UWSP IACUC SOP #5
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.
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Approved suppliers currently used by UWSP faculty and staff include:

Members of the Wisconsin Aquaculture Association: www.wisconsinaquaculture.com

Registered fish farms: http://datcp.wi.gov/Farms/Fish_Farms/index.aspx

Licensed or permitted commercial fishing operations: http://dnr.wi.gov/org/caer/cs/licenses.htm

Carolina Life Science: www.carolina.com

Nasco: www.enasco.com
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
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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 bodylengths 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: Leadbased 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. Shortterm 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.
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Tanks should be monitored for:
o Proper flow rate or filtration rate
o Water source or treatment (e.g. dechlorination)
o Temperature
o Dissolved oxygen levels/Gas supersaturation (TDG)
o Water pH
o Nitrogen levels (ammonia and nitrite)
o Photoperiod
o Mortalities
o 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 reintroduced, 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 (http://www.fda.gov/AnimalVeterinary/DevelopmentApprovalProcess/Aquaculture/ucm132954.htm).
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
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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 postchemical 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.
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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
Room
Responsibility
Manager of animal care facility
Phon
e
x 4823
222 TNR
Live animals in TNR
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
Greg Fischer
Professor: Fisheries
x 3228
442 TNR
Facility Manager
7793461
NADF
advising re: fish care
advising re: Northern Aquaculture
Demonstration Facility (NADF)
Contacts
Title
Emily Starke
Tammy Naczek
American Veterinary Medical Association (AVMA). 2007. AVMA Guidelines on Euthanasia.
www.avma.org/issues/Animal_Welfare/euthanasia.pdf
Fish Related Institutional Animal Care and Use Committees (IACUCs) Web Resources. National
Agriculture Library, USDA. www.nal.usda.gov/awic/pubs/Fishwelfare/iacuc.htm
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.
Public Health Service Policy on Humane Care and Use of Laboratory Animals (2002). Office of Laboratory
Animal Welfare (OLAW), National Institutes of Health (NIH).
http://grants.nih.gov/grants/olaw/references/phspol.htm
SOP for the Sampling of Fish in Wadeable Streams through the use of Electofishing (2002): electrofishing in
streams. Environmental Protection Agency. www.epa.gov/region1/lab/ecology/efishing.html and
http://www.epa.gov/region1/lab/reportsdocuments/wadeable/methods/ElectroFishing.pdf
Use of Fishes in Research Committee of the American Fisheries Society. 2004. Guidelines for the Use of
Fishes in Research. www.asih.org/files/fish%20guidelines.doc
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________________________________________________________________________________________
Table 1. Zoonotic diseases of fish
Pathogen
Bacteria
Streptococcus
Staphylococcus
Clostridium
Erysipelothrix
Mycobacterium
Nocardia
Vibrio
Plesiomonas shigelloides
Aeromonas
Pseudomonas
Escherichia
Salmonella
Klebsiella
Edwardsiella
Leptospirosis
Parasites
Anasakiasis
Eustrongyloides
Cestodes
Trematodes
Protozoa
Viruses
Calicivirus
Fungi
Candida
Ingestion of
fish tissue
Ingestion of
infected water
Dermal contact
w/infected fish
Dermal contact
w/infected water
*
*
+
+
*
*
+
+
+
+
*
+
*
+
+
*
+
+
+
?
+
?
+
+
+
+
*
*
+
+
?
+
?
*
*
+ reported cases in humans
* no known cases in humans but the potential risk exists
? exposure route of reported human disease unknown
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