care
Greg Fischer / Facility Manager / 779-3461 / NADF / advising re: Northern Aquaculture Demonstration Facility (NADF)

American Veterinary Medical Association (AVMA). 2007. AVMA Guidelines on Euthanasia.

Fish Related Institutional Animal Care and Use Committees (IACUCs) Web Resources. National Agriculture Library, USDA.

This website contains links to many Standard Operating Procedures, protocols, and guidelines for the use of fish in research. Many of these documents could be easily modified to SOP for UWSP faculty and investigators.