CCAC Fish Guidelines English - AAALAC

Canadian Council on Animal Care Guidelines on: The Care and Use of Fish in Research, Teaching and Testing *This reference was adopted by the Council on Accreditation with the following clarification and exceptions:

1. The Three Primary Standards and the AVMA Guidelines for the Euthanasia of Animals: 2013 Edition will prevail.

2. The document contains a number of guidance based on regional regulations which may not be applied globally.

The reference should be used as guidance. Any mandatory aspects will be considered by Council in conjunction with a performance based assessment.

Reference Resources

Caveats from AAALAC’s Council on Accreditation regarding this resource:

This Reference Resource begins on the next page....

Canadian Council on Animal Care

guidelines on:

the care and use offish in research,

teaching andtesting

This document, the CCAC guidelines on: the care and use of fish in research, teaching and testing, has beendeveloped by the ad hoc subcommittee on fish of the Canadian Council on Animal Care (CCAC)Guidelines Committee.

Mr John Batt, Dalhousie UniversityDr Kristina Bennett-Steward, BionicheMr Cyr Couturier, Memorial UniversityDr Larry Hammell, University of Prince Edward IslandDr Chris Harvey-Clark, University of British Columbia (Chair)Mr Henrik Kreiberg, Fisheries and Oceans CanadaDr George Iwama, Acadia UniversityDr Santosh Lall, National Research CouncilDr Matt Litvak, University of New Brunswick at St JohnDr Don Rainnie, University of Prince Edward IslandDr Don Stevens, University of GuelphDr Jim Wright, University of CalgaryDr Gilly Griffin, Canadian Council on Animal Care

In addition, the CCAC is grateful to former members of CCAC Council: Ms Susan Waddy, Fisheriesand Oceans Canada; Dr Jack Miller, University of Western Ontario; and Dr Choong Foong, DalhousieUniversity; and to Dr David Noakes, University of Guelph who provided considerable assistance inpreliminary phases of this project. CCAC thanks the many individuals, organizations and associationsthat provided comments on earlier drafts of this guidelines document. In particular, thanks are extend-ed to representatives of Fisheries and Oceans Canada, Environment Canada, the CanadianAquaculture Institute, the Canadian Food Inspection Agency and the Canadian Society of Zoologists.

© Canadian Council on Animal Care, 2005

ISBN: 0–919087–43–4

Canadian Council on Animal Care1510–130 Albert StreetOttawa ON CANADA

K1P 5G4

http://www.ccac.ca

CCAC guidelines on: the care and use of fish in research, teaching and testing, 2005

TABLE OF CONTENTS

A. PREFACE . . . . . . . . . . . . . . . . . . . . .1

SUMMARY OF THE GUIDELINESLISTED IN THIS DOCUMENT . . . . . . . .3

B. INTRODUCTION . . . . . . . . . . . . . .13

1. Definition of Fish . . . . . . . . . . . . . . . . . . .13

2. Rationale for Guidelines on theCare and Use of Fish . . . . . . . . . . . . . . . .13

3. Ethical Overview . . . . . . . . . . . . . . . . . . .14

3.1 Principles of the Three Rs . . . . . . . .14

4. Responsibilities . . . . . . . . . . . . . . . . . . . . .15

4.1 Responsibilities of investigators . .15

4.2 Responsibilities of the animalcare committee . . . . . . . . . . . . . . . . .16

4.3 Role of the veterinarian . . . . . . . . .17

5. Government Regulations andPolicies on the Use of Fish . . . . . . . . . . . .17

5.1 International . . . . . . . . . . . . . . . . . . .17

5.2 Federal . . . . . . . . . . . . . . . . . . . . . . . .18

5.3 First Nations . . . . . . . . . . . . . . . . . . .20

5.4 Provincial/territorial . . . . . . . . . . . .20

5.5 Municipal . . . . . . . . . . . . . . . . . . . . .20

C. AQUATIC FACILITIES . . . . . . . . . .21

1. Water Supply . . . . . . . . . . . . . . . . . . . . . . .21

2. Water Quality . . . . . . . . . . . . . . . . . . . . . .21

3. Engineering and Design . . . . . . . . . . . . .22

3.1 Structural materials . . . . . . . . . . . . .23

3.2 Room ventilation and airflowin aquatic areas . . . . . . . . . . . . . . . . .24

3.3 Mechanical and electricalrequirements . . . . . . . . . . . . . . . . . . .25

3.4 Lighting . . . . . . . . . . . . . . . . . . . . . . .25

3.5 Redundancy in aquatic lifesupport systems . . . . . . . . . . . . . . . .26

4. Types of Systems . . . . . . . . . . . . . . . . . . . .26

4.1 Flow-through systems . . . . . . . . . . .27

4.2 Recirculation systems . . . . . . . . . . .27

4.3 Static systems . . . . . . . . . . . . . . . . . .27

4.4 Mesocosms . . . . . . . . . . . . . . . . . . . .28

5. Fish Housing . . . . . . . . . . . . . . . . . . . . . . .28

5.1 Fish well-being . . . . . . . . . . . . . . . . .28

5.2 Tank/enclosure design . . . . . . . . . .28

D. FACILITY MANAGEMENT,OPERATION ANDMAINTENANCE . . . . . . . . . . . . . . .31

1. Security and Access . . . . . . . . . . . . . . . . .31

2. General Maintenance of the Facility . . .31

3. Environmental Monitoringand Control . . . . . . . . . . . . . . . . . . . . . . . .32

3.1 Management of water quality . . . .33

3.2 Temperature . . . . . . . . . . . . . . . . . . .33

3.3 Oxygen . . . . . . . . . . . . . . . . . . . . . . .34

3.4 Supersaturation . . . . . . . . . . . . . . . .34

3.5 pH . . . . . . . . . . . . . . . . . . . . . . . . . . . .35

3.6 Nitrogen compounds . . . . . . . . . . .35

3.7 Carbon dioxide . . . . . . . . . . . . . . . . .36

3.8 Salinity . . . . . . . . . . . . . . . . . . . . . . . .36

3.9 Toxic agents . . . . . . . . . . . . . . . . . . .37

E. CAPTURE, ACQUISITION,TRANSPORTATION ANDQUARANTINE . . . . . . . . . . . . . . . .38

1. Capture of Wild Stock . . . . . . . . . . . . . . .38

2. Killed Specimens . . . . . . . . . . . . . . . . . . . .38

3. Piscicidal Compounds . . . . . . . . . . . . . . .38

4. Acquisition of Hatchery Fish . . . . . . . . .39

5. Transportation . . . . . . . . . . . . . . . . . . . . . .39

6. Quarantine and Acclimation . . . . . . . . . .40

6.1 Quarantine . . . . . . . . . . . . . . . . . . . .40

6.2 Acclimation . . . . . . . . . . . . . . . . . . . .41

F. HUSBANDRY . . . . . . . . . . . . . . . . .42

1. Record-keeping and Documentation . . .42

1.1 Standard Operating Procedures . .42

1.2 General checklists . . . . . . . . . . . . . .42

1.3 Assessment of fish well-being . . . .42

2. Density and Carrying Capacity . . . . . . .42

3. Food, Feeding and Nutrition . . . . . . . . .43

3.1 Nutrition . . . . . . . . . . . . . . . . . . . . . .43

3.2 Food and feeding . . . . . . . . . . . . . . .43

3.3 Feed quality and storage . . . . . . . .43

3.4 Larval weaning . . . . . . . . . . . . . . . .45

3.5 Use of medicated feeds . . . . . . . . . .45

4. Broodstock and Breeding . . . . . . . . . . . .46

4.1 Induction of spawning . . . . . . . . . .46

G. HEALTH AND DISEASECONTROL . . . . . . . . . . . . . . . . . . .47

1. Fish Health Program . . . . . . . . . . . . . . . .47

1.1 Disease prevention . . . . . . . . . . . . .47

1.2 Disease diagnosis andidentification of pathogens . . . . . . .47

1.3 Injuries and other disorders . . . . . .48

H. EXPERIMENTAL PROCEDURES . .50

1. Handling and Restraint . . . . . . . . . . . . . .50

1.1 Restraint of dangerous species . . .51

2. Restricted Environments . . . . . . . . . . . . .51

3. Surgery . . . . . . . . . . . . . . . . . . . . . . . . . . . .51

3.1 Surgical preparation andskin disinfection . . . . . . . . . . . . . . . .52

3.2 Water quality during surgery . . . .53

3.3 Anesthesia . . . . . . . . . . . . . . . . . . . . .53

3.4 Surgical equipment . . . . . . . . . . . . .54

3.5 Incisions . . . . . . . . . . . . . . . . . . . . . . .54

3.6 Suture materials and techniques . .54

3.7 Pathophysiology of surgeryand wound healing in fishes . . . . .55

3.8 Postoperative care . . . . . . . . . . . . . .55

4. Administration of Compoundsand Devices by Various Routes . . . . . . .56

4.1 Branchial diffusion("inhalation") . . . . . . . . . . . . . . . . . . .56

4.2 Oral . . . . . . . . . . . . . . . . . . . . . . . . . .56

4.3 Injection . . . . . . . . . . . . . . . . . . . . . . .57

4.4 Implants, windowsand bioreactors . . . . . . . . . . . . . . . . .57

5. Tagging and Marking . . . . . . . . . . . . . . . .57

5.1 Tissue marking . . . . . . . . . . . . . . . . .58

5.2 Tagging . . . . . . . . . . . . . . . . . . . . . . .58

6. Collection of Body Fluids . . . . . . . . . . . .58

7. Use of Infectious Disease Agents,Tumorigenic or Mutagenic Agents,and Toxic and Noxious Compounds . . .59

8. Endpoints and Criteria for EarlyEuthanasia . . . . . . . . . . . . . . . . . . . . . . . . .59

8.1 Recognition of "pain", "distress"and "stress" . . . . . . . . . . . . . . . . . . . .59

8.2 Choosing an appropriateendpoint . . . . . . . . . . . . . . . . . . . . . .60

9. Monitoring . . . . . . . . . . . . . . . . . . . . . . . . .62

10. Negative Reinforcement Modalities . . .62

11. Exercise to Exhaustion . . . . . . . . . . . . . . .62

12. Environmental Extremes . . . . . . . . . . . . .62

13. Genetically Modified Fish . . . . . . . . . . . .62

I. EUTHANASIA . . . . . . . . . . . . . . . .64

J. DISPOSITION OF FISHAFTER STUDY . . . . . . . . . . . . . . . .65

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1. Consumption of Fish . . . . . . . . . . . . . . . .652. Release of Fish to Wild . . . . . . . . . . . . . . .653. Fish as Pets . . . . . . . . . . . . . . . . . . . . . . . . .654. Transfer of Fish Between Facilities . . . . .655. Disposal of Dead Fish . . . . . . . . . . . . . . .65

K. REFERENCES . . . . . . . . . . . . . . . .66

L. GLOSSARY . . . . . . . . . . . . . . . . . .73

M. ABBREVIATIONS . . . . . . . . . . . . .75

APPENDIX ARELEVANT GUIDELINESAND ORGANIZATIONS . . . . . . . . .76

APPENDIX BZOONOTIC DISEASE-TRANSMISSION OF FISHDISEASES TO MAN . . . . . . . . . . . .77

APPENDIX CGUIDELINES FOR CONTAINMENTFACILITIES (FOR PATHOGENSTUDIES) . . . . . . . . . . . . . . . . . . . .79

APPENDIX DWATER QUALITY CRITERIA FOROPTIMUM FISH HEALTH – FORCOLDWATER, WARMWATER ANDMARINE SPECIES OF FISH . . . . .84

iv

The Canadian Council on Animal Care (CCAC)is the national peer review agency responsiblefor setting and maintaining standards for thecare and use of animals used in research, teach-ing and testing throughout Canada. In additionto the Guide to the Care and Use of ExperimentalAnimals, vol. 1, 2nd ed., 1993 and vol. 2, 1984,which provide the general principles for thecare and use of animals, the CCAC also publish-es detailed guidelines on issues of current andemerging concerns. The CCAC guidelines on: thecare and use of fish in research, teaching and testingis the seventh of this series. This documentsupersedes Chapter I - Fish, Guide to the Care andUse of Experimental Animals, vol. 2 (CCAC,1984).

These guidelines aim to provide information forinvestigators, animal care committees, facilitymanagers and animal care staff that will assistin improving both the care given to fishes andthe manner in which experimental proceduresare carried out.

The present document has drawn substantiallyfrom the work of organizations listed inAppendix A. Their contributions to the devel-opment of these guidelines are gratefullyacknowledged.

The guidelines have been developed by theCCAC subcommittee on fish and werereviewed by a total of 69 experts. A preliminaryfirst draft was agreed on by the subcommitteeand circulated to experts in June 2002 (includingrepresentatives of the organizations listed inAppendix A), and a second draft was circulatedfor widespread comment in June 2003. A finalreview was carried out in August 2004 involv-ing all individuals who had previously provid-ed significant input to the development process.The development of these guidelines alsoinvolved consultation with the CanadianAssociation for Laboratory Animal Science(CALAS) and the Canadian Society ofZoologists (CSZ) through workshops held atannual meetings in Québec City (June 2003),Acadia University (May 2004), and Hamilton(June 2004). Consultations were also held at theAquaculture Association of Canada andAquaNet annual meetings in Québec City(October 2004), and at the CCAC Workshop onthe Fish Guidelines in Vancouver (April 2005).

The guidelines have been organized in a formatthat should facilitate easy access to relevant sec-tions. Early sections provide an ethicaloverview relevant to the use of fishes inresearch, teaching and testing. This is followed

the care and use offish in research,

teaching andtesting

A. PREFACE

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by a brief overview of regulations and responsi-bilities relevant to the care and use of fishes inscience in Canada. The remainder of the docu-ment provides guidelines to assist in caring forfishes in laboratory facilities, followed byguidelines to help in the development andreview of experimental protocols. An overviewof the CCAC guidelines on: the care and use of fishin research, teaching and testing is providedthrough a summary of the guidelines listed in

this document prior to the beginning of themain text.

The refinement of animal care and use guide-lines is a continuous process. These guidelinesare intended to provide assistance in the imple-mentation of best practices, and should not beviewed as regulations. Where regulatoryrequirements are involved or where it isabsolutely imperative to adhere to a particularguideline, the term must has been used.

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B. INTRODUCTION

3. Ethical Overview

Guideline 1:

Fishes used in research, teaching and testingmust be treated with the respect accorded toother vertebrate species.

p. 14

4. Responsibilities

Guideline 2:

Projects involving the use of fishes for research,teaching or testing should be described within aprotocol. Protocols should be approved by ananimal care committee prior to the commence-ment of the work.

Section 4.1 Responsibilities of investigators, p. 15

Guideline 3:

Before working with fishes, investigators, techni-cal staff and post-graduate students must be prop-erly trained and have their competency evaluated.


Guideline 4:

Investigators are responsible for, and must com-ply with, occupational health and safety regula-tions regarding the protection of personnel fromknown or suspected physical and biologicalhazards.


Guideline 5:

Investigators should be aware of the potentialrisks associated with zoonotic agents present infishes.


5. Government Regulations and Policies onthe Use of Fish

Guideline 6:

Anyone acquiring or transporting fishes, or con-ducting research on fishes, must be familiar with,

and comply with, relevant international, federaland provincial/territorial legislation and policiesgoverning the capture of fishes and/or theirtransfer from one water body or jurisdiction toanother.

p. 17

C. AQUATIC FACILITIES

Guideline 7:

Aquatic facilities are complex systems that must be well designed to minimize stress to thefishes, promote efficient operation of the facility,and ensure a safe working environment for personnel.

p. 21

2. Water Quality

Guideline 8:

If fresh or sea water is drawn from an open bodyof water or a municipal source, it should be test-ed for, and treated to remove, contaminants andpathogens.

p. 21

3. Engineering and Design

Guideline 9:

In designing and constructing aquatic facilities,assistance should be sought from people withexperience in this field.

p. 22

Guideline 10:

Construction materials for facilities housing fish-es should be selected carefully for resistance tocorrosion and water damage.

Section 3.1 Structural materials, p. 23

Guideline 11:

Materials in aquatic facilities which are potential-ly toxic to fishes should be reduced to the mini-

SUMMARY OF THEGUIDELINES LISTED IN THIS DOCUMENT

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mum. Any toxic material should be listed, andthe list must be available to staff.

Section 3.1 Structural materials, p. 24

Guideline 12:

Air handling systems should be engineered toensure that aquatic areas are well ventilated andhumidity is controlled, and to ensure that aerosoltransfer between tanks and through the facilitityis minimized.

Section 3.2 Room ventilation and airflow in aquatic areas,p. 24

Guideline 13:

All electrical systems must be professionallyinstalled to appropriate code standards (federal,provincial/territorial and municipal buildingcodes) for operation in moist environments, andmust include proper grounding and ground-fault interrupters on all circuits. Extension cordsshould be avoided, and electrical wires should befixed safely, away from water and from person-nel circulation areas.

Section 3.3 Mechanical and electrical requirements, p. 25

Guideline 14:

Electrical components and equipment should belocated outside the splash zone, and should behoused in moisture-proof enclosures. Electricalfixtures should be secured with gaskets to pre-vent incursion of water, and should be locatedabove pipe runs.


Guideline 15:

Machinery that produces noise and vibrationshould be isolated from areas housing fish.


Guideline 16:

Light should be phased on and off, and shouldincorporate wavelengths and intensities appro-priate for the species where this is known. Wheretask lighting is needed for people working in theroom, it should be restricted in its dispersionthroughout the room or be placed at a lowerlevel than the tank surface.

Section 3.4 Lighting, p. 25

Guideline 17:

All aquatic facilities should have an emergencycontingency capacity, capable of maintaining

aerated and filtered water and assuring the con-tinuation of life support.

Section 3.5 Redundancy in aquatic life support systems, p. 26

Guideline 18:

Critical systems, including pumps, should beduplicated to ensure that failures cause onlyminimal interruptions in service.

Section 3.5 Redundancy in aquatic life support systems, p. 26

4. Types of Systems

Guideline 19:

An adequate water supply of suitable qualityshould be provided for the fish at all times.

p. 27

5. Fish Housing

Guideline 20:

Aquatic environments should be designed tomeet the established physical and behavioralrequirements of the fishes in terms of shelter,social grouping, overhead cover and lighting.

Section 5.1 Fish well-being, p. 28

Guideline 21:

The shape, colour, depth, and volume of tanksshould be appropriate for the species and lifestage being held.

Section 5.2 Tank/enclosure design, p. 28

Guideline 22:

Tanks should have smooth, inert, sealed interiorsurfaces.


Guideline 23:

Tanks should be self-cleaning, or adequatemeans for the regular cleaning of tanks should beincorporated into the design.


Guideline 24:

Tanks should be equipped with a covering thatprevents fishes from jumping from the tank, e.g.,tank nets or rigid coverings.


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D. FACILITY MANAGEMENT, OPERATIONAND MAINTENANCE

1. Security and Access

Guideline 25:

Access to fish facilities should be designed tominimize traffic through the area. Access shouldbe restricted to those personnel required formaintenance of the facility and the care of thefishes, and those using the facilities for experi-ments or teaching.

p. 31

2. General Maintenance of the Facility

Guideline 26:

All architectural and engineering specificationsand drawings of the facility should be availableto those in charge of running the facility, asshould all operating manuals for special equip-ment such as pumps, chillers and computer con-trol systems.

p. 31

Guideline 27:

Aquatic facilities must have written maintenanceschedules developed specifically for the facility.

p. 31

Guideline 28:

Facilities should be kept in a clean and orderlymanner. Tanks should be disinfected before andafter every experiment.

p. 31

Guideline 29:

The staff responsible for operating an aquaticfacilitiy should have the specialized knowledge,experience and training for proper function,operation and maintenance of the water system.

p. 31

Guideline 30:

Sufficient numbers of staff must be available foranimal care and facility management and main-tenance 365 days a year for both routine andemergency needs.

p. 31

3. Environmental Monitoring and Control

Guideline 31:

An environmental monitoring system is essentialfor aquatic facilities and should be designed tosuit the water management system.

p. 32

Guideline 32:

Water quality monitoring systems should be ableto detect and react to changes in water qualitybefore they become life-threatening to animalshoused in the system.

p. 32

Guideline 33:

Water quality parameters should be monitored atan appropriate frequency for the facility, andshould allow predictive management of waterquality, rather than only reactive management ofcrises in water quality.

p. 32

Guideline 34:

Good water quality measuring equipmentshould be available, regularly calibrated andwell maintained. Records of water qualityshould be maintained and should be retrievablefor retrospective analysis in the event ofproblems.

p. 33

Guideline 35:

Water quality must be monitored and main-tained within acceptable parameters for thespecies being held.

Section 3.1 Management of water quality, p. 33

Guideline 36:

Fishes should not be subjected to rapid changesin temperature, particularly to rapid increases intemperature.

Section 3.2 Temperature, p. 33

Guideline 37:

Fishes should be kept in water with an adequateconcentration of oxygen.

Section 3.3 Oxygen, p. 34

Guideline 38:

Aquatic systems are susceptible to acute orchronic supersaturation. Individuals responsible

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for operating aquatic systems should understandthe causes of gas supersaturation and how tomitigate potential problems.

Section 3.4 Supersaturation, p. 34

Guideline 39

Water pH should be maintained at a stable andoptimal level as changes in pH may influenceother water quality parameters.

Section 3.5 pH, p. 35

Guideline 40:

Free ammonia and nitrite are toxic to fishes andtheir accumulation must be avoided.

Section 3.6 Nitrogen compounds, p. 35

Guideline 41:

Salinity changes are inherently stressful for fish-es, and should be conducted slowly and withattention to the physical status of the fishes.

Section 3.8 Salinity, p. 36

Guideline 42:

When there is reason to believe hazardous mate-rials or infectious agents have accidentallyentered the water system, that system should beisolated and tested.

Section 3.9 Toxic agents, p. 37

Guideline 43:

Chemical products should be safely stored awayfrom the aquatic housing area and the watersupply.

Section 3.9 Toxic agents, p. 37

E. CAPTURE, ACQUISITION,TRANSPORTATION AND QUARANTINE

1. Capture of Wild Stock

Guideline 44:

Wild fishes should be captured, transported andhandled in a manner that ensures minimal mor-bidity and mortality.

p. 38

Guideline 45:

Where exotic fishes are obtained from aquariumsuppliers or collection sources, local, provin-cial/territorial and federal authorities should beconsulted to determine the risk of escape, acci-

dental introduction, exotic diseases and otherdetrimental outcomes, and how to minimizethese risks.

p. 38

3. Piscicidal Compounds

Guideline 46:

Alternatives to the use of piscicidal compoundsshould be sought, such as anesthetic agents withminimal environmental and non-target speciesimpacts.

p. 38

4. Acquisition of Hatchery Fishes

Guideline 47:

Fishes should come from hatcheries with definedhealth status and preferably known genetic his-tory. Hatcheries should be encouraged to devel-op husbandry and management practices consis-tent with those used in the production of otherlaboratory animals.

p. 39

6. Quarantine and Acclimation

Guideline 48:

After transport and before use in experiments,fishes should be acclimated to laboratory conditions during a period of quarantine andacclimation.

p. 40

Guideline 49:

As far as possible, fish from various sourcesshould not be mixed.

p. 40

Guideline 50:

Quarantine areas should be subject to extra vigi-lance in monitoring fish and good record keep-ing to detect and respond to any health problemsin quarantined fish.

Section 6.1 Quarantine, p. 40

Guideline 51:

The duration of quarantine should be appropri-ate to assure the health of the fishes.


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Guideline 52:

Quarantine areas should be managed accordingto rigorous infectious agent control practices.


F. HUSBANDRY

1. Record-keeping and Documentation

Guideline 53:

Detailed Standard Operating Procedures shouldbe developed for the maintenance and care of allfishes and for sanitation of tanks, rooms andequipment.

Section 1.1 Standard Operating Procedures, p. 42

Guideline 54:

Checklists should be used for each group of fishso that records are maintained of all cleaning,maintenance and experimental procedures.

Section 1.2 General checklists, p. 42

Guideline 55:

Basic physical and behavioral parameters indica-tive of well-being in fishes should be monitoreddaily and written records should be maintained.Any perturbation of these parameters should be investigated and the causes identified andcorrected.

Section 1.3 Assessment of fish well-being, p. 42

2. Density and Carrying Capacity

Guideline 56:

Each species should be housed at a density thatensures the well-being of the fish while meetingexperimental parameters. However, in somecases, the ideal environment for maintaining agiven species will have to be developed usingperformance-based criteria such as growth rate.Established maximum densities should not beexceeded.

p. 42

3. Food, Feeding and Nutrition

Guideline 57:

Fish feed should be purchased from sources thatmanufacture feed according to standardsemployed in the feed industry for fish and other domestic animals, and according to pub-

lished nutrient requirements for the species, ifavailable.

Section 3.2 Food and feeding, p. 43

Guideline 58:

Feed bags should be labeled with date of manu-facture and guaranteed analysis information.Small aliquots of feed should be retained forindependent testing when large feed lots arereceived.

Section 3.2 Food and feeding, p. 43

Guideline 59:

Feed should be stored in dedicated areas that aredark, temperature and humidity controlled andpest-free to ensure its nutritional quality. Feed forimmediate use and feed in feeders should besimilarly protected. Feed used for daily feedingshould be kept in sealed-top containers to protectit from humidity and light, and frequentlyreplaced with feed from storage.

Section 3.3 Feed quality and storage, p. 43

Guideline 60:

Fishes must be fed at appropriate intervals andwith a nutritionally adequate, properly sizedfeed. Optimal feeding techniques are essentialfor good health and well-being, and to preventthe fouling of water with uneaten feed.


Guideline 61:

Whether fishes are fed manually or automatical-ly, they should be observed regularly to deter-mine whether they are responding as expected,and whether the ration is sufficient or overfeed-ing is occurring.


Guideline 62:

Medicated feeds must only be used under veteri-nary prescription and supervision.

Section 3.5 Use of medicated feeds, p. 45

4. Broodstock and Breeding

Guideline 63:

Holding systems and environmental conditionsfor broodstock should be appropriate for thespecies. Particular attention should be paid to theimportance of environmental cues for the main-

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tenance (or manipulation) of endogenous repro-ductive rhythms.

p. 46

Guideline 64:

Where possible, rational genetic management ofbroodstock should be used. For broodstock, astrict disease and health control program shouldbe implemented with veterinary advice to ensurethe production of healthy progeny and preven-tion of disease transfer through water sources,fish or eggs.

p. 46

G. HEALTH AND DISEASE CONTROL

1. Fish Health Program

Guideline 65:

All facilities must have a fish health monitoringprogram.

p. 47

Guideline 66:

Strategic measures for disease prevention shouldinclude: 1) a formal written agreement with afish health professional (usually a veterinarian)responsible for the management of morbidityand mortality problems at the facility; 2) a pro-gram for the detection and management of dis-ease conditions and water quality problemsrelated to physiological stress; 3) strategic appli-cation of disease control measures, such as quar-antine, immunization, and prophylactic treat-ments; and 4) a system of regular monitoring andreporting for health assessment purposes.

Section 1.1 Disease prevention, p. 47

Guideline 67:

A health management program should focus onearly diagnosis and identification of the causalagents, stressors and mechanisms so that correctcontrol measures can be initiated.

Section 1.2 Disease diagnosis and identification ofpathogens, p. 47

Guideline 68:

Fish health management programs should striveto identify both clinical and subclinical/adventi-tious pathogens which may occur as a result ofexperimental stressors.


Guideline 69:

Particular attention should be paid to monitoringfishes following any potentially stressful event.


Guideline 70:

Handling procedures should be carried out onlyby competent individuals using techniques thatminimize the potential for injury. Efforts shouldbe made to minimize morbidity and mortalitycaused by osmoregulatory compromise, sys-temic acidosis, and opportunistic infections ofdamaged skin that can result from handling andtraumatic injuries.

Section 1.3 Injuries and other disorders, p. 48

Guideline 71:

Health management measures should be used toensure that behavioral interactions with negativeconsequences such as aggression are avoided.


Guideline 72:

A Standard Operating Procedure should beestablished for any standard treatments, andinclude the definition of endpoints should fish beadversely affected.


H. EXPERIMENTAL PROCEDURES

1. Handling and Restraint

Guideline 73:

Fishes should be fasted prior to handling.

p. 50

Guideline 74:

Personnel involved in handling fishes shouldundergo training in methods to ensure theirexpertise and to minimize injury and morbidityto fishes in their care.

p. 50

Guideline 75:

Fishes should be handled only when necessary,and the number of handling episodes should beminimized.

p. 50

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Guideline 76:

Fishes should be handled in a fashion that mini-mizes damage to their mucus-skin barrier.

p. 50

Guideline 77:

Restraint and handling of fishes should be car-ried out in a manner to minimize visual stimula-tion. Where feasible, fishes should be protectedfrom direct light and rapid changes in lightingwhile being restrained.

p. 51

Guideline 78:

In general, fishes should not be kept in air contin-uously for more than 30 seconds.

p. 51

Guideline 79:

Those who work with dangerous species must betrained and competent to do so. Appropriateemergency items (e.g., antivenom, an appropri-ate first aid kit, etc.) must be on hand.

Section 1.1 Restraint of dangerous species, p. 51

2. Restricted Environments

Guideline 80:

Every effort should be made to provide fishesheld in restricted environments with as non-stressful an environment as possible, within theconstraints of the experimental design.

p. 51

3. Surgery

Guideline 81:

Surgery should be performed by individualswith appropriate training.

Section 3.1 Surgical preparation and skin disinfection, p. 52

Guideline 82:

Before surgery is attempted on living animalsthat are expected to recover, suture and surgicaltechniques should be practiced on inanimatematerials or dead specimens until competency isattained.

Section 3.1 Surgical preparation and skin disinfection, p. 52

Guideline 83:

Surgical sites should be prepared in a fashionthat minimizes tissue damage and contamina-tion of wound areas.Section 3.1 Surgical preparation and skin disinfection, p. 52

Guideline 84:

Attention should be paid to the use of asepsis,disinfection and the use of sterile instruments tominimize wound contamination and maximizethe healing response.Section 3.1 Surgical preparation and skin disinfection, p. 52

Guideline 85:

During prolonged surgery, water quality shouldbe maintained at a high level, with minimal bac-terial and organic burden. Water for anesthesiashould be from the same source as the tank waterto minimize shock caused by differences in tem-perature, pH, electrolytes, etc.Section 3.2 Water quality during surgery, p. 53

Guideline 86:

Anesthetics should be used in experimentswhere there is expected to be noxious stimuli,and in experiments entailing extensive handlingor manipulation with a reasonable expectation oftrauma and physiological insult to the fish.Section 3.3 Anesthesia, p. 53

Guideline 87:

Anesthetics should be chosen on the basis oftheir documented ability to provide predictableresults, including immobilization, analgesia andrapid induction and recovery, while allowing fora wide margin of safety for the animals and theoperators.Section 3.3 Anesthesia, p. 53

Guideline 88:

Regardless of the application, anesthetics shouldbe tested on a small sample of fish, as the effectof an anesthetic can vary with local water condi-tions, as well as the species, life stage, and size ofthe fish.Section 3.3 Anesthesia, p. 54

Guideline 89:Personnel working with anesthetic agents in fishmust be adequately trained and protected withpersonal protective equipment.Section 3.3 Anesthesia, p. 54

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Guideline 90:

Any incisions should avoid the lateral line andshould follow the longitudinal axis of the fish.

Section 3.5 Incisions, p. 54

Guideline 91:

In general, strong, inert, non-hygroscopic monofil-ament suture material and atraumatic needlesshould be used for closure of incisions in fish skin.

Section 3.6 Suture materials and techniques, p. 54

Guideline 92:

In laboratory or applicable field situations, fishmust receive careful attention and monitoringfollowing surgery.

Section 3.8 Postoperative care, p. 55

Guideline 93:

Fish should be held in a manner that reduces oreliminates intraspecific interactions in tanks, andmeets appropriate living conditions for thespecies.


Guideline 94:

The costs and benefits of the use of prophylacticantibiotics post surgery should be carefullyconsidered.


Guideline 95:

Social factors, such as size differences, ability tofeed or exclude other fish from feed, and agonis-tic behavior, should be considered in experimen-tal design and when maintaining social groupsof recovering fish.


4. Administration of Compounds andDevices by Various Routes

Guideline 96:

If a treatment compound is to be administeredorally, the volume dose rate should not exceed1% body weight (1 mL/100 g).

Section 4.2 Oral, p. 56

Guideline 97:

Care should be taken during injection to intro-duce the needle in spaces between the scales.Intramuscular injections may be made into thelarge dorsal epaxial and abdominal muscles, tak-ing care to avoid the lateral line and ventralblood vessels. Intraperitoneal (IP) injectionsshould avoid penetrating abdominal viscera assubstances that cause inflammation may lead toadhesion formation.

Section 4.3 Injection, p. 57

Guideline 98:

Implanted materials should be biocompatibleand aseptic, and should be implanted using ster-ile techniques.

Section 4.4 Implants, windows and bioreactors, p. 57

5. Tagging and Marking

Guideline 99:

Investigators must aim to minimize any adverseeffects of marking and tagging procedures on thebehaviour, physiology or survival of individualstudy animals. Where such effects are unknown,a pilot study should be implemented.

p. 57

Guideline 100:

Marking techniques which cause significant tis-sue injury, such as branding, tattooing or clip-ping important fins, should only be used if evi-dence is provided to an animal care committeeindicating that alternative methods cannotachieve the desired result.

Section 5.1 Tissue marking, p. 58

6. Collection of Body Fluids

Guideline 101:

Sedation or anesthesia should be used to restrainfish for collection or cannulation purposes. It isimportant to realize that both restraint and anes-thesia may alter physiological parameters suchas serum glucose and various hormone levels.

p. 59

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8. Endpoints and Criteria for Early Euthanasia

Guideline 102:

Investigators should eliminate, mitigate orminimize potential pain and distress wheneverfeasible and consistent with good scientificpractice.

Section 8.1 Recognition of “pain”, “distress” and “stress”,p. 59

Guideline 103:

A defined endpoint should be established forstudies which involve potential pain and/or dis-tress to the animal. A pilot study should be usedto identify clinical signs to be used as the end-point and to establish appropriate monitoring ofthe animals.

Section 8.2 Choosing an appropriate endpoint, p. 60

Guideline 104:

When conducting research with defined, earlypre-lethal endpoints, a list of parameters shouldbe established to permit an objective assessmentof health status.


Guideline 105:

In any study where there is expected morbidityand mortality, the criteria for early euthanasiashould be clearly defined.


9. Monitoring

Guideline 106:

Depending on the study and the time of morbid-ity, monitoring should be done at least daily.Frequency of monitoring should allow for thetimely removal of fish before severe morbidityoccurs. Frequency of monitoring should beincreased where mortality is expected to behigh.

p. 62

10. Negative Reinforcement Modalities

Guideline 107:

Pilot studies and literature searches should beused to establish the least invasive method of

obtaining a consistent response when using neg-ative reinforcement modalities in fishes.

p. 62

11. Exercise to Exhaustion

Guideline 108:

Studies involving the forced swimming of fishesto the point of exhaustion, often in conjunctionwith negative reinforcement, should be conduct-ed with strict adherence to guiding principles ofminimization of distress of animals. Fishes usedin exercise to exhaustion studies should be mon-itored continuously.

p. 62

12. Environmental Extremes

Guideline 109:

Studies involving the exposure of fishes to envi-ronmental extremes should select the earliestendpoint possible.

p. 62

13. Genetically Modified Fish

Guideline 110:

Genetically modified fishes may have changesin physiology and anatomy as the result of their genetic alteration, and should be closelymonitored.

p. 63

Guideline 111:

Genetically modified fishes must not be permit-ted to enter the food or feed chain unless theyhave undergone a thorough safety assessmentand have received authorization for sale, manu-facture and/or import as a food or feed byHealth Canada and the Canadian FoodInspection Agency.

p. 63

I. EUTHANASIA

Guideline 112:

Where feasible, the euthanasia of fishes shouldconsist of a two-step process, with initial anes-thesia to the point of loss of equilibrium, fol-

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lowed by a physical or chemical method to causebrain death.

p. 64

Guideline 113:

If a physical technique of euthanasia is usedwhen killing fishes, it should entail the physicaldestruction of brain tissue by pithing or crushingthe brain.

p. 64

J. DISPOSITION OF FISH AFTER STUDY

1. Consumption of Fish

Guideline 114:

Fishes destined for food and subjected to seda-tion or anesthesia should be held for the desig-nated withdrawal time before being killed.

p. 65

2. Release of Fish to Wild

Guideline 115:

In general, research fishes that have been kept incaptive environments must not be released intothe wild. Release into the wild is only permissi-ble under appropriate licence under the Fisheries(General) Regulations or similar provincial/ter-ratorial regulations.

p. 65

4. Transfer of Fish Between Facilities

Guideline 116:

Fishes should undergo health assessment beforebeing transported between facilities. Appropri-ate regulatory approval and permits must be inplace before any transfer.

p. 65

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The greatest challenge in providing guidelines on:the care and use of fish is the wide variety of fishesused in Canada and the diversity of their habits,behavior, life history, and environmental andhusbandry requirements. In addition, the scien-tific information required to define the preferredconditions for fish well-being is limited. Whileconsiderable research has been conducted onculture strategies and environmental and waterquality requirements, such studies have general-ly been aimed at determining conditions thatoptimize production in aquaculture systems,rather than improving the welfare of fishes, andhave not usually addressed the differencebetween tolerance and preference (Fisher, 2000).

An important consideration in these guidelinesis the naturally high mortality rates of juvenilesin species whose ecological strategies include thegeneration of large numbers of progeny toensure adequate survival in the wild. In addition, many experimental populations ofspecies with usually high survival contain indi-viduals that will not thrive to adulthood evenunder the best environmental conditions. Insome situations, a population-based (or a groupof study fish) approach to well-being may beappropriate, but individuals that are not likely tothrive should be euthanized as soon as they areidentified.

Another consideration for these guidelines is thegeneral acceptance by the public of the currentkilling methods used in harvesting wild fishes orin recreational angling. In general, the publicappears to be willing to accept these killingmethods for food production but not when fish-es are used for research. These guidelines acceptthat for research, teaching, and testing use of anyanimal, including fishes, more emphasis will beplaced on individual well-being than is general-ly accepted for the commercial harvesting or pro-duction of animals for food. It is recognized,however, that in some instances investigatorsmay obtain fishes from people involved in com-mercial or recreational harvesting and have littleinfluence over the capture methods.

These guidelines apply to fishes held in facilitiesfor research, teaching and testing, as well as tofishes that are studied in their natural habitats.

1. Definition of Fish

For the purpose of these guidelines, fishes aredefined as all bony and cartilaginous fish genera(classes Chondrichthyes [cartilaginous fishes],Agnatha, and Osteichthyes [bony fishes]). Fisheggs, embryos or larvae that have not developedbeyond exclusive reliance on their own yolknutrients are not covered by these guidelines.Similarly, invertebrates (except cephalopods) arenot covered under the CCAC system of surveil-lance, but institutions are encouraged to fosterrespect for these animals by ensuring that hold-ing facilities and levels of husbandry meet stan-dards equivalent to those used for fishes.

2. Rationale for Guidelines on theCare and Use of Fish

The use of fishes as experimental subjects hasincreased substantially over the past twodecades. This increase in use is a result of therapid development of the aquaculture industry,requirements for testing involving fishes as indi-cators of environmental change, and the use offishes as a replacement for mammals in biomed-ical, pharmacological and genetic research(DeTolla et al., 1995; Fabacher & Little, 2000). Thetrend toward the use of fishes as a replacementfor studies that would previously have usedmammals as experimental subjects is not dis-couraged. However, it must also be recognizedthat fishes have the capacity to perceive noxiousstimuli. Noxious stimuli are those stimuli thatare damaging or potentially damaging to normaltissue (e.g., mechanical pressure, extremes oftemperature and corrosive chemicals). Whetheror not fishes have the capacity to experience anyof the adverse states usually associated with painin mammals is subject to a great deal of debate inthe scientific literature (FAWC, 1996; FSBI, 2002;Rose, 2002; Braithwaite & Huntingford, 2004).Nonetheless, fishes are capable of behavioral,

B. INTRODUCTION

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physiological and hormonal responses to stres-sors (including noxious stimuli) which can bedetrimental to their well-being. These CCACguidelines both support the leadership role thatCanadians play in fish research, and ensure thatthe welfare of fishes is carefully considered dur-ing the use of fishes for research, teaching andtesting, recognizing that better welfare will resultin better science.

3. Ethical OverviewGuideline 1:

Fishes used in research, teaching and testingmust be treated with the respect accorded toother vertebrate species.

The CCAC's surveillance system for animalsused in research, teaching and testing is based onthe principles of humane science, i.e. the ThreeRs of Russell and Burch (Russell & Burch, 1959) -Reduction, Replacement and Refinement. For theCCAC, these principles are laid out in its policystatement on: ethics of animal investigation (CCAC,1989). The ethics of animal investigation applies toall species covered by the CCAC system, i.e. allvertebrates and cephalopods.

In addition, the CCAC system takes a "moralstewardship" approach to the use of animals in science as explained in the CCAC Experi-mental Animal User Training Core Topics -Module 2, Ethics in Animal Experimentation(www.ccac.ca/en/CCAC_Programs/ETCC/Module02E/TableofContentsModule02.htm).The first guideline statement in the CCAC guide-lines on: institutional animal user training (CCAC,1999a) states, "Institutions must strive throughtheir training programs to sustain an institution-al culture of respect for animal life".

3.1 Principles of the Three RsAccording to the CCAC policy statement on: ethicsof animal investigation (CCAC, 1989), it is theresponsibility of the local animal care committee(ACC) to ensure that fishes are used only if theinvestigator's best efforts to find a non-animalmodel have failed.

As for any other species covered by the CCACsystem, investigators using fishes are required touse the most humane methods on the smallest

number of animals necessary to obtain validinformation. This requires the use of a soundresearch strategy, including: identification of keyexperiments that determine whether a particularline of enquiry is worth pursuing; use of pilotstudies; staging of in vitro to in vivo experimentswhere possible; and implementation of stagedincrease in test stimuli where possible (Balls et al.,1995). The numbers and species of animalsrequired depend on the questions to be explored.Field studies, aquaculture studies and laboratorystudies require different statistical designs; fieldstudies and aquaculture production typicallyrequire the use of larger numbers of animals. Thelife stage of the fishes used in each study willalso affect the numbers of animals needed.Studies of early life stages typically require largenumbers of individuals. In all cases, studiesshould be designed to use the fewest animalsnecessary. Heffner et al. (1996) and Festing et al.(2002) provide discussions on the appropriatetreatment of samples and experimental units.Investigators are encouraged to consult with astatistician to develop study designs that havethe appropriate statistical power to accomplishthe research objectives (Nickum et al., 2004).

The CCAC policy statement on: ethics of animalinvestigation (CCAC, 1989) also requires adher-ence to the following principles:

• animals must be maintained in a manner thatprovides for their optimal health and well-being, consistent with the demands imposedby the experimental protocol;

• animals must not be subjected to pain and/or distress that is avoidable and that is not required by the nature of the relevent protocol;

• expert opinion must attest to the potentialvalue of studies with all animals, includingfishes (e.g., scientific merit for research, seeCCAC policy statement on: the importance ofindependent scientific merit of animal basedresearch projects [CCAC, 2000a]; pedagogicalvalue for teaching; and the appropriateness ofthe method to provide data for testing accord-ing to current regulatory requirements);

• if pain or distress is a justified component of

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the study, the intensity and duration ofpain/distress must be minimized; and

• an animal observed to be experiencing severe,intractable pain and/or distress shouldimmediately be killed using an approvedmethod of euthanasia.

Meeting the principles outlined above requiresthat fishes be accorded the same degree of care asother animals under the CCAC system. There aretwo main ethical drivers for CCAC guidelines: tomaximize animal well-being, and to minimizepain and/or distress. Any factor that disturbs thenormal physiological balance of an animal has aneffect on the studies being conducted, and there-fore should be avoided or minimized for scientif-ic as well as ethical reasons, unless the factoritself is the subject of investigation.

Fishes comprise a great number of species, eachwith specific anatomical, physiological andbehavioral characteristics. Investigators and ani-mal care staff should therefore acquaint them-selves with the characteristics of the species pro-posed to ensure that appropriate facilities andhusbandry procedures are in place prior toobtaining the animals.

4. Responsibilities

Descriptions of the responsibilities of investiga-tors, animal care committees (ACCs) and veteri-narians are provided here; however, moredetailed information is given throughout theseguidelines to assist both investigators and mem-bers of ACCs to meet their responsibilities.

4.1 Responsibilities of investigators

4.1.1 Protocols involving the useof fish

Guideline 2:

Projects involving the use of fishes forresearch, teaching or testing should bedescribed within a protocol. Protocols shouldbe approved by an animal care committeeprior to the commencement of the work.

Investigators are responsible for obtaining ACCapproval before beginning any animal-basedwork. For further details concerning the informa-

tion that should be included in a protocol form tobe submitted to an ACC, see CCAC guidelines on:animal use protocol review (CCAC, 1997a); andCCAC policy statement on: terms of reference for ani-mal care committees (CCAC, 2000b) or most recentrevisions. Investigators obtaining fishes from thewild or carrying out field studies should alsoconsult the CCAC guidelines on: the care and use ofwildlife, Section B 3.1.1.1 Protocols involving theuse of wildlife (CCAC, 2003a).

When working outside of Canada, Canadianinvestigators are subject to the same guidelinesthat apply to work within Canada, as well as tothe relevant legislation, regulations and guide-lines pertaining to animal care in the countrywhere the work is conducted. This also applies tocollaborative research projects, whether the workis conducted in Canada or elsewhere (see CCACpolicy statement on: animal-based projects involvingtwo or more institutions [CCAC, 2003b]).

4.1.2 Studies and activities requiringprotocols

4.1.2.1 Work requiring protocols and inclusion in animal use inventories

These guidelines provide recommendations forfishes when they are being used by investigators.Fishes should be treated humanely whether ornot they are to be included in animal use proto-cols or inventories.

The following require protocols and inclusion inanimal use inventories (i.e. CCAC Animal UseData Form, see Reporting of Animal Use Data atwww.ccac.ca/en/CCAC_Programs/Assessment/AUDFen.htm):

• fishes held live in confinement for any periodof time (even hours) for research, display,teaching or testing;

• fishes lethally sampled in the field forresearch, teaching or non-routine testingpurposes;

• fishes caught, sampled or otherwise manipu-lated and released in the field for research,teaching and testing purposes; and

• genetically modified fishes.

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4.1.2.2 Work not requiring protocols orinclusion in animal use inventories

The following will not require protocols or inclu-sion in animal use inventories:

• fish eggs, embryos or larvae that have notdeveloped beyond exclusive reliance on theirown yolk nutrients;

• wild source or hatchery fishes that have notbeen assigned to research studies, and whosepropagation is sufficiently understood to beconsidered routine;

• fishes being observed in the field that are notbeing handled or interfered with in any way;

• fishes being counted at installations such ascounting fences and traps;

• fishes being lethally sampled under govern-ment or other regulatory mandate for estab-lished fish inspection procedures, abundanceestimates, and other population parametersrequired for assessing stocks and for routinemonitoring of contamination/toxin levelsand disease; and

• fishes already killed in the course of estab-lished aquaculture industry or commercialfishing purposes.

Guideline 3:

Before working with fishes, investigators,technical staff and post-graduate studentsmust be properly trained and have their com-petency evaluated.

According to CCAC guidelines on: institutionalanimal user training (CCAC, 1999a), investigatorsand students should complete the CoreComponents of the Recommended Syllabus for anInstitutional Animal User Training Program(CCAC, 1999b) and should have completed therelevant hands-on training to meet the Syllabusrequirements on the use of fish as a research animal. "Students" refers to post-graduate stu-dents; undergraduate students are expected to besupervised by a properly qualified individual.See the CCAC website (www.ccac.ca/en/CCAC_Programs/CCAC_Programs-ETC.htm)for further information on relevant courses for

investigators using fish as a research animal.Animal users should receive refresher trainingon a five-year basis, and additional trainingshould be given as needed in order to be able tocarry out procedures competently.

Guideline 4:

Investigators are responsible for, and mustcomply with, occupational health and safetyregulations regarding the protection of per-sonnel from known or suspected physicaland biological hazards.

As with any other laboratory, animal care facili-ties (including aquatic facilities) should have anoccupational health and safety program. All per-sonnel using the facility should be familiar withthe requirements of relevant federal, provincial/territorial and municipal legislation. ChapterVIII of the CCAC Guide to the Care of ExperimentalAnimals (CCAC, 1993a) provides additionaldetails on occupational health and safety.

Guideline 5:

Investigators should be aware of the potentialrisks associated with zoonotic agents pres-ent in fishes.

A brief review of fish zoonotic agents is provid-ed in Appendix B of this document.

4.2 Responsibilities of the animalcare committee

The CCAC Terms of Reference for Animal CareCommittees (CCAC, 2000b, or most recent version)should be consulted for detailed information onthe roles and responsibilities of institutionalACCs. In particular, ACCs are responsible forreviewing all studies conducted by investigatorsbelonging to their institution, whether the work isconducted in-house or elsewhere. ACCs shouldensure that appropriate care will be provided forall animals at all stages of their life and under allexperimental situations. ACCs are responsible forensuring that there is appropriate management ofthe facilities housing the animals. In particular,ACCs should verify that there is a person clearlydesignated to be in charge of animal care andmanagement of the facilities who should also be amember of the ACC. Additionally, members ofthe ACC should visit the animal facilities andareas in which animals are used on a regular

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basis, in order to better understand the workbeing conducted within the institution.

ACCs are responsible for ensuring that veteri-nary care is available to all animals being usedfor experimental purposes within the institution.

4.3 Role of the veterinarian

The CCAC uses the CALAM/ACMAL Standardsof Veterinary Care (CALAM/ACMAL, 2004) asthe Canadian standards for the role and responsi-bilities of the veterinarian within an institutionusing animals for research, teaching or testing, andassesses participants in its program based on thesestandards. Veterinarians working at institutionswith large populations of fishes are encouraged tohave special training in fish health management inresearch, teaching or testing environments.

5. Government Regulations andPolicies on the Use of Fish

Guideline 6:

Anyone acquiring or transporting fishes, orconducting research on fishes, must be famil-iar with, and comply with, relevant interna-tional, federal and provincial/territorial legis-lation and policies governing the capture offishes and/or their transfer from one waterbody or jurisdiction to another.

It is important to verify current regulatory infor-mation with the regulatory agencies identifiedbelow to ensure compliance with current legalrequirements.

5.1 International

There exist several international agreements,codes, and conventions that relate to the intro-ductions and transfers of aquatic organisms.Requirements are typically incorporated throughdomestic legislation. Therefore, for activitiesoccurring in or otherwise pertaining to Canada,verification with Canadian authorities and com-pliance with Canadian laws should ensure com-pliance with international standards. Someexamples of international agreements, codes andconventions include:

Convention on Biological Diversity (CBD)

• Signed and ratified by Canada in 1992, CBDsets out broad commitments to the protectionof biological diversity. Categories of pro-grams of work under CBD include Marineand Coastal Biodiversity and FreshwaterBiodiversity. www.biodiv.org/convention

Convention on International Trade in EndangeredSpecies of Wild Fauna and Flora (CITES)

• CITES, in force since 1975, has 167 membercountries (as of 2005), including Canada.Member countries ban commercial trade inendangered species and regulate and moni-tor trade in other species that might becomeendangered. CITES applies not only to liveanimals, but also to "Parts and parts thereof",which includes all types of biological sam-ples (skin, hair, bones, blood, serum, etc.).www.cites.org

Code of Practice for Introduction and Transfer ofMarine Organisms

• The International Council for the Explorationof the Sea (ICES) requests early notification of planned introductions which may affectjoint water bodies, in order to carry out inter-national review. www.ices.dk/reports/general/2004/ICESCOP2004.pdf

International Aquatic Animal Health Code forFinfish, Molluscs and Crustacea

• To facilitate international trade, l'OfficeInternational des Epizooties (OIE) uses this Code (updated every two years) whichdefines minimum health requirements to avoid the risk of spreading aquatic ani-mal diseases. www.oie.int/eng/normes/en_acode.htm

Sanitary/Phytosanitary (SPS) Agreement

• This provides agreed-upon rules for GeneralAgreement on Tariffs and Trade (GATT) andWorld Trade Organization (WTO) in the useof SPS measures in international trade.www.wto.org/english/tratop_e/sps_e/spsagr_e.htm

Canada-USA Free Trade Agreement (FTA) andNorth American Free Trade Agreement (NAFTA)

• These agreements provide SPS measures con-sidered acceptable for trade between Canada,

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USA and Mexico. www.nafta-sec-alena.org/DefaultSite/index_e.aspx?CategoryId=42

North American Commission (NAC) (Canada andUSA) of the North Atlantic Salmon ConservationOrganization (NASCO)

• The NAC establishes protocols for the intro-duction and transfer of salmonids on theAtlantic seaboard. www.nasco.int

International Joint Commission

• This Commission prevents and resolves dis-putes between US and Canada under the 1909Boundary Waters Treaty. In particular, theCommission relies on applications forapproval of projects affecting boundary andtransboundary waters, and may regulate theoperation of these projects. www.ijc.org/en/home/main_accueil.htm

Organisation for Economic Cooperation andDevelopment (OECD)

• The OECD has developed test guidelines forhuman health and for environmental health.Investigators conducting studies to generatedata to be submitted for regulatory acceptanceshould refer to Environment Canada or HealthCanada to confirm compliance with the cur-rent OECD guidelines. www.oecd.org/home

5.2 Federal

Fisheries and Oceans Canada (DFO) is responsi-ble for administering the Fisheries Act and theOceans Act, and is a responsible minister underthe Species at Risk Act (SARA) for aquatic organ-isms. DFO's program responsibilities include,but are not limited to, the management of com-mercial, recreational and aboriginal fisheries and fisheries habitat protection (includingregulation of the movement and introduction offish), and protection and recovery of aquaticspecies at risk. Certain pollution prevention sec-tions (e.g., S36) of the Fisheries Act are adminis-tered by Environment Canada (for further infor-mation see Section H.7. Use of Infectious DiseaseAgents, Tumorigenic or Mutagenic Agents, andToxic and Noxious Compounds). Although fish-eries management is a federal responsibility,there are delegation agreements with a numberof provinces regarding inland waters.

Fish cannot be caught or removed without a per-mit from the appropriate regulatory body.Investigators seeking to capture fishes for use inresearch, teaching or testing should contact theirlocal DFO office to determine the proper licens-ing requirements for their respective region.

DFO also administers the Fish Health ProtectionRegulations (FHPR) under the federal FisheriesAct. The objective of the FHPR is to minimize therisk of introducing or spreading diseases of con-cern. A license is required in order to release livefish into any fish habitat, or to transfer live fish toany fish rearing facility. It is important that any-one seeking to import or transport fish contactDFO for advice on regulatory requirements.

Canada now has a National Code on Introduc-tions and Transfers of Aquatic Organisms (I&TCode) (Fisheries and Oceans Canada, 2003) thatincludes all species of finfish, molluscs, crus-taceans, echinoderms, and other invertebrates.The I&T Code is an agreement between federal,provincial and territorial governments (signedby the Canadian Council for Fisheries andAquaculture Ministers in 2003) to conduct intro-duction and transfer approval processes in astream-lined manner through I&T Committeesestablished in each province and territory inCanada. The I&T Code process implements vari-ous federal and provincial regulations. LocalDFO offices or the relevant provincial/territorialgovernment should be contacted to determinethe application and approval requirements forintroductions of fish into fish habitat and/ortransfers of fish into fish rearing facilities.

The Wild Animal and Plant ProtectionRegulations of International and InterprovincialTrade Act (WAPPRIITA) is the enabling legisla-tion for CITES in Canada. The import or exportof any animal (including fishes) on the CITES listrequires a CITES permit from the CanadianWildlife Service (CWS) of Environment Canadaunder WAPPRIITA and the appropriate importor export permit for the provincial or territorialagency responsible for wildlife.

WAPPRIITA also provides the authority to protectCanadian ecosystems from the introduction of list-ed harmful invasive species by requiring permits,and makes it an offence to transport an animal orplant from one province or territory to another, or

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export from a province or territory, without therequired provincial or territorial permits.

The Committee on the Status of EndangeredWildlife in Canada (COSEWIC) develops andmaintains a national listing of Canadian speciesat risk, based on the best scientific evidenceavailable (www.cosewic.gc.ca). COSEWIC con-sists of representatives from: the wildlife depart-ments of all 13 Canadian provincial and territori-al governments; federal departments and corpo-rations concerned with wildlife, including CWS(which provides the Secretariat), Parks Canada,DFO and the Canadian Museum of Nature; andthree non-governmental conservation organiza-tions. It is the responsibility of the respectiveprovincial and territorial jurisdictions where thespecies occurs to take whatever actions areappropriate to address the threats and limitingfactors placing a species at risk. The federal gov-ernment implements their responsibility throughSARA which, upon legal listing, makes it illegalto kill or harm species designated as endangeredor threatened.

Environment Canada and Health Canada areresponsible for administering the NewSubstances Notification Regulations (NSNR)under CEPA, 1999. The purpose of the NSNR is toensure that no new substance is introduced intothe environment before an assessment has beenconducted to determine potential risks to theenvironment or human health. New substancesinclude live aquatic organisms that meet theCEPA 1999 definition of an "animate product ofbiotechnology" and can either be naturally occur-ring or genetically modified. Currently, notifica-tion is not required under the NSNR when thefish is to be used for research and developmentonly and there is no release from the researchfacility of the living organism, the genetic materi-al of the organism or material from the organisminvolved in toxicity. DFO has recently takenresponsibility for administering the NSNR foraquatic organisms with novel traits. Investigatorsimporting or using these organisms should con-tact the biotechnology office of DFO for regulato-ry advice. The New Substances NotificationBranch of Environment Canada can provide moreinformation on the regulatory requirements relat-ed to other aquatic new substances.

5.2.1 Reintroduction and release

In most instances, fishes may not be releasedinto the environment. However, in special casessuch as telemetry studies involving the captureand release of wild fishes, appropriate permit-ting is required before capture and release iscarried out through the I&T Code process.Fishes involved in such studies may alsorequire permanent marking or tagging if theyhave been subjected to treatment with anesthet-ic agents or other drugs with withdrawal timerequirements.

In each province and territory, there are I&TCommittees that evaluate requests to introduceor transfer fishes in accordance with the Fishery(General) Regulations. Regulations applied bythe I&T Committees in each province/territorymay differ. As a watershed may have a diseaseprofile different from other watersheds, fishescan be moved only between watersheds of simi-lar disease status and the permit must accompa-ny the shipment (one for each shipment of livefish, fertilized or unfertilized sex products, orproducts of fish). I&T Committees also reviewthe potential genetic and environmental impactsof such transfers (see above section on NationalCode on Introductions and Transfers of AquaticOrganisms).

5.2.2 Containment

There are several requirements for containmentunder various federal and provincial/territorialregulations. I&T Committees may issue transferlicenses with conditions listed (e.g., containmentrequirements). DFO and the Canadian FoodInspection Agency (CFIA) are currently develop-ing containment guidelines for fish pathogenswhich will be implemented through regulations.In addition, information regarding containmentrequirements for research involving animateproducts of biotechnology including geneticallymodified fish under the NSNR are further out-lined in Section H.13 Genetically Modified Fish.

Facilities conducting research involving fishpathogens are required to consult CFIABiohazard Containment and Safety Division,

to ensure appropriate levels of biocontainmentare in place (see Appendix C). Where thisinvolves the holding of live aquatic animalhosts, pathogen challenges or treatment trials,DFO provides science advice to CFIA as statedin their Memorandum of Understanding onBiologics.

5.3 First Nations

The Aboriginal Communal Fishery LicensesRegulations (laws.justice.gc.ca/en/F-14/SOR-93-332/index.html) describe communal licensesfor aboriginal communities for fishing and relat-ed activities. These regulations are under theFisheries Act and are referenced in variousprovincial and territorial regulations.

5.4 Provincial/territorialProvinces and territories also have legal require-ments that may need to be met in relation toactivities involving fish and aquatic ecosystems.Investigators who are uncertain of which agen-cies they are required to contact for appropriateregulations and permits should contact theirprovincial/territorial governments.

5.5 MunicipalMany municipal governments have regulationsgoverning the holding and use of fish withinmunicipal boundaries, as well as regulations per-taining to effluent and disposal of hazardouswaste. Investigators must ensure that theyadhere to appropriate municipal by-laws.

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Guideline 7:

Aquatic facilities are complex systems thatmust be well designed to minimize stress tothe fishes, promote efficient operation of thefacility, and ensure a safe working environ-ment for personnel.

The most basic principle in the design of fish-holding facilities is that a healthy fish populationis dependent on a stress-free environment, whichin turn requires the best possible facilities andwater system (Wedemeyer, 1996a). Water pro-vides the supporting medium for all aquaticspecies and serves two essential purposes: theprovision of oxygen for all life processes, anddilution and removal of metabolic wastes. Bothof these functions must be thoroughly addressedin any fish-holding system. Section C. AquaticFacilities is primarily targeted to land-basedholding systems, rather than to outdoor ponds oropen water cages; however, the provision of astress-free environment is equally applicable tothese types of facilities.

1. Water Supply

Aquatic facilities should be designed to mini-mize pathogens in the water intake and mayrequire methods of preventing biofouling inwater intake and distribution systems. Waterintakes located in bodies of water should be posi-tioned in areas where they will not pick updebris, recycled wastewater or shipping waste(i.e. bilge water and fuel spills), or be subject tobreaking waves or ice damage.

2. Water QualityGuideline 8:

If fresh or sea water is drawn from an openbody of water or a municipal source, it shouldbe tested for, and treated to remove, contam-inants and pathogens.

A comprehensive analysis of water qualityparameters (ions, pH, metals, etc.) should be con-

ducted before a fish holding/testing facility isplanned within a particular location or buildingto ensure that the water supply is suitable. Thisanalysis is usually the responsibility of the engi-neer in charge of designing the facility, in consul-tation with a knowledgeable fish health profes-sional. Laboratories capable of carrying out theinitial comprehensive analysis of the proposedwater supply can be found through the CanadianAssociation for Environmental AnalyticalLaboratories (CAEAL), www.caeal.ca. Watersources can be tested for a wide range of bacteri-al and chemical contaminants at most regionalhospital laboratories, and some larger municipal-ities may also have water testing laboratories.Water quality guidelines for the protection ofaquatic life have been developed by the waterquality task group of the Canadian Council ofMinisters of the Environment (www.ec.gc.ca/C E Q G - R C Q E / E n g l i s h / C e q g / Wa t e r /default.cfm#dri) and may provide guidance onacceptable levels of contaminants. Appendix D,although drafted for on-going water qualityevaluation, provides a list of the parameterswhich should be tested. Institutions that havecommissioned the facility should ensure that thisanalysis has been carried out. Depending on thereliability of the water source, further testingmay be needed, for example on an annual basis.The supply should also be evaluated to ensurethat there is sufficient capacity, including periodsof maximum demand or emergency situations.To protect fish from potential contaminants,other measures, such as a carbon filtering systemor a reverse osmosis system, may be requiredwhere problems from the water source have beendetected (Huguenin & Colt, 2002).

Tests to determine the chemical composition andpresence of contaminants/toxins will determinethe treatment necessary to make the water suit-able for use. Seasonal factors such as phyto- orzooplankton blooms, tidal cycles, seasonal watermass turnovers and lake turnovers can haveperiodic effects (scales of hours, days or months)for both sea water and fresh water, and theseneed to be anticipated.

C. AQUATIC FACILITIES

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Fresh water often requires treatment to removechlorine or other disinfectants, reduce suspend-ed sediments, release supersaturated gases, oreliminate pathogens (Fisher, 2000). With munic-ipal water supplies, chlorine or chloraminesmust be removed; activated charcoal (for largevolume systems) and sodium thiosulfate (forsmaller systems) are commonly used for thispurpose (see Hodson & Spry, 1985).

Another potential problem with municipal wateris that the pipes through which it passes canrelease metals, which in relatively high concen-trations can be toxic to fishes. The extent ofrelease and toxicity will vary with water quality.There are generally no problems associated withblack iron or plastic supply pipes from municipalsources. Galvanized iron piping, where used,may cause problems of zinc toxicity, and copperpiping or copper and brass fittings may causecopper toxicity if levels exceed 5µg/L. In addi-tion, problems with cadmium toxicity may beencountered if excessive amounts of solder havebeen used on copper fittings.

Well water does not require dechlorination, butit is often depleted of oxygen and may havehigh levels of metal ions or carbon dioxide,nitrogen and other gases. Other potential prob-lems include the presence of ammonia andexcessively high or low alkalinity (Stickney,1994). If well water is to be used, a pressuredrop test should be conducted to ensure that thesupply is adequate and reliable. Water takenfrom a well has undergone filtration during per-colation through the source geological forma-tion, and water temperature may be fairly con-stant throughout the year. Depending on thesystem requirements, this can reduce systemcomplexity and cost by reducing the need forsome filtration, as well as sterilizing and heat-ing equipment. The major problem with seawa-ter wells is that site conditions and geology areoften unfavourable, high flow rates can be diffi-cult to obtain if wells are not carefully situated,and they can be subject to draw-down duringperiods of high usage. On the other hand, sea-water wells have the advantage that they do notrely on pipe systems subject to storm and icedamage and blockage.

3. Engineering and Design

Guideline 9:

In designing and constructing aquatic facili-ties, assistance should be sought from peo-ple with experience in this field.

It is not possible in these guidelines to providethe engineering details required for aquatic facil-ities. However, several sources are available thatprovide information on the important engineer-ing and design principles of the various types ofaquatic systems, e.g., Stickney (1994), Pennell &Barton (1996), Ostrander (2000) and Huguenin &Colt (2002). The CCAC guidelines on: laboratoryanimal facilities - characteristics, design and develop-ment (CCAC, 2003c) should also be consulted forconsideration of the general principles of facilitydesign.

The design of the facility is critical, and signifi-cant input from investigators and operationalpersonnel is required during the design and con-struction phase to ensure it meets the require-ments of the fishes being held and the needs ofthe investigators and facility managers.

In aquatic facilities, it is particularly important toensure that the floor is sufficiently strong to sup-port the weight of the intended tanks plus water.

In addition to requirements for additional space,requirements for services (water, air, electricalpower, etc.) usually increase during the life of afacility. Therefore, the flexibility to permit laterretrofits should be included in the originaldesign.

The facility design should ensure easy access toall systems for operation and maintenance,including cleaning. Supply lines, drain lines andother critical components should not be buriedor otherwise made inaccessible.

Modular construction is desirable to allow forthe easy removal of components with minimaldisruption of operations.

Considerable care should be taken with all fittingsand equipment on the suction side of pumps toprevent air leaks that will cause nitrogen supersat-uration. Facilities should be designed to be able to

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add in desaturation equipment when the poten-tial for supersaturation exists.

Facilities should be designed with dedicated sep-arate quarantine areas for the isolation of newstock as appropriate.

Conventional facilities should incorporate trans-fer areas, foot and hand cleaning stations,restricted access and other common sanitarymeasures to prevent the introduction and spreadof aquatic animal pathogens of concern. Workrequiring the containment of aquatic animalpathogens requires special design considerations(see Appendix C).

Water supply lines should be secure, protectedfrom disruption and comply with local regula-tions. Where possible, water lines to and fromaquatic holding tanks should consist of hard,permanently fixed pipes to prevent problemsdue to hose flattening, air locks, fouling, etc. Thismay not be possible in a research facility whereflexibility of the facility is of paramount impor-tance, but the use of hard plumbed lines beforethe main water pump are strongly recommend-ed. All lines should be prominently labeled toprevent confusion and potentially lethal effectson fishes.

Water supply and drain lines should be designedto facilitate cleaning by simple, low technologymethods.

Pressure gauges and flow meters should beinstalled at points throughout the system tomonitor the condition of the lines and the per-formance of the pumps and filters. Performanceshould be monitored by flow, concentration ofdissolved gases, temperature, pH, salinity, dis-solved oxygen, etc.

All compressors providing gases to the systemshould have devices to remove moisture, and oiltraps to prevent any oil that leaks from compres-sors from entering the fish tanks through the aer-ation system. Food-grade lubricants should beused wherever possible. Intakes to compressorsshould be located so that only clean air is used,free of engine exhaust, tobacco smoke or otherairborn contaminants.

Main drains should be over-sized to handle tran-sient large flows of water. Gutters should havecovers that are flush with the floor and that per-mit water to drain quickly. Drains and guttersshould be designed to self-clean under normalflow, and to permit the use of a 'cleaning pig' toremove any buildup of waste in the lines. Wherefeasible, drains on all tanks should have trapsand easily accessible clean-out ports.

It is imperative to incorporate appropriate efflu-ent treatment, following regulatory requirementsfor effluent, into the design of the facility. If theeffluent is untreated, it should be discharged in alocation that is remote from the system intake tominimize the chance that effluent disease agentswill be re-circulated through the system. In addi-tion, it is essential to take into account the poten-tial impacts on wild aquatic organisms in waterreceiving the effluent. For the most part, dis-charge to a municipal sewer will provide ade-quate treatment of fish lab effluents. Calculationsof dilutions should indicate that potentially nox-ious materials in fish lab effluents (e.g., disinfec-tants) will be diluted to non-toxic concentrationsin sewers before the sewage reaches the sewagetreatment plant.

When effluent treatment is required, an appro-priate back-up system must be in place to ensureeffluent treatment remains valid during times ofpower outages.

3.1 Structural materialsGuideline 10:

Construction materials for facilities housingfishes should be selected carefully for resist-ance to corrosion and water damage.

It is critical that suitable construction materialsbe chosen for an aquatic facility, including mate-rials used for tanks, tank covers and stands, aswell as plumbing, mechanical and electrical com-ponents. Surfaces should be sealed, smooth, eas-ily cleaned and made of impervious material.Floors should be non-skid, especially when wet.

Porous materials, including wood, other organicmaterial and materials prone to de-laminationshould be avoided as they provide locations forsome pathogens/parasites and can increase theorganic matter load in tanks, leading to stressors

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and disease problems. If these materials are used,they must be properly sealed with nontoxic,opaque sealants.

In particular, the use of wood should be eliminat-ed in areas that are constantly wet (e.g., standsfor tanks) to prevent the growth of toxigenicmoulds and harbourage of pathogens. Wood islikely to rot and weaken in humid conditions.

Many materials dissolve or corrode in salt water.Selecting materials for marine applications is anengineering specialty and such expertise shouldbe sought when building a marine facility.

Guideline 11:

Materials in aquatic facilities which are poten-tially toxic to fishes should be reduced to theminimum. Any toxic material should be listed,and the list must be available to staff.

Many common construction materials are toxicto fishes. Huguenin & Colt (2002) provide anexcellent review of the biological constraints tothe selection of construction materials. Materialsmay leach out or release specific ions, chemicals,or corrosion by-products from their surfaces. Allmetals should be sealed or be inert.

The application of metals in marine facilitiesrequires consideration of the type of metals andtheir interactions with both sea and fresh water.The effects of that interaction and the influenceof fish in the systems should also be considered.Aluminum, titanium, stainless steel, steel andcast iron are all commonly used in aquatic facili-ties, but each has its own unique characteristicswhich must be considered. Reviews of some ofthese characteristics can be found in Huguenin &Colt (2002).

If concrete structures are used, care must betaken to ensure the type of concrete is appropri-ate for its intended use. Marine grades of con-crete should be used and should be sealed to pre-vent the incursion of salt, which can penetrateand weaken concrete and internal support mate-rials such as steel rebar. Salt incursion is a majorcause of failure in submerged concrete.

Pipes, fittings and valves should not contain cop-per, nickel, brass, zinc or galvanizing treatmentsas their use can result in toxic concentrations of

heavy metals. Fisher (2000) provides concentra-tion ranges generally regarded as safe for fishculture. Polyvinyl chloride (PVC) pipes andother materials must meet human drinkingwater standards and must be adequately flushedto eliminate acetone, methylethylketones andtetrahydrofurans that are released following glu-ing (DeTolla et al., 1995). High-impact PVC cancontain lead and other toxicants, and should beavoided in fish holding facilities.

Any silicone sealant must be labelled as beingsuitable for use in aquaria (or have this clearlystated in other information available for theproduct) as there is a high risk of toxicity fromcompounds added to regular sealants to controlcuring and mould growth. If silicone sealant isused, it must be allowed to cure to release anyvolatile toxins.

Less obvious sources of toxicity include paints,untreated fiberglass surfaces, insulating materi-als and wood preservatives (Huguenin & Colt,2002). Toxicity can also be introduced into thewater directly from the air through condensationor aerosols. Creosoted pilings can cause prob-lems if located near water intakes. Overheadmaterial (air ducts, pipes, paints, galvanizedducts, etc.) must be chosen carefully as conden-sation can cause them to drip toxic products intothe water. Pressure treated wood must not beused inside due to its release of toxic vapoursand condensates.

Because of the difficulty in identifying potentialtoxicants, it may be useful to test new facilities oradditions with small groups of sentinel fish, suchas trout, before subjecting the majority of thefishes to the new conditions.

3.2 Room ventilation and airflow inaquatic areas

Guideline 12:

Air handling systems should be engineeredto ensure that aquatic areas are well ventilat-ed and humidity is controlled, and to ensurethat aerosol transfer between tanks andthrough the facilitity is minimized.

The science of air quality management in aquat-ic facilities is not highly developed. However, theairflow should be sufficient to ensure that sur-faces dry properly. Excess humidity hastens

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structural damage to tanks, room fixtures, etc.and encourages growth of pathogenic bacteriaand fungi. The ventilation should ensure a com-fortable working environment for personnel.Airflow directions should be designed to mini-mize the spread of potential aerosols and toallow for the safe handling of any dangeroussubstances.

Installation of any heating, ventilation and airconditioning (HVAC) system has the potential tointerfere with the ventilation in the other areas ofthe building and requires experienced profes-sional advice prior to and during installation.

3.3 Mechanical andelectrical requirements

Guideline 13:

All electrical systems must be professionallyinstalled to appropriate code standards (fed-eral, provincial/territorial and municipal build-ing codes) for operation in moist environ-ments, and must include proper groundingand ground-fault interrupters on all circuits.Extension cords should be avoided, and elec-trical wires should be fixed safely, away fromwater and from personnel circulation areas.

Guideline 14:

Electrical components and equipment shouldbe located outside the splash zone, andshould be housed in moisture-proof enclo-sures. Electrical fixtures should be securedwith gaskets to prevent incursion of water,and should be located above pipe runs.

Some equipment is designed to be submersible;however, any electrical component or equipmentmust have Canadian Standards Association(CSA) approval for its intended use. All groundfault interrupters should be tested on a frequent,regular basis.

Electric power and sea water are a dangerouscombination due to the extreme corrosivenessand the high electrical conductivity of sea water.All electrical equipment must be properlychecked for safe working condition before use inand around sea water. Care should be taken toavoid salt build-up on floors, and floors shouldbe pitched to promote drainage and avoid pud-dles. Electrical equipment should not be placed

under water pipes or tanks as condensation cancause water to drip on the equipment.

Guideline 15:

Machinery that produces noise and vibrationshould be isolated from areas housing fish.

Pumps and fans can be quite noisy and can causevibration. Where possible, all such equipmentshould be located outside the immediate locationwhere the fishes are housed, with sound andvibration attenuation. Reasonable access tomachinery for servicing should be incorporatedinto the designs. Ideally, all servicing of equip-ment should be conducted outside the facility tominimize excessive noise, disturbance and con-tamination of the environment around the fishes(Popper, 2003; Smith et al., 2004).

3.4 LightingGuideline 16:

Light should be phased on and off, andshould incorporate wavelengths and intensi-ties appropriate for the species where this is known. Where task lighting is needed forpeople working in the room, it should berestricted in its dispersion throughout theroom or be placed at a lower level than thetank surface.

Fishes are easily startled when light switches areactivated at night and may jump out of opentanks or incur injuries by bumping into the wallsof the tank. Having lights on automated dimmercontrols that allow the lights to be graduallybrought up in intensity over several minutes isimportant (Stickney, 1994; DeTolla et al., 1995).Lowering the daytime light intensity and havingsome residual light at night may eliminate theneed for dimmer controls. Care should be takento ensure that fish are not disturbed by night-time security lighting entering through windowsin the fish holding facilities.

Light influences, either directly or indirectly,almost all physiological and behaviouralprocesses in fishes, including growth, develop-ment (e.g., rate of smoltification in salmon), andreproduction. The response of fishes to light iscomplex and light often acts synergistically withother environmental factors, such as tempera-ture, making it difficult to predict the effect that

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inappropriate lighting regimes can have on thefishes or the experimental results (Stickney,1994). Also, while the influences of light are rea-sonably well understood in some species, e.g.,salmonids (Pennell & Barton, 1996), in many oth-ers little is known about how light affects behav-ior and physiology.

The natural history of the species, in particularthe normal swimming depth, can provide cluesto help meet the species preferences for thewavelength and intensity of light. Some speciesof fishes reportedly experience adverse effectsfrom some wavelengths of light. The output offluorescent lights can be diminished by usingdummy bulbs to reduce light levels. Securityand night lighting should not create visible shadows at night in fish tanks, as this can impact natural biorhythms. Screening or otherremedies should be used to protect the fishes'natural photoperiod.

3.5 Redundancy in aquatic lifesupport systems

Guideline 17:

All aquatic facilities should have an emer-gency contingency capacity, capable of main-taining aerated and filtered water and assur-ing the continuation of life support.

Generators or other emergency power sourcesshould be available to support vital functionsduring power shortages and should be testedregularly. Plans for longer-term power outagesmust also be in place.

Considerable planning is necessary to anticipatethe problems that develop in any aquatic facility,and to develop systems and strategies to mini-mize the consequences of failures. The strategiesemployed will depend on the size and type offacility. Knowledge of the length of time that thewater quality is maintained in the event of apower failure is necessary, in order to evaluatethe level of redundancy required. In addition,this will be dependent on the staff response timein the event of an emergency. As a minimum,back-up life support must be available for thelength of time it would require for staff to rectify the problem or to euthanize the fishes ifnecessary.

Guideline 18:

Critical systems, including pumps, should beduplicated to ensure that failures cause onlyminimal interruptions in service.

Main system water pumps, filters and otheressential life support components should haveback up so that they may be replaced withoutaffecting the supply of water or operation of the system. It is often better to use spare equip-ment regularly to ensure its operational efficien-cy. If water lines are critical, they should beduplicated.

Depending on the complexity of the system andthe survival time of the fishes within the systemin the event of an emergency, facilities may relyon observation or alarm systems for alerting staffto changes in critical parameters (see Section D.3.Environmental Monitoring and Control).

Once the monitoring system detects a failure, thecause(s) should be corrected as soon as possible.

4. Types of Systems

The choice of system (i.e. flow-through, recircu-lation or static tanks) should take into considera-tion the types of studies which are intended to becarried out in the system.

It is recognized that some research will requireestablishment of simulated ecosystems. Thisshould be justifiable and have direct benefits toresearch.

Aquatic systems are either static or flowing.Flowing systems can be either recirculation orflow-through systems. Flow-through or single-pass systems are ones in which water continu-ously enters, flows over and exits the areas occu-pied by fishes. Recirculation systems are systemswhere a proportion of the water exiting the sys-tem is recycled after it receives treatment torestore its quality. An intermediate type of sys-tem involves the constant addition of new wateralong with the re-use of some percentage of thewater in the system (Fisher, 2000).

The engineering requirements for flow-throughand recirculating systems differ substantiallybecause the influent (source water) and effluent

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water quality requirements vary (Fisher, 2000;Huguenin & Colt, 2002). Fisher (2000) provides aflow chart of the elements involved in the twotypes of systems.

Guideline 19:

An adequate water supply of suitable qualityshould be provided for the fish at all times.

Water flow within a system should be sufficientto remove suspended solids and wastes and toensure that water quality parameters are main-tained within acceptable levels. Water flow alsoshould be appropriate to enable fishes to swimcorrectly and to maintain normal behavior.

For static tank systems, debris should besiphoned off on a regular basis, in order to main-tain water quality parameters within the prede-termined range for species-specific requirements.In addition, water should be regularly removedand replaced to avoid the build up of nitroge-nous material, which can become toxic.

4.1 Flow-through systemsFlow-through systems offer many advantagesfor fishes used for research or testing, and areless complex to build and operate than recirculat-ing systems (Fisher, 2000). However, they requirea continuous supply of large quantities of waterof a consistently high quality. Potential sources ofwater include municipal supplies, wells (eitherfresh or sea water) or natural bodies of water.Caution should be exercised in use of municipalsupplies as the presence of chlorine can kill fish-es in culture. Section C.2. Water Quality, discuss-es water quality and the parameters which areimportant to measure and control irrespective ofthe type of system.

Systems pumped directly from the water source(e.g., ocean, lake or river), rather than from awell, present additional challenges for ensuringstable water quality. Depending on the seasonand tidal cycles, there may be problems such astemperature fluctuations, organic/biotic content,corrosion/fouling of supply system, presence ofcontaminants, salinity fluctuations, pathogensand disease, toxins from algal blooms, etc.

Effluent from flow-through systems may requiretreatment before release to ensure that biological

and chemical waste does not exceed environ-mental standards and that potential diseaseorganisms are not released (Ackefors et al., 1994;Fisher, 2000). It is expected that all municipal,provincial and federal waste disposal standardswill be met.

4.2 Recirculation systemsWater management in closed systems is complex.Pennell & Barton (1996) and Huguenin & Colt(2002) provide useful reviews and bibliographiesof water re-use systems; however, since recircula-tion technology is changing rapidly, system man-agers need to stay up-to-date on innovations.

Recirculating systems involve the complete orpartial re-use of water by circulating it through atreatment system that reduces biological andchemical waste loads to levels comparable to theinfluent water (Fisher, 2000). Recirculation sys-tems use a series of processing stages that pro-vide some or all of the following: 1) separation oflarge suspended solids (uneaten food and fae-ces); 2) fine filtration; 3) processing to reduce sus-pended solids, chemical oxygen demand anddissolved organic carbon, and to re-oxygenatethe water; 4) removal of dissolved organic mate-rials (foam fractionation); 5) disinfection; 6) bio-logical filtration/nitrification/denitrification;and 7) toxicant removal with activated charcoal(Fisher, 2000). While stages 1 to 7 are ideal, itshould be noted that some partial re-use systemsdo not (and cannot) use all these treatment orprocessing stages.

Filters, particularly for marine systems, shouldbe as large as necessary to maintain appropriatewater quality, even up to the same volume as theholding tanks, depending on stocking densityand species. Regular changing of charcoal filtersis required before saturation occurs to ensurethat toxicants are not released back into thewater.

4.3 Static systemsStatic systems are systems in which no newwater is continuously added from outside orrecirculation sources. For this reason, these sys-tems are sometimes called 'bath systems'. Staticsystems are often challenging to maintain, havevery low inherent biological carrying capacityand are prone to water quality changes.

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To assure water quality is maintained in staticsystems, the following measures should be taken:

• acceptable limits for water quality parametersshould be pre-defined;

• water quality parameters and animal condi-tion must be frequently monitored;

• regular partial volume water changes shouldbe used to improve water quality, along withdevices which create water movement suchas corner filters, undergravel filters, hang fil-ters and internal impeller or jet pumps;

• establishment of beneficial bacterial commu-nities should be encouraged by housing asmall number of fish initially to mature thewater. These bacterial communities should bepreserved where possible by rinsing filters inseasoned water, rather than in chlorinated tapwater which can kill the bacteria;

• regular tank cleaning and vacuuming todecrease bioburden from food and faeces; and

• the use of low stocking densities of aquaticorganisms, as the nitrogen loading of staticsystems is rapid and the denitrification poten-tial is low.

4.4 MesocosmsWhen dealing with large volumes of water (usu-ally 50,000L and above) to recreate aquaticecosystems (mesocosms), it should be recog-nized that they may not operate in the same wayas smaller tanks usually found in aquatic facili-ties. In particular, special considerations areneeded depending on the material the mesocosmis made from (e.g., dug ponds and floatingmarine bag systems have very different require-ments): water turnover rates may not be calculat-ed the same way as smaller holding tanks; spe-cial requirements may be needed for cleaningand removal of debris and for catching the fish;and considerations need to be given to theimpact of solar exposure (e.g., build up of algae).It is therefore important that these systems beoperated by individuals with the relevant experi-ence. As for any other aquatic facility, theyshould be operated in a manner that does notnegatively impact on the health and well-beingof the animals. Therefore, the general guidelines

concerning the use of fishes for research and test-ing also apply to mesocosms.

5. Fish Housing

5.1 Fish well-beingGuideline 20:

Aquatic environments should be designed tomeet the established physical and behavioralrequirements of the fishes in terms of shelter,social grouping, overhead cover and lighting.

Many fishes have requirements for environmen-tal conditions that will provide optimal welfare,and where feasible, these should be included inthe design of aquatic environments for fishes.Social behavior and social influences on behaviorcan be quite complex, and require investigatorsand animal care staff to have a good understand-ing of the species-specific requirements of theanimals. For example, many species of flatfishand eel do best in long-term holding when pro-vided with burrow type environments. Inaggressive species, the provision of vertical bar-riers or shelters may eliminate or curb aggressivebehavior.

Where the environmental requirements of fishare not well known, as far as possible the holdingconditions should be designed to approximatethe source environment. Long-term holding ofsuch species should be considered experimentalrather than routine, and should be conductedwith increased oversight and monitoring of indi-cators of well-being such as feed intake andgrowth.

Consideration should also be given to popula-tion densities, water flow rates or other physicalfeatures that may have an effect on social interac-tions. However, adding complexity to the aquat-ic environment should be balanced againstthe need to maintain a high standard of waterquality.

5.2 Tank/enclosure designGuideline 21:

The shape, colour, depth and volume of tanksshould be appropriate for the species and lifestage being held.

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Tank design should take into account speciespreferences. For some larger species, particularlybottom dwellers, the surface area of the tank ismore important than the volume or depth ofwater. However, fishes that occupy the entirewater column depend more on the volume ofwater in the tank. Fishes that maintain their posi-tion by orienting themselves to a directional cur-rent or flow require oval or round tanks, whilespecies that need considerable space for swim-ming, such as the common carp, prefer an oblongtank. The depth of the tank can also be importantas some species will not feed in shallow tanks.Smaller species, such as zebra fish are commonlyhoused in rectangular glass aquariums.

The natural habitat and behavior of the fishesshould be considered in selecting the appropriatecolour of the tank. For example, fishes that areadapted to camouflage with the bottom of thetank require tank colours within their naturalrange of experience in the wild.

Most species adapt well to fiberglass (FRP), plas-tic, metal or concrete tanks or raceways, andmany species can be reared without difficulty incages or net-pens with mesh bottoms (Stickney,1994). Concrete is an excellent material for fishtanks, but should have an appropriate liningwhich protects the fish from leachants and pre-vents water from penetrating the concrete. Woodshould not be used as a tank material in contactwith system water as it is a porous material thatmay contain toxic elements (in particular, pres-sure treatment or glue in plywood), is subject torot, and requires the use of sealants, which can betoxic. Vinyl tanks are only suitable for temporaryholding, as the plasticizers can be toxic and vinyloften contains contaminants. Glass is also suit-able for tanks, but as with other clear materials,particular consideration should be given to theeffect of external stimulation of the fishes. Forexample, some fishes are stressed if placed inglass tanks with no substrate on the bottom.Nonetheless, glass tanks are particularly usefulfor studies where good visibility is essential (e.g.,studies with a behavioral component or toxicolo-gy studies).

Newly manufactured tanks require a condition-ing/depuration period to flush out solvents(Schreck & Moyle, 1990).

Guideline 22:

Tanks should have smooth, inert, sealed inte-rior surfaces.

The need for a smooth, inert, sealed surface tofacilitate tank cleaning should be balanced withthe preferences of the fish for a more complexenvironment. For example, in some experiments,investigators may use gravel at the bottom of thetank to replicate the natural environment, andthis can be removed to permit thorough cleaningof the tank and substrate.

Guideline 23:

Tanks should be self-cleaning, or adequatemeans for the regular cleaning of tanksshould be incorporated into the design.

Good water conditions should be maintained, asretention of wastes in fish tanks promotes theproliferation of pathogenic bacteria, protozoa andfungi, and leads to oxygen depletion. The accu-mulation of ammonia is a prime issue in deter-mining the frequency of cleaning necessary, asun-ionized ammonia is extremely toxic to fishes(See Timmons et al., 2001, particularly Chapter 5;and Section D.3.1 Management of water quality).

The turnover time of water in a holding tank isimportant from the perspective of tank hygiene.The recommended turnover time is a function ofspecies, stocking density, social behavior, dis-solved gas levels in influent water, the tank con-figuration and feeding frequency. Tank turnovertimes should also consider rates of oxygen con-sumption and ammonia production. Sprague(1969) provided a nomograph to estimate 90 to99% molecular replacement times based on flowrate and tank volume. Simple arithmetic divisionof tank volume by water inflow rate does notyield replacement time; replacement time is con-siderably longer (Sprague, 1969). Turnover timesshould ensure that water quality is maintained.Where increasing the water flow is not possible,aeration of the water and regular cleaning of theholding tank should be performed.

Guideline 24:

Tanks should be equipped with a coveringthat prevents fishes from jumping from thetank, e.g., tank nets or rigid coverings.

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Tank lids also serve as an important barrieragainst the introduction of any foreign objects,animals or chemicals. The height between thewater surface and lid should be such that it min-imizes the risk of damage to the fish if they jump.

The ability to visually assess all fishes in tanksand incoming water flows to each tank is essen-tial. Where tanks are covered, visual accessshould be easily attained through removal or

partial removal of the lid. Lids may also be con-structed of materials such as plexiglass or clearacrylic which permit visual observation and aresuitable for contact with aquatic animals.

Tank supports must be properly designed,strong, sturdy and durable, given the consider-able weight of water and the potential catastoph-ic effects of any collapse on staff, animals and thebuilding. Transfer of weight to the floor structureshould be taken into account.

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Well managed aquatic facilities will have a regu-larly scheduled preventive maintenance pro-gram for all life support systems, as well as anannual maintenance program for all other equip-ment and for surfaces. The day-to-day operationof the facility, such as scheduled sanitation meas-ures, feeding schedules, and environmental andfish health checks, should be conducted in a stan-dard fashion. The development of standard oper-ating procedures (SOPs) for management offacilities is strongly encouraged to ensure consis-tency (CCAC, 2000b).

1. Security and AccessGuideline 25:

Access to fish facilities should be designedto minimize traffic through the area. Accessshould be restricted to those personnelrequired for maintenance of the facility andthe care of the fishes, and those using thefacilities for experiments or teaching.

A security system should be in place that isappropriate for an aquatic system. Environmentswith high humidity and salt in the air can causeproblems for card access and keypad securitysystems.

Personnel should access fish facilities only whennecessary. Movement through the facility shouldbe from the cleanest areas to the dirtiest areas.Facility-specific clothing and footwear should besupplied in the entry area, and personnel shouldwash their hands as soon as they enter the facility.

2. General Maintenance ofthe Facility

Guideline 26:

All architectural and engineering specifica-tions and drawings of the facility should beavailable to those in charge of running thefacility, as should all operating manuals forspecial equipment such as pumps, chillersand computer control systems.

Inventories of necessary spare parts for all essen-tial facility components should be maintained.

Guideline 27:

Aquatic facilities must have written mainte-nance schedules developed specifically forthe facility.

A documented preventive maintenance programis required for all life support systems.

Both routine maintenance and equipment over-haul and replacement should occur while theequipment is still operating normally. All electri-cal equipment, life support equipment and airand filtration systems should be checked andserviced at regular intervals. Checklists shouldbe available to ensure the duties have been com-pleted and that there is a record of all service(Shepherd & Bromage, 1988).

Guideline 28:

Facilities should be kept in a clean and order-ly manner.Tanks should be disinfected beforeand after every experiment.

Guideline 29:

The staff responsible for operating an aquat-ic facility should have the specialized knowl-edge, experience and training for proper func-tion, operation and maintenance of the watersystem.

Guideline 30:

Sufficient numbers of staff must be availablefor animal care and facility management andmaintenance 365 days a year for both routineand emergency needs.

The inherent nature of aquatic facilities requiresthat there be constant maintenance, repair andupgrading. Trained competent staff need to be oncall and accessible 24 hours a day, 7 days a week.In particular, facilities must have a method forassuring a rapid emergency response by staffoutside of normal operating times.

D. FACILITY MANAGEMENT, OPERATIONAND MAINTENANCE

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3. Environmental Monitoringand Control

Guideline 31:

An environmental monitoring system is essen-tial for aquatic facilities and should be designedto suit the water management system.

Water management involves a great manymechanical and electrical components, the mal-function of which can quickly result in stressfuland possibly lethal consequences for fishes.

Many facilities have simple systems where unex-pected environmental changes are unlikely tooccur. These facilities should not need to estab-lish costly water monitoring systems for a rareevent, and in these situations monitoringthrough regular visits by custodial staff is usual-ly sufficient. For small scale aquatic facilities,routine monitoring may simply consist of dailyvisual system and animal checks, and limitedtesting with hand-held equipment such as a ther-mometer, dissolved oxygen (DO) meter and a pHmeter.

Large sophisticated systems, however, willrequire extensive, generally computer-based,monitor and control systems with redundant andfail-safe modes and automated emergency con-tact systems. Remote monitoring of water quali-ty can reduce the number of visits per day intoholding rooms, which is particularly relevant forquarantine rooms and during experiments.

Guideline 32:

Water quality monitoring systems should beable to detect and react to changes in waterquality before they become life-threatening toanimals housed in the system.

Early detection of physiologically significantfluctuations in environmental conditions isimportant through automated or repeated manu-al testing. Environmental change is more likelyto occur in the following circumstances: waterquality is known to fluctuate based on previoustesting; previous occurrences of compromisedfish in studies with similar holding conditions; orwhen large complex systems depend on manycontinual water quality adjustments (e.g., recir-culated systems for large numbers of fish). It is

critical that all malfunctions be immediately rec-ognized by staff, or that staff are alerted by auto-mated systems, so that corrective action can betaken. Remote monitoring of water quality canreduce the number of visits per day into holdingrooms, which is particularly relevant for quaran-tine rooms and during physiologically-sensitiveexperiments.

Guideline 33:

Water quality parameters should be moni-tored at an appropriate frequency for thefacility, and should allow predictive manage-ment of water quality, rather than only reac-tive management of crises in water quality.

Parameters that need to be measured and the fre-quency of measurement vary greatly, dependingon whether the system is an open or a recircula-tion system, and whether it is a seawater orfreshwater system (Fisher, 2000; Huguenin &Colt, 2002). At a minimum, environmental moni-toring systems should provide information onwater flow or oxygen saturation and water tem-perature. Examples for frequency of testing arelisted in Appendix D; however, these are sug-gested measurements only. For example, whilethere may be no need to measure nitrites/nitratein a high volume flow-through system (depend-ing on the source of the water), such measure-ments are critical with recirculation systems.Parameters measured should also be relevant tothe health of the species housed in the system,and taken at a frequency that allows adjustmentsto be made well in advance of catastrophic mor-bidity and mortality (Wedemeyer, 1996a). Inaddition, the ability to conduct rapid tests isimportant when a change in the water quality issuspected.

In general, recirculation systems should be mon-itored for a larger number of parameters includ-ing, but not restricted to, dissolved oxygen, tem-perature, salinity (marine systems), pH, ammo-nia, nitrite, nitrate and total dissolved solids(Fisher, 2000).

When water quality analysis is done, it should beat a time reflecting greatest demand on the sys-tem (usually after feeding) to identify potentialproblems.

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Guideline 34:

Good water quality measuring equipmentshould be available, regularly calibrated andwell maintained. Records of water qualityshould be maintained and should be retriev-able for retrospective analysis in the event ofproblems.

3.1 Management of water qualityGuideline 35:

Water quality must be monitored and main-tained within acceptable parameters for thespecies being held.

Water quality, in the context of fish holding sys-tems, refers to all the factors—physical, chemicaland biological—that influence the well-being ofthe animals. The term "quality" implies that nofactor should exceed concentrations likely to betoxic in the context of the facility or study, or failto remain within the species-specific range forlife-sustaining factors (Ackefors et al., 1994).

In experiments, it is important that water qualityparameters remain relatively constant through-out the initial holding period and the experimen-tal period (except, of course, for the variablesthat are being manipulated experimentally).

The most common water quality factors knownto affect fishes are temperature, dissolved oxy-gen, pH, suspended solids/sediment, carbondioxide, nitrogen supersaturation, ammonia,nitrite, nitrate (Wedemeyer, 1996a; Kreiberg,2000) and chlorine. These must be monitored ona regular basis; close monitoring is particularlyimportant in closed recirculation systems.

The definition of acceptable range is complicatedby the fact that appropriate conditions are notwell-defined for many species and the require-ments of individual species may vary betweendifferent life stages (e.g., larvae, juveniles andadults) or according to physiological status (e.g., spawning, feeding and previous history ofexposure).

Water quality is the most important factor inmaintaining the well-being of fishes and inreducing stress and the risk of disease. Fishesshow varying degrees of flexibility to changingwater quality conditions. Some degree of accli-

mation may be necessary when transferring fish-es and this should be carried out over as long aperiod as possible.

Preferences and tolerances for some species offishes are known. For other species, preferencesmay be unknown, and may require pilot studiesto determine the appropriate conditions. Thesepilot studies should be overseen by the veteri-narian or experienced fish health expert and theACC.

3.2 TemperatureGuideline 36:

Fishes should not be subjected to rapidchanges in temperature, particularly to rapidincreases in temperature.

Fishes are ectothermic, meaning that their bodytemperature is similar to that of their environ-ment. Therefore, water temperature is a veryimportant water quality parameter and highlyspecies specific. All vital functions are influencedby body temperature, and the rates of these func-tions increase or decrease according to the sur-rounding water temperature. All species of fish-es have a specific temperature range in whichthey function normally and maintain goodhealth. The temperature tolerance ranges of fish-es vary greatly, both among species and life his-tory stages, and fishes are often referred to ascold (0 to 10ºC), cool (10 to 20ºC) or warm (20 to30ºC) water species, depending on the thermalregime to which they are naturally adapted.Most fish species can tolerate a range of temper-atures, although each fish species has its ownStandard Environmental Temperature.

The term "rapid changes" is very species specific.Even within the same species it varies, as it isdependent on the temperature of the water inrelation to the thermal maximum for the fish.This is different between summer and winter. Inpeak summer temperatures, a change of 5ºC maybe too much, whereas in the middle of winterwhen the ambient temperature is low, adjust-ment may be easier. As a general rule of thumb,temperature changes should not exceed morethan 2ºC per 24-hour period.

Changes in ambient temperature for fishes aremuch more significant to many vital functions

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than in the case of land animals. Susceptibility todiseases, parasites, and toxicants is greatly affect-ed by temperature. The further the temperatureshifts in either direction from the optimal range,the greater the potential for stress and disease.

The term "acclimation" is used widely to describeany "adaptation" to changed circumstances.However, it must be understood that true accli-mation of fishes to a new temperature is aprocess that involves production of new variantsof many metabolic enzymes, changes in lipidtypes and actual cellular restructuring. Thisprocess will generally have advanced substan-tively after 24 hours at the new temperature, butmay require as much as 6 to 8 weeks for comple-tion. The duration of acclimation is a tempera-ture-dependent process. At lower temperatures,the actual change in physiological processes willoccur at a slower rate. One to two weeks acclima-tion may be fine for salmon at 10°C, but at 5°Cacclimation is likely to take longer. It will varywidely among species as to rate and scope; somespecies simply lack the ability to acclimate to thenew temperature. Hochachka & Somero (1971)provide a comprehensive overview of thisprocess.

3.3 OxygenGuideline 37:

Fishes should be kept in water with an ade-quate concentration of oxygen.

In most species, water O2 saturation levelsshould be above 90%, although some speciesthrive at lower O2 concentration. Some species(air-breathing fishes) have evolved methods ofextracting oxygen from air, and a subset of thesefishes will drown if not allowed access to air,underlining the importance of understanding thespecific requirements for the species of fish to beused. In general, cold water fishes have lowertolerance for low oxygen levels than warm waterspecies of fish.

Oxygen concentration will vary according totemperature, atmospheric pressure and salinity.As the temperature increases, the water's capaci-ty to carry oxygen decreases; in addition, thefishes' demand for oxygen increases due to anincrease in metabolic rate. A variety of other factors can dictate the amount of oxygen

required by a fish; for example, the age, healthand activity rate of the fish, as well as any han-dling procedures.

The congregation of fish at the tanks' water inletor gasping behavior at the surface is an indica-tion of insufficient oxygen.

In some instances, low O2 levels can be remediedby aeration, reducing the stocking density anddecreased feeding. Balancing these variables isessential to prevent low O2 levels. Airstones canbe used to improve the aeration of the water;however, placement and type of stone should bechosen so as not to disrupt the self-cleaningaction of the tank. Better quality airstones pro-duce smaller bubbles with less agitation of thewater column and are superior in oxygen trans-fer. Where necessary, supplementary oxygena-tion of tank water should be provided.

3.4 SupersaturationGuideline 38:

Aquatic systems are susceptible to acute orchronic supersaturation. Individuals respon-sible for operating aquatic systems shouldunderstand the causes of gas supersatura-tion and how to mitigate potential problems.

Supersaturation of water is a possibility in anyfish holding system; therefore, fish care staffshould be aware of the acute signs of supersatu-ration, both in the fish and within the facility.Supersaturation is a condition where the totalgas pressure in a body of water exceeds the baro-metric pressure in the overlying atmosphere. Itmay arise due to an excess of any one or moregases present in the water, and is best measuredby devices which assess the sum of all partial gaspressure in water (e.g., saturometers). Supersatu-ration is discussed in detail by Colt (1984, 1986).

Water can become supersaturated with dissolvedgases under a variety of conditions, includingwhen water is heated in a closed vessel or expe-riences a pressure change, or when gas (includ-ing air) is compressed into water. Water may besupersaturated in a source location which canfurther complicate the situation. Although oxy-gen and carbon dioxide can become problematicwhen supersaturated, dissolved nitrogen is per-haps the most dangerous. It is advisable that

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total gas levels in fish holding tanks be moni-tored on a regular basis.

Gas bubble disease can occur when fishes areexposed to supersaturated water; fishes absorbthe gas and it is then released from suspension inbody fluids, forming internal bubbles (Speare,1998a). Sub-clinical supersaturation can be achronic stressor for fishes; therefore, using acutegas bubble disease as an endpoint for tolerablelevels is inadequate. The biological response togas supersaturation varies with species, life stage,water quality, and the animal's depth in the watercolumn (Colt & Orwicz, 1991). Affected fishesmay show a variety of signs, including bubbles ofgas under the skin, between fin rays, in fin tipsand in gill tissue. Gas bubble formation in capil-lary beds causes ischaemia and tissue necrosis,and bubbles in the circulatory system, includingin the heart and brain, cause rapid death.

Supersaturation can be reduced in a number ofways. The preferred methods of addressingsupersaturation involve passing the waterthrough trickle columns packed with surfacearea rich media such as pea gravel or bio-rings,vacuum degassing or O2 injection. Other meth-ods include vigorously breaking up the water toallow for the escape of excess gases into theatmosphere or the use of spray bars.

Facilities should have the ability to rapidly checkthe dissolved gas saturation levels in the event ofacute morbidity/mortality incidents. It is criticalthat staff understand the causes of supersatura-tion and the means to mitigate the problem.Several good references are available on thetopic, e.g., Colt & Orwicz (1991), Pennell &McLean (1996) and Huguenin & Colt (2002).

3.5 pHGuideline 39:

Water pH should be maintained at a stableand optimal level as changes in pH may influ-ence other water quality parameters.

The pH of water will vary greatly according toits source and composition. In addition to theinfluence of naturally occurring minerals (e.g.,contact with silicates will lower the pH, whereaswater flowing through carbonate rock will havea higher pH), industrial pollution will alter pH

levels of water supplies. A number of factors caninfluence pH, including the addition of sulphurdioxides and nitrogen oxides, sewage and agri-cultural run off, compounds that are added orbecome introduced to the water, and CO2 fromthe atmosphere and from the respiration of thefishes.

Most fishes can adapt to a wide range of pH, pro-vided that any pH change is gradual. The major-ity of freshwater species live in waters with pHvalues ranging from pH 6 to 8. Outside the rangeof pH 6 to 9, freshwater fishes become stressed,grow slowly and are prone to infectious diseases.The range for saltwater fishes is narrower: pH 7.5to 8.5. The optimal pH range for maintainingfreshwater fishes is between 6.5 and 7.5. Theoptimal pH range for maintaining marine fishesin natural seawater is between 8.0 and 8.5. ThepH range for maintaining marine fishes in syn-thetic seawater is 7.5 to 8.5. Although seawaterhas a high buffering capacity, marine animalsproduce acidic waste, reducing the pH value ofthe water over time. The fall can be limited bycarrying out partial water changes (i.e. 10% overa two-week period). An alternate but less effec-tive method is the addition of sodium bicarbon-ate to add carbonate buffering capacity. It is alsoimportant to consider the optimal pH for growthof bacteria within the biological filters in recircu-lation systems.

The pH of the water has a considerable effect onother important parameters of water quality.Most importantly, water pH and ammonia levelsare closely related, which is especially relevant inrecirculation systems. Ammonia is much lesstoxic at lower pH levels. For this reason, it isadvisable to maintain the systems at the lowestpH suitable for the species.

3.6 Nitrogen compoundsGuideline 40:

Free ammonia and nitrite are toxic to fishesand their accumulation must be avoided.

Ammonia toxicity is extremely pH (H+) depend-ent, and therefore, control of pH and feed man-agement are critical in minimizing ammoniaaccumulation.

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One of the main excretory products of fishes isammonia which is discharged into the waterthrough the gills and urinary tract. Dissolvedurea, as well as particulate wastes (feed and fae-ces), are converted to inorganic compounds suchas ammonia and phosphate. Other sources ofammonia may be from contamination of waterby organic compounds such as antibiotics, paintfumes, fumes from ammonia-based cleaningcompounds and insecticides.

In aqueous solution, ammonia is present in twoforms: ionized (NH4

+), and un-ionized or "free"(NH3). The term ammonia refers to the sum ofthe total concentration of the ammonium ion(NH4

+) plus the concentration of free ammonia(NH3). NH3 and NH4

+ are in constant equilibri-um. It is much more important to know the con-centration of un-ionized ammonia, as only theun-ionized (non-ionized) form is toxic to fishes.For a review of allowable limits of ammonia forfish, see US Environmental Protection Agency(1999).

Avoiding ammonia accumulation, in particularwithin recirculation systems, is very difficult.However, more important than the accumulationof nitrogenous products (ammonia and nitrite) isthe balance of these products in relation to thepH of the water (Ip et al., 2001). The physiologi-cal effects of ammonia and nitrite toxicity havebeen reviewed by Speare (1998b). Other meas-ures of minimizing ammonia include increasedflushing rate, biofiltration (i.e. filtering the waterthrough a matrix of nitrifying bacteria to convertammonia to nitrite and then immediately tonitrate), fish density or temperature reductions,or use of ammonia-absorbent compounds infresh water.

Aside from control of pH and good feed manage-ment practices, other measures are usually anessential element of recirculation systems.Biological filters provide the substrate necessaryfor colonization by bacteria (for exampleNitrosomonas and Nitrobacter) that oxidize ammo-nia. The substrate can be media such as zeolite,crushed oyster shell, sand, dolomite, pea gravel,or synthetic substances (e.g., styrofoam beads,plastic rings or fiber filters). In general, the larg-er the surface area to volume ratio of the filter,the better the filter, bearing in mind that an effec-tive filter needs to optimize the balance between

water flow and surface area. However, smallparticles such as sand can clog the system andtherefore sand-based filters are more difficult tomaintain.

Nitrifying bacteria are sensitive to sudden pHchanges and do not generally grow well outsidethe range of pH 7.2 to 8.5. Their growth canalso be affected by chemicals used for diseasetreatment, as well as by sudden changes intemperature.

Ammonia can be stripped from fresh water usingan ion exchange apparatus. This involves pass-ing the water through a bed of zeolite crystals totrap the ammonia which is subsequentlyreleased as gas.

3.7 Carbon dioxide

Carbon dioxide is produced by fishes during res-piration and dissolves in water to form carbonicacid, thus lowering the pH and increasing thepotential for hypercapnea. Although high CO2concentration can be fatal to fishes, in generalthis is not likely to be a problem in aquatic facil-ities, provided there is adequate ventilation.However, when recirculation technology isapplied to high density fish culture systems, CO2may become the major limiting factor.

3.8 SalinityGuideline 41:

Salinity changes are inherently stressful for fishes, and should be conducted slowlyand with attention to the physical status ofthe fishes.

Salinity requirements of fishes vary according towhether they are marine or freshwater in origin.Some species are able to tolerate a wide range ofsalinity, others can tolerate only narrow ranges ofsalinity. In others, salinity tolerance may varyaccording to life stage (e.g., Atlantic salmon).Maintaining fishes at a suboptimal salinity canresult in osmoregulatory stress, impaired growthrates and reduced disease resistance. However, itshould be noted that rapid, dramatic, short-termsalinity shifts are sometimes used as therapeutictreatements.

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3.9 Toxic agentsGuideline 42:

When there is reason to believe hazardousmaterials or infectious agents have acciden-tally entered the water system, that systemshould be isolated and tested.

A host of infectious and toxic agents exist that arepotentially harmful to fishes. Susceptibility toboth chronic and acute toxicants vary withspecies, life stage, acclimation conditions, andother environmental conditions (e.g., tempera-ture, water hardness, etc.) (Barton, 1996).

Common problems encountered in water sys-tems include toxicity from chlorine and otheradditives, copper from copper pipes, and gassupersaturation. Systems that rely on ambientfresh water or seawater may undergo seasonalincreases in bacterial burden and the presence ofpathogenic bacteria often originating in sewage.Other problems can result from the use of flysprays, paints, solvents, etc.

For most toxic agents, there is often no localexpertise in regulatory or university depart-

ments to recognize when such problems exist, toperform the analyses needed to verify and quan-tify the problem, or to recommend or implementsolutions in a timely fashion. When a toxic agentis known to have entered the system, there arerarely defined solutions, other than to "flush thesystem". The laboratory personnel are most like-ly to be able to deduce what the problem is (e.g.,copper from a new source of copper pipe); how-ever, other experts should be consulted foradvice or help, including veterinarians, fishhealth experts, regulatory personnel or analyticallaboratory personnel.

Guideline 43:

Chemical products should be safely storedaway from the aquatic housing area and thewater supply.

As many paints, insecticides, cleaners, fixatives,adhesives, caulking agents or solvents are toxic,great care should be taken in their use aroundaquatic facilities.

Occupational health and safety guidelines andother regulations (e.g., dangerous goods regula-tions) must be followed when storing chemicals.

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Irrespective of the source of fish, investigatorsshould ensure that the relevant regulations forcapture and acquisition are followed (see SectionB.5. Government Regulations and Policies on theUse of Fish).

1. Capture of Wild Stock

Whether fishes are being collected for live study,preserved for study in a museum, or beingprocessed to obtain data needed for fisheriesmanagement field studies, investigators shouldobserve and pass on to students and employeesa strict ethic of habitat conservation and respect-ful treatment of the animals. Research goals willgenerally dictate the appropriate samplingmethod; however, investigators should select themethod that has the least impact on the fishesand on the local ecosystem.

Guideline 44:

Wild fishes should be captured, transportedand handled in a manner that ensures mini-mal morbidity and mortality.

Investigators should be aware that the stress ofcollecting, handling and transporting fishesfrom the wild can make them susceptible to dis-ease. Modifications to capture techniques maybe required to improve survivability of the fish-es. In particular, electrofishing is a stressful pro-cedure, leading to acute physiological distur-bances, increased susceptibility to predation(Schreck et al., 1976) and significant effects onother taxa in the vicinity (Bisson, 1976). Lessstressful procedures should be used wheneverpossible.

The largest number of fishes used in research areobtained from private or government hatcheries,but other genera of wild or exotic fishes may berequired. Where fishes are to be taken from thewild, all necessary collecting permits and author-izations must be obtained before starting the

project. Local fisheries/conservation officersmust also be notified.

Guideline 45:

Where exotic fishes are obtained from aquar-ium suppliers or collection sources, local,provincial/territorial and federal authoritiesshould be consulted to determine the risk ofescape, accidental introduction, exotic dis-eases and other detrimental outcomes, andhow to minimize these risks.

Investigators should be aware of the list of nativespecies that are endangered, threatened or vul-nerable. Lists of protected fishes in Canada can befound at: www.speciesatrisk.gc.ca/default_e.cfm.

2. Killed Specimens

The numbers of fishes collected should be theminimum number required to accomplish thestudy, and each animal collected should serve asmany types of study as possible. The mosthumane euthanasia techniques should be used,bearing in mind the health and safety of the per-sonnel (see Section I. Euthanasia). Fish should beeuthanized prior to immersion in formalin orother preservatives.

3. Piscicidal Compounds

Guideline 46:

Alternatives to the use of piscicidal com-pounds should be sought, such as anesthet-ic agents with minimal environmental andnon-target species impacts.

On rare occasions, it may be necessary to use pis-cicidal compounds in field situations to capturedead specimens. If piscicidal compounds are tobe used, an impact analysis should be carried outprior to use to determine the potential localeffects (e.g., by-catch).

E. CAPTURE, ACQUISITION, TRANSPORTATIONAND QUARANTINE

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4. Acquisition of Hatchery FishGuideline 47:

Fishes should come from hatcheries withdefined health status and preferably knowngenetic history. Hatcheries should be encour-aged to develop husbandry and managementpractices consistent with those used in theproduction of other laboratory animals.

In the interests of obtaining high quality researchanimals, high quality fishes should be sourcedfrom reputable fish suppliers. Where possible,site visits of the hatcheries should be carried out,in order to provide quality assurance of theirprocesses and practices. Institutions are encour-aged to develop a list of reputable fish suppliers.

5. Transportation

High survival rates should be obtained evenwhen fishes are transported long distances.Success in transporting fishes requires prevent-ing the physiological problems which can becaused when relatively large numbers of fishesare held in relatively small volumes of water(Wedemeyer, 1996a; Wedemeyer, 1996b). Themajor challenge in transporting fishes is themaintenance of appropriate water quality. A life-support system must be provided that will pre-vent adverse water quality changes and meet thephysiological requirements of the fishes.

Shepherd & Bromage (1988), FAWC (1996) andWedemeyer (1996a; 1996b) provide detailedinformation on fish transportation.

Some of the critical elements required for fishtransportation are:

• The transportation container should be wellinsulated to minimize temperature changesduring transport; in some cases heat or refrig-eration may be required to ensure tempera-ture is maintained within the appropriaterange for the species.

• All containers should have opaque lids tominimize slop and loss of fishes, and toreduce light levels within.

• Whenever possible before transport, fishesshould be fasted for 12 to 48 hours, depend-

ing on species, age and water temperature, toensure an empty gut and minimize nitroge-nous waste and water pollution, and to con-serve metabolic energy.

• It is common practice to cool the water tem-perature to reduce fish activity and metabo-lism during transport.

• Once loaded, and at regular intervals duringtransport, the behavior of the fishes, transporttank temperature and oxygen levels shouldbe checked to ensure there are no problems.On arrival, particular care should be taken tocheck water temperature to ensure that fishesare not exposed to temperature shock duringtransfer.

• For transport of large fish shipments or wheremore than minimal distances are being trav-eled, it is advisable to use fish transport vehi-cles that are constructed to supply high qual-ity oxygenated water and have on-boardmonitoring of life support systems.

• Auxiliary aeration or oxygenation systemsshould be installed to ensure oxygen satura-tion and serve as backup should a failureoccur with the water circulation system.

• Whenever possible, water testing instru-ments, such as dissolved oxygen meters,should be used throughout the transport.

• For long hauls when fish densities are veryhigh, it may be necessary to remove nitroge-nous products using circulation and filtration.

• Small quantities of fishes can be transportedin plastic or polyethylene bags under anatmosphere of pure oxygen. These bagsshould be transported in a cooler to maintainwater temperature as close as possible to thefishes' initial/starting temperature. Smallbags of fishes are likely to heat up quickly,and fishes could become thermally stressed.

• All tanks and pipes in the transportation sys-tems should be disinfected between ship-ments, followed by thorough soaking andrinsing to remove all traces of potentiallylethal disinfectants.

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Any stressful event, such as handling or trans-portation, causes a rapid increase in adrenaline.Adrenaline causes temporary changes in gill per-meability. In freshwater, this results in dilution ofblood by excessive entry of water, and vice-versain seawater. Blood levels of important elec-trolytes are pushed out of the normal ranges foras much as 24 hours following a brief stress suchas dip netting (Wedemeyer, 1972). For these rea-sons, brackish water (i.e. water with an osmoticpressure similar to blood) has been recommend-ed as an effective tool for the transport of a num-ber of fish species (Kreiberg, 2000).

Sedation of fishes prior to and during transportmay be useful in reducing sensory awareness,and hence mitigating the stress of transport. Thelevel of sedation should be sufficiently light toallow the fishes to maintain equilibrium, swim-ming and breathing (Wedemeyer, 1996b). Studieshave shown that the initial crowding stage dur-ing capture and transportation is the most stress-ful for fishes. Therefore, fishes should be sedatedprior to transportation (Kreiberg, 1992). Thechoice of anaesthetic agent for sedation is impor-tant, as some anaesthetics which are effective inthe rapid induction of deep anaesthesia (e.g.,TMS and 2-phenoxy ethanol) have an excitatoryeffect during initial absorption, which defeats thepurpose of calming the fishes (Kreiberg, 2000).Metomidate is the most appropriate choice forsedation during transport.

6. Quarantine and Acclimation

Guideline 48:

After transport and before use in experi-ments, fishes should be acclimated to labora-tory conditions during a period of quarantineand acclimation.

A combined approach for acclimation and quar-antine should be used as far as possible so that both are accomplished simultaneously (seeSection 6.1 Quarantine).

Guideline 49:

As far as possible, fish from various sourcesshould not be mixed.

Some of the factors that influence the responsesof fishes are genetic differences, age, growth rate,

environmental and nutritional history, and exposure to biogenic compounds. It is importantto document the strain used in each experimentand to use the same strain throughout an experi-ment. For these reasons, investigators need to ensure that fishes to be used for experimentalpurposes are obtained from a reputable supplierwith good health management, and that new arrivals are carefully screened and quaran-tined, and are healthy before entering the mainpopulation.

6.1 QuarantineGuideline 50:

Quarantine areas should be subject to extravigilance in monitoring fish and good recordkeeping to detect and respond to any healthproblems in quarantined fish.

The purpose of quarantine after receipt of ship-ments of fish is to isolate those fish from the mainpopulations in the facility to permit observationand testing until such time as the newly arrivedfish are determined to be healthy and free fromcommunicable disease. Thereafter, these fish canbe integrated into the populations of the facility.

Quarantine can also be used to isolate popula-tions of fish that become sick some time afterentry into the facility. Quarantine is primarily ameasure to ensure that fish are isolated and san-itary measures are put in place to ensure there isno escape of viable pathogens or their hosts fromthe facility and into the surrounding waters, ortransfer of pathogens to other animals in thefacility. Information specifically for containmentof marine and freshwater organisms used in dis-ease studies or incidentally infected with trans-missible diseases is given in Appendix C.

Ideally, quarantine should involve isolation offishes being studied or held for different purpos-es in separate areas. However, for facilities withonly one room, plastic sheeting may be used towall off a quarantine tank to prevent splash oraerosol transmission. The water supply shouldbe separate so that water from quarantined tanksis not circulated to other tanks, and effluentshould also be separate when there is a need totreat the water, prior to recirculation or release.In addition, rigorous SOPs for disinfectionshould be in place.

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Guideline 51:

The duration of quarantine should be appro-priate to assure the health of the fishes.

In instances in which the fish are received from ahealth certified source (such as a salmonid hatch-ery that is in compliance with DFO’s Fish HealthProtection Regulations), the duration of the quar-antine period may be decreased depending onthe health status, but quarantine is highly advis-able for any fish introduction regardless of thesource. In other instances, such as receipt of wildfishes captured in the field, a longer duration ofquarantine is more appropriate. This is especial-ly important if the newly acquired fishes will beadded to healthy existing populations (DeTolla etal., 1995). Minimum quarantine time should beestablished based on the holding temperature,source of fishes, and the anticipated timeframefor expression of the pathogens of concern. Newstock should undergo routine health screening,including necropsy examination, if they are to bemixed with existing stocks.

Guideline 52:

Quarantine areas should be managed ac-cording to rigorous infectious agent controlpractices.

Particular vigilance should be paid to practicessuch as effluent disinfection, footbaths, handwashing stations, dedicated accessories (such asnets) and hand implements, and clean to dirtytraffic flow in the quarantine area, in order toavoid the potential transfer of pathogens to themain areas of the facility.

Newly arrived fishes may bring pathogenicorganisms with them, either in an active or carri-er state, to which the resident populations havenot been exposed. As a result of the stress of han-dling and crowding in tanks, the fishes' immunesystem may be depressed and a disease outbreakmay occur. The danger of introduction of newpathogens is especially relevant if fishes fromwild populations with unknown disease histo-ries are used. Prior to bringing wild fishes intothe lab, fish health professionals should be con-

sulted for advice on appropriate prophylacticdisinfection procedures to be used, in order todetermine appropriate treatments.

6.2 AcclimationOn arrival in a facility, new fishes should be han-dled as little as possible, and care should betaken to prevent thermal shock (Wedemeyer,1996a). For practical purposes, thermal shockmay be defined as an abrupt change in tempera-ture of more than 2 or 3°C. If fishes have beentransported in plastic bags, the bags should befloated in the receiving tank until the tempera-ture has equilibrated. Ideally, fishes transportedin tanks should be adapted to their new environ-ment by slowly transferring water from the newsystem into the transport tank. When fishesarrive in poor quality water, where the stress ofstaying in the poor water exceeds the physiolog-ic impact of the transition to good quality water,the fishes must be removed immediately. SeeSection D.3.2 Temperature, for notes aboutchanging the water temperature.

Acclimation involves ensuring a gradual adjust-ment of the living conditions for the fishes. Ingeneral, fishes brought into a facility should beallowed to adjust to their new environment(including water quality, temperature, illumina-tion and diet). This period should also ensurethat any problems related to the stress of trans-port (e.g., anorexia, unanticipated morbidity andmortality) have been resolved.

Fishes should be gradually reintroduced to feed-ing during acclimation. It is common for newlytransported fishes to refuse to feed, particularlywhen a new feed is introduced. Where possible,samples of the feed previously used by the orig-inal supplier should be obtained to permit tran-sition to the new feed source by blending. Returnto feeding is a good indication that the fish areacclimating successfully.

As a last resort, larger fishes such as recentlyacquired wild-captured fishes can be sedatedand force fed to initiate digestive processes andencourage return to feeding.

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Good fish husbandry requires attention to detailand the rigorous and consistent performance ofroutine chores. The importance of a high stan-dard of husbandry cannot be over emphasizedand should be regularly reinforced.

1. Record-keeping andDocumentation

1.1 Standard Operating ProceduresGuideline 53

Detailed Standard Operating Proceduresshould be developed for the maintenance andcare of all fishes and for sanitation of tanks,rooms and equipment.

Each facility should prepare a facility StandardOperating Procedures (SOPs) manual for accept-able fish husbandry practices and standards. Themanual should be reviewed and updated regu-larly, and the ACC and facility managementshould ensure that users follow the SOPs. In par-ticular, SOPs should be developed to ensure thattanks are properly disinfected and kept cleanbetween experiments.

1.2 General checklistsGuideline 54:

Checklists should be used for each group of fish so that records are maintained of all cleaning, maintenance and experimentalprocedures.

As a minimum, the following records should bemaintained on all captive fishes.

1. On the tank or holding area:

• source of fish and date of arrival;• species and sex (if identifiable);• estimated age and weight;• name of principal investigator and list of

emergency contacts;• animal-use protocol number, including expi-

ration date;

• transfer history of fish (i.e. where they havebeen housed within the facility);

• number of fish in tank;• daily records of husbandry (including feed-

ing schedule), maintenance, experimentalprocedures and water quality parameters asrequired (see Appendix D); and

• morbidity/mortality.

2. Readily accessible in the facility records:

• history of the fish, including disease andongoing health status;

• documentation of regulatory compliance; and• copy of animal-use protocol.

1.3 Assessment of fish well-beingGuideline 55:

Basic physical and behavioral parametersindicative of well-being in fishes should bemonitored daily and written records shouldbe maintained. Any perturbation of theseparameters should be investigated and thecauses identified and corrected.

It is important to be aware of an animal's state ofwell-being, both before and during the conductof any studies. The objective evaluation of keybehavioral and physical variables should help inthe detection of abnormalities, whether related toenvironmental/husbandry factors or to theeffects of experimental procedures themselves.The assessment of well-being in fishes is chal-lenging because their responses to adverse con-ditions are not always displayed, as occurs inmammals, and because significant observationalrestrictions are imposed by the rearing environ-ment itself.

2. Density and Carrying CapacityGuideline 56:

Each species should be housed at a densitythat ensures the well-being of the fish whilemeeting experimental parameters. However,in some cases, the ideal environment for

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maintaining a given species will have to bedeveloped using performance-based criteriasuch as growth rate. Established maximumdensities should not be exceeded.

The number of fish that can be carried in a givenwater supply is extremely variable and dependson the species, water temperature, pathogenload, dissolved oxygen level, metabolic rate ofthe fish, feeding rate, and how fast the water isbeing exchanged.

It is important to recognize that there are pro-found effects of both maximal and minimal den-sities; below certain densities territorial behaviormay increase (for example, in salmonids housedbelow minimal densities, feeding is diminished).Wedemeyer (1996a) has reviewed the physiolog-ical responses of fish to crowding.

To prevent problems in feeding due to territorial-ity and aggression when dissimilar sized fish arehoused together, the fish should be graded peri-odically to ensure similar sizes within groups.

3. Food, Feeding and Nutrition

Most species display daily and seasonal feedingrhythms, and may be specialized to feed on spe-cific types of food (Groot, 1996; Madrid et al.,2001). Although fishes brought in from the wildgenerally prefer live feed to formulated feed,most learn to feed effectively on pellets and showremarkable flexibility in their ability to ingestand digest formulated feeds. The acceptance offeed depends upon chemical, nutritional andphysical characteristics of ingredients selectedfor feed formulation as well as feed processing.The structure and function of their digestive sys-tems influences the patterns of food intake anddigestive efficiency; meal sizes and feeding fre-quencies should be set accordingly (Goddard,1996; Alanärä et al., 2001).

3.1 NutritionNutritionally balanced diets and appropriatefeeding regimes are critical in ensuring that fish-es remain healthy. Commercially manufacturedfish feeds contain nutrients and energy sourcesessential for growth, reproduction and health.Essential nutrients include protein and aminoacids, lipid and fatty acids, vitamins and miner-

als. Deficiency of these nutrients can reducegrowth rate and feed consumption, and lead todiseases (NRC, 1993; Conklin, 2000). As fishesare ectothermic, their metabolic rate is deter-mined by the water temperature. Therefore,feeding rates and quantities need to take temper-ature into consideration (Alanärä et al., 2001;Kestemont & Baras, 2001).

3.2 Food and feedingGuideline 57:

Fish feed should be purchased from sourcesthat manufacture feed according to standardsemployed in the feed industry for fish andother domestic animals, and according topublished nutrient requirements for thespecies, if available.

If fish are to be introduced into the food or feedchain (see Section J. Disposition of Fish AfterStudy), the fish feed must be in compliance withthe Feeds Act and Regulations (laws.justice.gc.ca/en/F-9).

Guideline 58:

Feed bags should be labeled with date ofmanufacture and guaranteed analysis infor-mation. Small aliquots of feed should beretained for independent testing when largefeed lots are received.

3.3 Feed quality and storageGuideline 59:

Feed should be stored in dedicated areas thatare dark, temperature and humidity con-trolled, and pest-free to ensure its nutritionalquality. Feed for immediate use and feed infeeders should be similarly protected. Feedused for daily feeding should be kept insealed-top containers to protect it fromhumidity and light, and frequently replacedwith feed from storage.

All feeds, whether moist, semi-moist or dry, aresusceptible to degradation with time. Moistfeeds containing minced raw fish or ensilagedfish should be fed within a few hours or frozen(Goddard, 1996). Dry feeds should be stored attemperatures < 20°C and humidity < 75%. Highhumidity increases susceptibility to mould, andhigh temperatures destroy certain vitamins and

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enhance the degradation of lipids. Vitamins infeeds can also be destroyed by oxygen, ultravio-let light and lipid peroxidation.

Feed can be frozen to extend its shelf life. This isan option when relatively low amounts of feedare required for a specific research project.However, certain micronutrients such as B com-plex vitamins are degraded by freezing andthawing, and therefore supplements may berequired.

Oxidative rancidity is one of the most seriouschanges than can occur in stored feed(Wedemeyer, 1996a; O'Keefe, 2000). In theabsence of antioxidant protection, lipids rich inpolyunsaturated fatty acids, including the essen-tial fatty acids, are highly susceptible to auto-oxidation, which produces harmful breakdownproducts that include free radicals (Hardy &Roley, 2000). The pathological effects of feedingoxidized oils are summarized by Tacon (1992).

Under no circumstances should mouldy feed beused, as it is highly toxic (Wedemeyer, 1996a).

Guideline 60:

Fishes must be fed at appropriate intervalsand with a nutritionally adequate, properlysized feed. Optimal feeding techniques areessential for good health and well-being, andto prevent the fouling of water with uneatenfeed.

Guideline 61:

Whether fishes are fed manually or automati-cally, they should be observed regularly todetermine whether they are responding asexpected, and whether the ration is sufficientor overfeeding is occurring.

When automated feeders are used, the equip-ment should be regularly serviced and the rate of intake of the fish checked as frequently as possible.

The practice of feeding involves determining theproper size and appropriate properties of thefood for the species (e.g., bottom feeders are fedmore easily with sinking pellets). The ration sizeneeds to be determined, as well as the feedingfrequency, the preferred time for feeding, and themost efficient means of distributing the feed.

When new feed is introduced, it should be mixedwith the accepted feed until the transition ismade between the two feeds.

Feeding techniques for captive fishes have thegeneral aim of encouraging rapid consumption,thus increasing feed ingestion, preventing leach-ing of water-soluble nutrients, and reducingwastage. Not only is a suitable diet important toobtain a good feeding response, but the cultureenvironment also influences the feedingresponse. For example, temperatures at the lowand high end of the tolerance range inhibit feed-ing, as do stressful conditions such as low oxy-gen levels and the development of social hierar-chies within the population (Kestemont & Baras,2001).

Feed or feces retention in the environment is aparticular concern in situations of overfeeding,and especially in recirculation systems. Theinfluence of feed quality and quantity on waterquality should be addressed in the study design.

Fish must not be overfed, except in experimentswhere fish are fed ad libitum. When fish are fed adlibitum, however, they should be monitored andexcess feed should be removed soon after thefeeding period. Most fish can survive for longperiods without feed and, in most instances, lackof food for a few days will not be overly distress-ful (De Silva & Anderson, 1995; Carter et al.,2001). Overfeeding, on the other hand, causesserious problems because of its effects on waterquality and the stimulation of potentially harm-ful bacterial and fungal growth (see Section 3.4Larval weaning, for the exception to this rule).

Feeding and fish size-sorting practices should beoptimized to ensure all fish have the opportuni-ty to feed. In the event of prolonged feed refusal,alternative plans should be in place, includingconsultation with a feed manufacturer, a fishnutritionist or a veterinarian. The primary modesof feed detection by fish are through olfactionand sight, but the taste and texture of the feed isthe key factor that determines whether the feedwill be swallowed or rejected. When changingfeed sizes, a mixture should be fed for a week toallow fish to make the adjustment. Certain feed-ing stimulants added to fish feeds enhancepalatability and feed acceptability.

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In some cases, for instance where wild fish havebeen brought into captivity, pelleted rations maynot be recognised as food. In addition, manysmall aquarium species, as well as the larvalstage of many larger marine fish species, areeither unable or reluctant to feed on preparedfeeds. It may also be necessary to feed live preyfor studies on fish foraging, and for short-termholding of fish from the wild. While live feeds(principally rotifer and brine shrimp) can giveexcellent results, in most cases, provision of livefoods requires culture of the prey item in addi-tion to the fish. The ethical cost of feeding livefeed (in particular live fish) to fish must be con-sidered, in addition to other potential disadvan-tages, such as the possibility of variable nutri-tional planes or the potential for introduction ofdisease. Additional information, including a gen-eral overview of nutrient requirements can befound in other publications (e.g., NRC, 1993;Conklin, 2000; Halver & Hardy, 2001).

Studies involving food restriction should under-go careful consideration. The CCAC policy state-ment on: ethics of animal investigation (CCAC, 1989)states that for "experiments requiring withhold-ing of food and water for periods incompatiblewith the species-specific physiological needs,such experiments should have no detrimentaleffect on the health of the animal". In general, fishshould not be permitted to lose more than 15% ofbody weight during periods of food restriction(Home Office, 2003). As with any other studies,the ACC is responsible for approving the end-point of the proposed study, in consultation withthe investigator and veterinarian or fish healthspecialist. It should be noted that for fish thathave naturally ceased to feed (e.g., spawningsalmon), it is not necessary to attempt to feed.

3.4 Larval weaningIt is recognized that early life stages of manyspecies have high natural mortality (see SectionB. Introduction). Failure to begin feeding or toacquire sufficient food has often been suggestedas a major cause of early mortality (Robin &Gatesoupe, 1999).

The switch to an exogenous feed supply is a cru-cial stage for fish, particularly for marine fish.This transition is the major feature used to definethe end of the embryonic period (Noakes &Godin, 1988). In general, highly fecund fish pro-

ducing small eggs, for example cod, haddockand flounder, are small at hatching, the yolk sacstage is shorter, and they are more difficult torear on artificial diets (Watanabe & Kiron, 1994).For all fish species, the change from endogenousfeeding to exogenous feeding, and again whenweaning from a live diet, are critical periodswhere large numbers of fish may die. In general,this is not a welfare concern, but individualsjudged unlikely to thrive should be euthanized.

The timing of first feeding and availability ofsuitable prey is critical. Altricial young, whichare not highly developed prior to first feeding,require a higher concentration of food to com-pensate for their underdeveloped gut and rapidgut transit time. Precocial young, which are moredeveloped at the first feeding stage, are able tofeed more efficiently on scattered individualprey items (Noakes & Godin, 1988).

The most crucial factor in the weaning process isthe provision of live invertebrates, such asrotifers and Artemia of the preferred size.Generally these food organisms are enrichedwith limiting nutrients (e.g., essential fatty acidsand amino acids) to provide larval growth andsurvival. Young fish, if deprived of exogenousfood, will reach a point of no return when theeffects of food deprivation become manifest asirreversible starvation.

3.5 Use of medicated feedsGuideline 62:

Medicated feeds must only be used underveterinary prescription and supervision.

Medicated feeds may be used to treat clinicalconditions, such as bacterial infections, or toinvestigate models of disease control. Medicatedfeeds must only be used under veterinary pre-scription with accompanying caution for with-drawal times, particularly in the case of antibi-otics. The use of medicated feeds for therapeuticreasons should be conducted under veterinarysupervision and with due care to the develop-ment of antibiotic resistant bacteria. Rationalantibiotic selection should be the result of clinicaljudgement and the use of antimicrobial sensitiv-ity testing. As far as possible, only agentsapproved for use in fishes should be used.

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Medicated feed may be less palatable and thefish may refuse to eat it. It is important whenstarting medicated feeds to monitor the feedingbehaviour of the fish and to be prepared to useadjunct measures, such as gradual mixing withnon-medicated feed, to encourage consumption.Use of diet palatability agents for medicatedfeeds (e.g., coating the diets with krill or marinefish hydrolysate) may be considered to inducefeeding. Records should be maintained of theduration of the treatment and its effects.

In recirculation systems, the use of chemothera-peutic agents should be carefully considered dueto the possible detrimental effects of these com-pounds on nitrifying biofiltration flora.

4. Broodstock and BreedingGuideline 63:

Holding systems and environmental condi-tions for broodstock should be appropriatefor the species. Particular attention should bepaid to the importance of environmental cuesfor the maintenance (or manipulation) ofendogenous reproductive rhythms.

Environmental factors, such as temperature,photic environment, habitat/tank design, nutri-

tion, holding density and species mix, are criticalto reproductive success.

Guideline 64:

Where possible, rational genetic manage-ment of broodstock should be used. Forbroodstock, a strict disease and health con-trol program should be implemented with vet-erinary advice to ensure the production ofhealthy progeny and prevention of diseasetransfer through water sources, fish or eggs.

4.1 Induction of spawningThe induction of spawning through administra-tion of injectable hormones is a common practicein broodstock management. The literatureshould be consulted to ensure doses and regimesare consistent with established standard meth-ods. Treated broodstock fish should be retainedfor the necessary withdrawal times if they are tobe killed for food.

Induction of spawning may result in morbidityand mortality due to retention of ova and otherunforeseen effects.

Personnel working with chemically inducedspawning fish should be aware of the complica-tions associated with hormone treatment.

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Under conditions of confinement in an artificialenvironment, fish are sensitive to variations inwater quality, nutrition, presence of patho-gen(s) and management practices of the facility.The expression of disease, whether infectious or non-infectious, cannot be considered in isola-tion from any of these factors. Microorganismsare distributed throughout any aquatic envi-ronment; however, their presence may onlybe obvious under sub-optimal environmentalconditions.

1. Fish Health Program

Healthy fish are pre-requisites for reliable data(Jenkins, 2000). Fish used for research should befree of any notable disease agents that could leadto a diseased condition (unless it is part of theexperimental protocol).

If a disease condition is part of the experimentaldesign, the potential effects of the pathogen orparasite on the research results should be pre-dictable, or constitute a variable that is beingtested through the research protocol.

Guideline 65:

All facilities must have a fish health monitor-ing program.

Institutions housing fish for research, teachingand testing should have access to expertise infish health, and preferably to a veterinarian withaquatic medicine experience and training. Thisindividual can assist in the development of SOPsto limit the introduction of disease into the facil-ity, and should be available for consultation onmatters relating to the health of the fish (seeSection B.4.3 Role of the veterinarian).

1.1 Disease preventionGuideline 66:

Strategic measures for disease preventionshould include: 1) a formal written agreementwith a fish health professional (usually a vet-erinarian) responsible for the management ofmorbidity and mortality problems at the facil-

ity; 2) a program for the detection and man-agement of disease conditions and waterquality problems related to physiologicalstress; 3) strategic application of diseasecontrol measures, such as quarantine, immu-nization, and prophylactic treatments; and 4)a system of regular monitoring and reportingfor health assessment purposes.

Stress, nutritional problems, water quality prob-lems, disease outbreaks from infectious causes,cannibalism and predation can all cause majorproblems in captive fishes (Wedemeyer, 1996a).Methods for early detection of emerging healthproblems should be implemented to facilitatemitigation and restoration of a level of healthcompatible with the study objectives. Goodhealth management is required in studies usinglive fish because study results can be adverselyaffected or compromised by suboptimal healthor disease events.

Many diseases in fish holding systems can beprevented through good husbandry, startingwith stock that have been pre-screened for infec-tious disease agents, subjecting incoming ani-mals to a period of isolation, and low level han-dling to minimize stress. This may also involvequarantine where aquatic animals "down-stream" of the holding facility are considered tobe susceptible to any escaped pathogens.Appropriate holding conditions should also bemaintained as outlined elsewhere in theseguidelines.

1.2 Disease diagnosis and identification of pathogens

Guideline 67:

A health management program should focuson early diagnosis and identification of the causal agents, stressors and mechanismsso that correct control measures can be initiated.

Disease management protocols should include areliable system for the detection and reporting ofclinical signs, and criteria to distinguish between

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acceptable and unusual levels of mortality.Isolation and rapid removal of dead fish willhelp reduce the spread of disease.

If unexpected losses of fish occur, staff shouldimmediately take water, food and fish samplesfor later analysis should they be required.Samples of affected fish should be retained fordiagnostic purposes. It is advisable to develop anSOP with the assistance of an experienced fishhealth or veterinary clinician to standardize sam-ple collection methods.

With active clinical problems, consultation with aclinical veterinarian is preferred. Where possible,live untreated fish with and without symptomsshould be provided to diagnostic laboratories asthis is far more valuable than the provision ofdead specimens.

Guideline 68:

Fish health management programs shouldstrive to identify both clinical and subclinical/adventitious pathogens which may occur as aresult of experimental stressors.

The presence of infectious diseases in an exper-imental population, even one not showing clin-ical disease, may lead to results that are diffi-cult to interpret due to the potential confound-ing variables caused by sub-clinical disease.The scientific validity and reproducibility ofexperiments made in a morbid or sub-clinicallyaffected population is questionable. The appli-cation of treatments may also be an experimen-tal variable.

Guideline 69:

Particular attention should be paid to moni-toring fishes following any potentially stress-ful event.

All fishes in the facility should be monitored ona daily basis. However, fishes undergoingstressful procedures have an increased risk ofdeveloping opportunistic infections, thereforeadditional care should be taken in observing thefishes for one to five days following a potential-ly stressful event. Reluctance to eat, unusualbehavior, discoloration of the integument andlesions are signs of possible developing healthproblems.

Physical damage is one type of stress, but morecommon stressors are new introductions, crowd-ing, handling, transportation, and degradation ofwater quality (such as sublethal changes in tem-perature, reduced dissolved oxygen, andincreased ammonia) (Wedemeyer, 1996a; Reddy& Leatherland, 1998; Speare, 1998b). Such stresscan suppress the immune response, allowing dis-ease organisms to proliferate. However, stresscan also lead to mortality in the absence of infec-tious disease agents.

1.3 Injuries and other disorders

1.3.1 Handling injuries

Guideline 70:

Handling procedures should be carried outonly by competent individuals using tech-niques that minimize the potential for injury.Efforts should be made to minimize morbidi-ty and mortality caused by osmoregulatorycompromise, systemic acidosis, and oppor-tunistic infections of damaged skin that canresult from handling and traumatic injuries.

Traumatic injuries can result from handling pro-cedures or abrasions from contact with tanks andequipment, other fish or predators (Speare,1998b). Malfunctioning equipment or inexperi-enced fish handlers can turn routine proceduresinto events that cause disease outbreaks. Somefactors that can increase the risks to fishes duringhandling include:

• malfunctioning or improper equipment;

• inexperienced fish handlers;

• dry or abrasive surfaces which fish will con-tact during handling, such as measuringboards, balances, etc.;

• warmer water temperatures;

• prolonged handling times; and

• repetition of procedures on the same individual.

Information on handling and restraint is given inSection H.1. Handling and Restraint.

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1.3.2 Behavioral interactionscausing injury

Guideline 71:

Health management measures should beused to ensure that behavioral interactionswith negative consequences such as aggres-sion are avoided.

Various steps, such as size-sorting fish, adjust-ing density or providing visual sight barriers,can be employed to minimize aggressiveencounters.

Some fish exhibit territorial behavior, which canlead to wounds (Speare, 1998b). Social interac-tions and density have an effect on behaviouralinteractions (Speare, 1998b) and can lead to com-promised behaviors, for instance the suppressionof feeding.

1.3.3 Feed-related disorders

Nutrition can also influence the health of fish bycausing nutrient deficiencies, imbalances or toxi-coses, or by introducing infective agents (seeSection F.3. Food, Feeding and Nutrition).

1.3.4 Toxicities resulting from use ofchemotherapeutants and environmental toxins

Guideline 72:

A Standard Operating Procedure should beestablished for any standard treatments, andinclude the definition of endpoints shouldfish be adversely affected.

Treatment of fish should be carried out in consultation with a veterinarian. SOPs for stan-dard treatments should also be developed inconsultation with the veterinarian or fish healthprofessional.

Medications are usually delivered to groups offish rather than to individual sick fish, whichputs more animals at risk of unexpected effectsof the treatment. As far as possible, fish that needto be treated should be isolated until the treat-ment is completed. When bath treatments areadministered, there should be close observationand maintenance of water quality, as this is amajor source of problems. In cases where theanticipated effects are unknown, a small numberof fish should be tested before application to thegroup as a whole.

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1. Handling and RestraintGuideline 73:

Fishes should be fasted prior to handling.

Defecation and vomiting are responses made byfishes in the interest of conserving metabolicenergy stores when exposed to acute stress.Digestion requires body energy; when there isimmediate danger, animals get rid of undigestedfood so as to maximize the energy available toflee, fight or recover from injuries. Fasting fishesensures that digestion does not consume energyduring handling, and that the fishes are left withenergy stores to assist in recovery. Additionally,fasting somewhat reduces ammonia output fromthe fishes and lessens the risk of bath contamina-tion from gut contents. Gut emptying times arelonger for larger fishes and colder temperatures.High quality water for procedures and recoveryshould be provided so that any gut emptyingthat does occur does not cause welfare problemsfor the fishes; in particular, this strategy shouldbe used so that any diagnostic or therapeuticintervention is not delayed.

Guideline 74:

Personnel involved in handling fishes shouldundergo training in methods to ensure theirexpertise and to minimize injury and morbid-ity to fishes in their care.

Guideline 75:

Fishes should be handled only when neces-sary, and the number of handling episodesshould be minimized.

Even routine handling procedures may causemorbidity and mortality, if carried out by person-nel who have not been adequately trained (seeSection G.1.3.1 Handling injuries).

Nearly all fishes held in the laboratory have to bephysically handled. Handling and disturbanceappear to be stressful events for fishes, althoughthey can be conditioned to the handling(Kreiberg, 2000). Appropriate handling equip-ment, preferably a bucket (or alternatively knot-

free-nets) and sanitizable tables, should be usedto minimize damage to fishes during handling.Measurements of body condition, such as weightand length, which involve hands-on manipula-tion should be conducted quickly and in a man-ner that is minimally stressful. Procedures thatinvolve more than momentary restraint orrequire that large numbers of fish be handledshould be conducted under sedation, unless thefish have been conditioned to the handling. Inthe long term, the effects of stress can includeloss of appetite, inhibition of growth, impairedreproductive success and impaired immuneresponse (Reddy & Leatherland, 1998). Depend-ing on the species and the frequency and intensi-ty of the stressor involved in handling, it maytake from a few hours to several days to resumenormal feeding.

Where fish are to be handled repeatedly, a suit-able period of recovery should be permittedbetween handling procedures. Recovery fromstress can be prolonged. Repeated handling mayrequire an increased level of monitoring, and thestress may be alleviated by the use of previouslyproven sedation (see Kreiberg, 2000).

Guideline 76:

Fishes should be handled in a fashion thatminimizes damage to their mucus-skin barrier.

Prolonged physical restraint of unsedated fishesshould be avoided as damage to skin andmucous membranes may result, as well asmyopathy. This is particularly true for salmonidspecies; more sedentary fishes appear to be lessstressed by physical restraint.

Fishes are highly reliant on the integrity of theirmucus and epidermal body covering as a barrierto osmotic stress and infectious agents. As theskin is relatively delicate in many species, anes-thetics and sedatives are frequently used to pre-vent external damage during procedures whichwould not require anesthesia in other species.Polymeric water additives such as poly-vinyl-pyrrolidone (PVP) have been found useful intransport and handling of fish (Carmichael &

H. EXPERIMENTAL PROCEDURES

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Tomasso, 1988; Wedemeyer, 1996b). In general,such compounds are considered to bond tem-porarily to exposed tissue, serving as a short-term replacement for shed mucus and restoringthe protection that mucus provides.

Guideline 77:

Restraint and handling of fishes should becarried out in a manner to minimize visualstimulation. Where feasible, fishes should beprotected from direct light and rapid changesin lighting while being restrained.

Exclusion of light, wholly or in part, has beenrecommended as a practice to reduce stress infish undergoing handling (Wedemeyer, 1985;Hubbs et al., 1988)

Manual restraint may be a practical means ofperforming rapid, minimally stressful proce-dures, but requires skilled and careful handlers.Many fishes are sensitive to visual stimuli, espe-cially light, so handling in a dimly lit area mayhelp lessen handling stress.

Guideline 78:

In general, fishes should not be kept in aircontinuously for more than 30 seconds.

In general, the length of time fishes are held outof water should be minimized, and should notexceed 30 seconds (Ferguson & Tufts, 1992);however, some species such as eels and catfishcan tolerate longer periods out of water. Thedamaging effects of even brief periods out ofwater on gill epithelial tissue in some fish specieshas been described; therefore, when out of waterthe gill lamellae should be kept moist.

Large fishes such as broodstock are less respon-sive out of water if their head is covered with adamp cloth or foam rubber, which also preservesmoisture in the gill region.

1.1 Restraint of dangerous speciesGuideline 79:

Those who work with dangerous speciesmust be trained and competent to do so.Appropriate emergency items (e.g., antiven-om, an appropriate first aid kit, etc.) must beon hand.

In general, dangerous species will be encoun-tered only under field conditions; however, therecommendations are equally applicable to thelaboratory situation. Dangerous species shouldbe handled in a manner that is safe both for theinvestigator and for the animal being handled.Procedures should minimize the amount of han-dling time required and reduce or eliminate con-tact between the handler and animal.

Investigators should never work alone whenhandling dangerous species. A second person,knowledgeable in the capture and handling tech-niques and emergency measures, should be pres-ent at all times.

Prior consultation with colleagues experiencedin working with the species and review of anyrelevant literature is important (CCAC, 2003a;Nickum et al., 2004).

2. Restricted Environments

Guideline 80:

Every effort should be made to provide fishesheld in restricted environments with as non-stressful an environment as possible, withinthe constraints of the experimental design.

Fish are frequently kept in physically restrictedenvironments, such as metabolic chambers,swim tunnels and calorimeters, for long periods.These fish should be accustomed to the restrictedenvironment before the study, and should bekept in such environments for the shortest dura-tion possible. Fish that fail to thrive in these envi-ronments should be removed.

3. Surgery

For a thorough review of fish surgical tech-niques, see Johnson (2000). CCAC Guide to theCare and Use of Experimental Animals, vol. 1,Chapter IX Standards for Experimental AnimalSurgery (CCAC, 1993b) should also be consultedfor general guidance, bearing in mind the differ-ence in environmental conditions necessary forthe successful conduct of surgery on fishes.

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3.1 Surgical preparation andskin disinfection

Guideline 81:

Surgery should be performed by individualswith appropriate training.

Surgery in fish can be complex and intricate.Anyone attempting any invasive surgery shouldbe properly trained in surgical aseptic technique,or should obtain the services of a veterinary sur-geon. Fish surgery should normally be coveredunder the institutional veterinary care program.

Guideline 82:

Before surgery is attempted on living animalsthat are expected to recover, suture and sur-gical techniques should be practiced on inan-imate materials or dead specimens until com-petency is attained.

Practice using cadavers and non-survival trialscan be useful in training investigators. Appropri-ate training and practice will help to minimizeanesthetic and surgical time, and contribute to afaster recovery of the animal.

Guideline 83:

Surgical sites should be prepared in a fash-ion that minimizes tissue damage and con-tamination of wound areas.

The removal of mucus and disruption of scalesthat occurs during surgical preparation is gener-ally thought to devitalize tissue and render thearea more subject to attack by saprophyticagents, particularly fungal and bacterial inva-sion. A variety of molecules with antibacterialproperties are found in the mucus and outer epi-dermal layers of fish, as are phagocytic andmononuclear cells. Beyond the gentle removal ofgrossly visible dirt and debris, preparationshould be limited in scope (Wagner et al., 1999).

The type of aggressive surgical scrub procedureused in mammalian surgery to render a site "sur-gically clean" is not generally used in fish sur-gery. There are also species differences, withspecies such as sharks having tough resistantskin, while others such as catfish have delicate,relatively unscaled skin. Dilute aqueous-basedpovidone-iodine solutions appear to be well tol-

erated by many species and can be irrigated overan area before draping and incision. Quaternaryammonium compounds and alcohol may be irri-tating and toxic when applied to the skin in somespecies, and should be avoided.

Although creating surgically clean skin (definedin mammals as having fewer than 10,000 bacteriaper gram tissue) is problematic in fish, the provi-sion of sterile occlusive drapes will help to main-tain a sterile to surgically clean operating field.Sterile plastic food wrap (saran wrap) is prefer-able to fabric drapes, as the latter is prone toabsorption of water and introduction of bacteriain the water. Surgical incisions can be made insterile fashion by cutting directly through thedrape into underlying tissue.

Guideline 84:

Attention should be paid to the use of asep-sis, disinfection and the use of sterile instru-ments to minimize wound contamination andmaximize the healing response.

Instruments should be cleaned and sterilizedbetween surgical procedures and if inadvertent-ly contaminated.

There is a lack of published information on theeffect of adventitious bacteria on the healing ofsurgical wounds in fish; however, it is reasonableto take precautions to minimize bacterial con-tamination and colonisation of wounds andbody cavities by using sterile techniques wherefeasible.

Instruments may be gas sterilised or autoclaved,or where this is not possible, cold sterilisation for10 minutes using benzalkonium chloride or relat-ed cold sterilants could be used, although thistechnique is unlikely to be sporicidal. These lat-ter agents are tissue toxic and the instrumentsshould be rinsed thoroughly in sterile waterbefore being used on tissue. In multiple surger-ies, two sets of instruments should be rotatedthrough a cold sterilizing solution. A hot beadsterilizer may also be used and is the most prac-tical method for many lab situations. Many sur-gical disinfectants, such as alcohol and glu-taraldehyde, cause devitalisation or excessivemucus production when applied to fish skin andshould be avoided.

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3.2 Water quality during surgeryGuideline 85:

During prolonged surgery, water qualityshould be maintained at a high level, withminimal bacterial and organic burden. Waterfor anesthesia should be from the samesource as the tank water to minimize shockcaused by differences in temperature, pH,electrolytes, etc.

Water used to irrigate fish gills during prolongedanesthetic procedures should be circulated andtreated to maintain proper anesthetic levels, oxy-gen, temperature, pH and salinity, and to removeparticulates. As water temperature can be affect-ed by room air temperature and use of surgicallights, it should be carefully monitored and controlled. Water quality can also be affected bythe production of mucus, urine or feces duringsurgery, and should be changed or renewedaccordingly.

In some instances, the addition of conditioningsolutions to the water may be justified, particu-larly for the replacement of electrolytes follow-ing trauma or stress. Proprietary mixtures con-taining electrolytes, artificial film materialsbased on polyvinylpyrrolidine and oxidativescavenger molecules are used to lessen stress andmorbidity following surgery; however, their effi-cacy has not been scientifically proven.

3.3 Anesthesia

Guideline 86:

Anesthetics should be used in experimentswhere there is expected to be noxious stimuli,and in experiments entailing extensive han-dling or manipulation with a reasonableexpectation of trauma and physiologicalinsult to the fish.

Anesthesia is generally defined as a state causedby an applied external agent, resulting in depres-sion of the nervous system, leading to loss of sen-sation and motor function.

The use of anesthetics facilitates work with fish-es and is required for invasive studies such assurgical preparations for physiological studies,where the fish must be held immobile for extend-ed periods of time. Sedation is also used for the

manipulation of animals during procedures suchas transport, grading or vaccination. Althoughthe use of anesthetics is primarily for holdingfishes immobile while being handled, it is alsoused to lower the level of stress associated withsuch procedures and may alleviate pain (Iwamaet al., 1988; Davis, 1992; Iwama, 1992). Anestheticoverdoses are also used routinely as an effectiveand humane means of euthanizing fishes.

Information on the characteristics of the majoranesthetics used on fishes, essential parametersfor their application, including optimum andlethal doses, as well as induction and recoverytimes is provided by Iwama et al. in Anesthetics, a supplement to these guidelines available on the CCAC website (www.ccac.ca/en/CCAC_Programs/Guidelines_Policies/GDLINES/Guidelis.htm). Possible physiological effects andcautionary notes are also given. Additional infor-mation is available in Iwama & Ackerman (1994).

Guideline 87:

Anesthetics should be chosen on the basis oftheir documented ability to provide pre-dictable results, including immobilization,analgesia and rapid induction and recovery,while allowing for a wide margin of safety forthe animals and the operators.

The investigator should ensure that the anesthet-ic selected has no toxic side-effects for the fish orthe handler, is biodegradable and can be clearedfrom the fish, and has no persisting physiologi-cal, immunological or behavioural effects.Investigators should also be aware that currentlyonly TMS (MS-222) and metomidate are regis-tered for veterinary use with fish in Canada.Investigators are individually responsible for theuse of anesthetic agents not approved for veteri-nary use in Canada.

Where anesthetics are to be used in the field, theCCAC guidelines on: the care and use of wildlifeshould be consulted for advice, in particular forrecommendations concerning drug residues(CCAC, 2003a).

Where anaesthesia will be of longer duration, arecirculation technique which ensures continu-ous delivery of oxygenated water and anestheticto the gills should be employed (Iwama &Ishimatsu, 1994).

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Guideline 88:

Regardless of the application, anestheticsshould be tested on a small sample of fish, asthe effect of an anesthetic can vary with localwater conditions, as well as the species, lifestage, and size of the fish.

The reaction of any fish being considered for sur-gery to the proposed anesthetic should be wellunderstood. Trials with healthy fish are recom-mended to ensure proper dosage and to accu-rately calculate the time to reach the necessaryanesthetic plane (Johnson, 2000).

Guideline 89:

Personnel working with anesthetic agents infish must be adequately trained and protect-ed with personal protective equipment.

Many of the anesthetics in use have the potentialto cause harm to humans if they are misused.

3.4 Surgical equipmentAn overview of the equipment used in fish sur-gery is provided by Brattelid & Smith (2000) andJohnson (2000).

3.5 IncisionsGuideline 90:

Any incisions should avoid the lateral lineand should follow the longitudinal axis of thefish.

Some fish scales may need to be removed follow-ing the skin preparation. Scales should beremoved individually by pulling in a posteriordirection to minimize damage. Only the scalesnecessary to create the incision should beremoved as the scales provide protection and sta-bility to the wound area. The epidermis and peri-toneum layers are easily torn, but the overall skinis tough due to a layer of dense collagen in mostspecies. Practice is necessary to obtain the opti-mal scalpel pressure to achieve a clean incisionand hence achieve rapid healing (Johnson, 2000).

Abdominal incisions may be made on the ventralmidline or lateral to this region. Midline inci-sions have been shown to minimize behavioralchanges in trout (Wagner & Stevens, 2000).Ventral midline approaches may intrude on

blood vessels and do not invade tissues such asmuscle, but the incision is likely to come in con-tact with the substrate in the tank, which maycause damage to the incision site. Lateralapproaches avoid this problem, but invade mus-cle and may result in accidental puncture ofunderlying organs.

Hemorrhage encountered during surgery mustbe controlled by direct pressure using swabs, byligation or by cautery. Gel foam can also be used.Cauterization should be used sparingly and withcare as it devitalizes tissue and predisposes it towound infection and breakdown.

3.6 Suture materials and techniquesGuideline 91:

In general, strong, inert, non-hygroscopicmonofilament suture material and atraumaticneedles should be used for closure of inci-sions in fish skin.

The skin of most teleost fishes consists of epider-mis with scales, dermis and hypodermis; all lay-ers are closely associated with one another andwith underlying muscle, peritoneum and otherlayers. Single interrupted sutures to appose alllayers are sufficient as epithelial cells migraterapidly to cover an incision, protecting the fishfrom the effects of the aquatic environment.Closure of individual layers is recommendedonly for very large fish.

Internal layers and organs can be closed usingsynthetic absorbable materials. Layers such asskin, that will be exposed to water and bacteria,should be closed with non-wicking, inert suturematerials, such as monofilament nylon (non-absorbable) or polydioxanone (absorbable).

Cyanoacrylate surgical adhesives may haveapplication in certain types of surgery on aquat-ic species; however, these materials have threemajor disadvantages in that they only bond drytissues, lack good holding strength in maintain-ing wounds closed in wet environments, andtend to promote inflammatory responses at thesite to which they are applied (Harvey-Clark,2002).

Summerfelt & Smith (1990) provide instruc-tions on suture technique. Suture patterns that

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give good tissue apposition and maximumsafety, such as simple interrupted and horizon-tal mattress, are recommended. These tech-niques have been further examined by Wagneret al. (2000).

3.7 Pathophysiology of surgery andwound healing in fishes

In general, fish skin heals faster than mammalianskin; however, fibrous proliferation can be slowand varies with temperature. The speed withwhich the fibrous proliferation closes the internalwound and provides sufficient strength toreplace the sutures is temperature dependent,and should be considered when selecting appro-priate suture material.

The basic physiology and histological responseto surgical wound healing of fishes has beencharacterised (Wagner et al., 1999). Factors thataffect wound healing include:

• water quality (hardness, levels of salt andother osmotically active compounds, andwater temperature), which regulates immuneresponse and tissue metabolism rate;

• presence of tank mates, cannibalism of surgi-cal wound area by conspecifics, and exclusionof animals with postoperative morbidity dur-ing competition associated with feeding;

• nutritional plane before surgery, good nitro-gen balance, anorexia after surgery (stressresponse), and speed of return to normalfeeding and other behaviours;

• hormonal status, especially smoltification insalmonids;

• changes in electrolyte balance due to openwounds and passive loss of water (marinefish) and of electrolytes (freshwater fish) tothe surrounding water;

• integrity of mucus layers and biofilms on fish;and

• presence/absence of opportunistic bacterialand fungal pathogens in water during andafter surgery.

3.8 Postoperative careLittle is known about the effect of analgesicdrugs on fishes. However, investigators areencouraged to use post-operative analgesiawhere appropriate as suitable analgesic agentsbecome available. Fishes do appear to produceopioid substances in response to pain and fear,similar to higher animals, i.e. Substance P,enkephalins and ß-endorphins (Vecino et al.,1992; Rodriguezmoldes et al., 1993; Zaccone et al.,1994; Balm & Pottinger, 1995), and the responseof goldfish to analgesia has been shown to besimilar to that of a rat (Jansen & Green, 1970).The response of carp (Cyprinus carpio) to electricshock, to the presence of alarm substance chemi-cals in water, and to hook and line fishing indi-cates that reactions to repeated shocks is graded,non-reflexive, and similar to that in mammals(Verheijen & Buwalda, 1988).

Guideline 92:

In laboratory or applicable field situations,fish must receive careful attention and moni-toring following surgery.

Although recovering fish may appear to be nor-mal, there may be prolonged metabolic effectsfollowing the stress of anaesthesia and surgery.In situations where monitoring is not possible,pilot scale evaluations of procedures should beconsidered. Where possible, fish should beallowed to recover from anesthesia until able toresume normal behavior. As anesthesia itselfcauses prolonged stress, careful procedures forrecovery are vital, for example, a quiet, wellaerated, possibly darkened tank will facilitaterecovery.

Fish require extra attention in the postoperativerecovery period. A number of common complica-tions may occur, including wound dehiscenceand infection, osmotic imbalances related to sur-gical incisions, and anorexia. Transient postsur-gical shock is a common problem in fish andincludes problems with oxygen debt, catabolicprocesses, fluid and electrolyte loss, and hor-monal imbalance. It is important to keep recov-ery water clean.

Guideline 93:

Fish should be held in a manner that reducesor eliminates intraspecific interactions in

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tanks, and meets appropriate living condi-tions for the species.

The return of the fish to the pre-surgery tank,including the presence of tank mates, has to beconsidered. The benefits of companion fish(social interaction, schooling fish and feedingactivity stimulation) have to be weighed againstthe disadvantages (predation, competition forfeed and biting of sutures).

Well oxygenated water, low lighting, shelterareas for recovering fish, and the use of waterconditioning agents to improve the bufferingability of the water and to supply lost electrolytesmay help speed recovery.

Guideline 94:

The costs and benefits of the use of prophy-lactic antibiotics post surgery should becarefully considered.

The indiscriminate use of antibiotics is not rec-ommended because of the possibility of encour-aging resistant strains of bacteria. In particular,antibiotics should not be administered to fishesin the wild following tagging or other minor sur-gical procedures.

Where surgical conditions cannot be made asep-tic, the early administration of broad-spectrumantibiotics of low toxicity to fish, chosen basedon knowledge of opportunistic flora in the spe-cific aquatic environment and on culture andsensitivity results in infected animals, may beappropriate (see CVMA, 2000).

A review of antibiotic properties and doses infishes can be found in Stoskopf (1993). However,a veterinarian should be consulted to determinethe appropriate treatment.

Guideline 95:

Social factors, such as size differences, abili-ty to feed or exclude other fish from feed, andagonistic behavior, should be considered inexperimental design and when maintainingsocial groups of recovering fish.

Recovery tanks should be designed to promotesmooth recovery with reduced risk of long-termeffects from anesthesia. Considerations for suit-able recovery tanks include opportunity to

observe subjects, good quality uncontaminatedwater (with removal of excreted anesthetic),avoidance of environmental stimulation, consis-tent temperature, and decreased exposure toother compromised fish which may be a sourceof infectious disease agents.

4. Administration of Compoundsand Devices by Various Routes

Morton et al. (2001) should be consulted for guid-ance on best practices for the administration ofsubstances. Although principally focussed onmammals, there are recommendations for fishesand a useful checklist to consult when planningprocedures. As with any procedure, administra-tion of compounds should be carried out by com-petent individuals under expert supervision,preferably a veterinarian.

4.1 Branchial diffusion ("inhalation")The most prevalent route of exposure of fish forchemical agents is via the gills. Fish gills have alarge surface area due to a series of lamellae pro-truding from the surface. The epithelium of thelamellae is extremely thin and designed to facili-tate the diffusion of respiratory gases. In addi-tion to gas transfer, fish gills also permit uptakeof other molecules. Diffusion or uptake efficien-cy of chemicals by the gills depends primarily ontheir hydrophobicity and molecular size (Black,2000a).

4.2 OralGuideline 96:

If a treatment compound is to be adminis-tered orally, the volume dose rate should notexceed 1% body weight (1 mL/100 g).

Fishes may be force-fed liquids and semi-solidsolutions using flexible rubber tubing and asyringe. Force-feeding is useful for the deliveryof stable isotope-labelled compounds and othertest substances. In some species, light anaesthe-sia is necessary to prevent struggling and vomit-ing. In others, such as some sharks, brief restraintand the use of a rigid speculum permits safe pas-sage of the tube.

Regurgitation may occur in some species afterforce-feeding. Fish should be carefully observed,

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particularly following resuscitation, to ensurethat the administered agent is retained.However, many fish have a J- or U-shaped stom-ach or pyloric flexure that prevents the intro-duced substance from being regurgitated, providing the tube is inserted into the stomachpast this flexure. In general, no more than 1%body weight should be administered orally in a single dose, although many species have high-ly distensible stomachs and can tolerate largerpercentages.

Electronic transmitters may be inserted via theoral route into the stomach using a hollow plas-tic tube with a central blunt trocar to push thetransmitter into the stomach.

4.3 InjectionGuideline 97:

Care should be taken during injection tointroduce the needle in spaces between thescales. Intramuscular injections may be madeinto the large dorsal epaxial and abdominalmuscles, taking care to avoid the lateral lineand ventral blood vessels. Intraperitoneal (IP)injections should avoid penetrating abdomi-nal viscera as substances that cause inflam-mation may lead to adhesion formation.

The most useful routes for injection in fish areintravascular, intraperitoneal and intramuscular.Details of injection techniques, suggested needlesizes, and injection volumes are available, e.g.,Summerfelt & Smith (1990), Stoskopf (1993) andBlack (2000b).

Chemicals to be injected should be dissolveddirectly in sterile physiological saline. However,hydrophobic chemicals should be dissolved invery small quantities of co-solvent (e.g., ethanol,methanol, or dimethyl sulfoxide [DMSO]) priorto dilution in saline. For chemicals that are notsoluble or stable at neutral pH, the pH of theinjection solution may be adjusted with an acidor base (Perry & Reid, 1994).

Final injection volumes should be as small aspossible to minimize physiological disturbancesto the fish. In addition, control fish (vehicleand/or sham injected) should be part of theexperimental protocol to correct for any effects ofthe injection procedure or the vehicle.

4.4 Implants, windowsand bioreactors

Guideline 98:

Implanted materials should be biocompatibleand aseptic, and should be implanted usingsterile techniques.

Bioabsorbable pellet implants of bioactive com-pounds in absorbable and nonabsorbable matrixvehicles are available from commercial sourcesor can be custom fabricated. These can be surgi-cally implanted in the peritoneal cavity orimplanted with a trocar introducer into musclemasses. Osmotic minipumps can be implanted ina similar fashion as can transmitters and teleme-try units. Windows to visualize visceral changes,such as splenic size change during blood loss,have been successfully used (Yamamoto et al.,1985).

5. Tagging and Marking

Tagging and marking techniques are used inboth field and laboratory studies. For field stud-ies, general principles are outlined in the CCACguidelines on: the care and use of wildlife (CCAC,2003a). As well, Concerted Action for Tagging ofFishes (www.hafro.is/catag/) provides detailedinformation on current best practices for taggingand telemetry in field research.

When choosing a marking method, primary con-sideration should be given to methodologies thatare not invasive, do not require recapture foridentification, and will remain visible for theduration of the study. Where possible, investiga-tors are encouraged to use natural features asmarks, rather than removing or damaging tis-sues or attaching auxiliary markers.

Guideline 99:

Investigators must aim to minimize anyadverse effects of marking and tagging pro-cedures on the behaviour, physiology or sur-vival of individual study animals. Where sucheffects are unknown, a pilot study should beimplemented.

The following criteria should be applied as far aspossible:

• marking should be quick and easy to apply;

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• marking code (numbers or colors) should bereadily distinguishable;

• markings should persist on animals until allresearch objectives are fulfilled;

• animals should experience no long-termadverse effects on health, behaviour, longevi-ty or social life;

• accurate records of the marking procedureshould be kept;

• marking of animals in the wild must complywith federal, provincial/territorial and otheragency regulations; and

• marking must allow for seasonal changes andgrowth of juvenile animals.

In choosing an acceptable marking technique,the investigator should consider the nature andduration of restraint, the amount of tissue to beremoved or damaged, the potential for painand/or distress, and the risk of infection. Wheninvasive procedures of more than short durationare proposed, or when species that are prone todamaging themselves during handling are con-cerned, anesthesia or sedation should be used.

Fish biologists use a variety of internal and exter-nal tags, ranging from fin clips to subcutaneousand abdominal transponders. No single tag ormarking approach is suitable for all fishes orapplications. Marks can range in longevity fromdays to decades. Some tags cause virtually noadverse reaction, such as visual implant tags,while others cause considerable cutaneous dam-age (Johnson, 2000).

5.1 Tissue markingGuideline 100:

Marking techniques which cause significanttissue injury, such as branding, tattooing orclipping important fins, should only be used ifevidence is provided to an animal care com-mittee indicating that alternative methodscannot achieve the desired result.

Any marking should be evaluated for its poten-tial effects on health and behavior.

5.2 Tagging

The methodology for tagging fish has been welldescribed (Neilsen, 1992). Tagging operationsmay involve stress and injury, both from han-dling the fish and the wound caused by applica-tion of the tag. Therefore, the effects of the mark-ing on fish behavior and health should be consid-ered (DeTolla et al., 1995). Where these areunknown, a pilot study should be run, using afew fish in the laboratory. The size, shape andplacement of tags should allow normal behav-iour, and should not cause entanglement inaquatic cover. Brightly colored tags may compro-mise an animal's camouflage or possibly act as apredator attractant in the wild. If fish arereleased, they must be in good health, able tofunction in the environment, and be releasedonly within their native range of distribution(DeTolla et al., 1995).

5.2.1 Genetic markers

The current use of genetic technology in theidentification of fish stocks entails the removal ofblood or tissue for identification.

5.2.2 Internal tags and marks

Information concerning the use of implantedwire tags, passive integrated transponder (PIT)tags, otolith marks and natural parasites to iden-tify fish is reviewed by Parker et al. (1990).Morton et al. (2003) and the CCAC guidelines on:the care and use of wildlife (CCAC, 2003a) providegeneral principles to be considered in theimplantation of telemetry devices. In particularthe effect of the weight, shape and size of thedevice on the physiology and behaviour of thefish must be considered.

6. Collection of Body Fluids

A review by Black (2000b) provides details oftechniques used to collect blood, urine, feces, andsperm from fish. In general when blood is col-lected, sample size is recommended to be up to1ml/kg body weight, although fish can sustainhigher percentages of blood volume removal.The fish must be permitted to recover theirhaematocrit level prior to subsequent blood col-lection. Hematocrit recovery times are tempera-

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ture dependent and highly variable betweenspecies.

Guideline 101:

Sedation or anesthesia should be used torestrain fish for collection or cannulation pur-poses. It is important to realize that bothrestraint and anesthesia may alter physiolog-ical parameters such as serum glucose andvarious hormone levels.

Blood collection should only be undertaken bytrained personnel using sterile equipment. Bloodmay be collected by a number of routes includingthe ventral tail vessels and dorsal aorta, and bycardiac puncture.

Longer term cannulation for repeated samples inconscious fish can be achieved in many teleostslarger than 150 grams by cannulating the dorsalaorta (Schreck & Moyle, 1990; Black, 2000b).

The use of indwelling catheters for urine collec-tion in teleosts has been described by Schreck &Moyle (1990) and Black (2000b).

7. Use of Infectious DiseaseAgents, Tumorigenic orMutagenic Agents, and Toxicand Noxious Compounds

As noted in Section B.5. Government Regulationsand Policies on the Use of Fish, regulations andguidelines are issued under Section 36 (5) of the Fisheries Act of Canada. The regulations for the pulp and paper industry, and for the metalmining industry, specify monthly acute lethalitytesting with rainbow trout on all effluentstreams, and the frequency of these tests. Theseregulations also specify Environmental EffectsMonitoring programs which require twice yearlysublethal toxicity tests with fish, invertebratesand plants. Guidelines for the petroleum refiningindustry and potato processing industry alsospecify similar acute lethality tests with rainbowtrout. Environment Canada conducts its ownaudit testing for these regulations and guide-lines. For the Fisheries Act and all the regulations(Pulp and Paper, Metal Mining, Potato Process-ing, etc.) see laws.justice.gc.ca/en/F-14/index.html. Environment Canada's Method Develop-ment & Applications Section has published a

series of peer-reviewed Biological Test Methods(for acute lethal, and sublethal tests) coveringfish, plants, bacteria, and invertebrates, devel-oped under the guidance of the InterGovernmental Environmental Toxicology Group(IGETG). These published methods, found atwww.etc-cte.ec.gc.ca/organization/spd_e.html,describe the care, disease prevention and use offish in testing in detail. Several provinces havesimilar fish testing requirements for industrialwastewater discharges, and these specify the useof the Environment Canada Biological TestMethods for this testing.

For protocols involving infectious disease agents,tumorigenic or mutagenic agents, or toxic andnoxious compounds, it is of particular impor-tance that protocols should include the use of theearliest endpoints that meet the scientific goals ofthe study; see CCAC guidelines on: choosing anappropriate endpoint in experiments using animalsfor research, teaching and testing (CCAC, 1998).

8. Endpoints and Criteria forEarly Euthanasia

8.1 Recognition of "pain", "distress"and "stress"

Guideline 102:

Investigators should eliminate, mitigate orminimize potential pain and distress when-ever feasible and consistent with good scien-tific practice.

Fishes have the potential to experience pain, andmanipulations that provoke stress or avoid-ance/escape behavior may be causes of distress.Fishes respond to noxious stimuli with alteredbehavioral, physiological and hormonal parame-ters. In general, the greater the intensity of stim-uli, the greater the deviation from normal. Inaddition, the pattern of response to nociceptiongenerally corresponds to the pattern seen inmore highly evolved vertebrates (Sneddon et al.,2003). Nonetheless, the recognition and evalua-tion of pain and/or distress in fishes is not easy.Many fish species are prey animals and aregenetically predisposed not to exhibit signs ofinjury or pain.

Although fishes lack some of the structures asso-ciated with pain perception in mammals (e.g.,

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well-developed cortex and neospinothalamictract), there exists evidence that fishes respond ina similar manner to noxious stimuli, learn toavoid "unpleasant" experiences and respondwith an amelioration of pain response after treat-ment with morphine (Jansen & Green, 1970).Fishes also react to aversive stimuli with a fullscale of endocrine and metabolic responses.Changes in corticosteroid and catecholamine lev-els, as well as increases in plasma glucose andlactic acid as demonstrated in some fish species,are generally recognized to be indicators of acutestress.

8.2 Choosing an appropriate endpoint

Guideline 103:

A defined endpoint should be established forstudies which involve potential pain and/ordistress to the animal. A pilot study should beused to identify clinical signs to be used asthe endpoint and to establish appropriatemonitoring of the animals.

Whenever live animals are used for research,teaching and testing, investigators have an ethi-cal obligation to minimize any pain or distressexperienced. The importance of identifying thescientific objectives for a study, and ensuring thatthese reflect a clear understanding of the mecha-nisms being studied and the consequences to theanimals used, is underlined in the CCAC guide-lines on: choosing an appropriate endpoint in experi-ments using animals for research, teaching and test-ing (CCAC, 1998).

Selection of appropriate endpoints that meet thescientific goals but minimize the adverse effectsfor the animals, requires the ability to identifysigns of "pain" and/or "distress" for fishes. Asdiscussed earlier, this can be a challenge in manyfish species. One way to address this challenge isto draw up a checklist of anticipated and poten-tial clinical signs. Each study may require its ownchecklist, depending on the protocol. Both 'pop-ulation' clinical signs (such as feed intake) and'individual' clinical signs (such as skin lesions)should be present on the check list. Informationaccompanying the checklist should includeinstructions concerning what actions should betaken when abnormalities are observed (in-

cluding who to contact) and instructions aboutadditional procedures to be taken if a fish is euthanized.

Guideline 104:

When conducting research with defined, earlypre-lethal endpoints, a list of parametersshould be established to permit an objectiveassessment of health status.

Establishing a checklist or scoring sheet mayinvolve a literature review and compilation ofdocumented clinical signs. In some cases, it maybe necessary to carry out a pilot study in order toobtain accurate information on clinical signs. It isthe responsibility of the investigator, workingwith the ACC and veterinarian, to decide when aclinical sign is a reliable predictor of an event(e.g., death) and what margin of error is accept-able. This is generally undertaken in consultationwith the veterinarian before approval for theendpoint to be used as part of the protocolreview process. Often it will be necessary to usea combination of clinical signs to make decisionsto terminate an experiment or euthanize anexperimental animal.

Recognizing the great physiological and behav-ioral diversity in fishes, the following parametersare given as suggestions for evaluation of clinicalsigns for fishes involved in research or testing.Other useful references include Goede & Barton(1990).

If the morbidity and clinical signs are known,then the parameters at which point an interven-tion (likely euthanasia) becomes essential can bedefined; for instance, anorexia of "x" days dura-tion alone or in combination with certain otherclinical signs (e.g., more than 10 parasites per fishin a parasite infection study) or a certain numberof days post-infection (e.g., parasite load numbertimes number of days infected).

The CCAC guidelines on: choosing an appropriateendpoint in experiments using animals for research,teaching and testing (CCAC, 1998) includes a use-ful discussion on clinical signs to establish pre-lethal endpoints for toxicology studies, clinicalobservations specific to fishes, and suggestionsfor quantifying pain and distress in finfish.

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Physical Appearance normal/abnormal

eye condition

fin and skin condition (Turnball et al., 1998)

mucus production

colour change (usually a darkening associated with disease or bilateral blindness)

Measurable Clinical Signs feed consumption

respiratory rate

posture in water column, i.e. the individual's position in the water (upright,upside down, tilted, etc.)

Unprovoked Behavior position in the water column (e.g., crowding near the inlet or outlet pipe, shoaling, etc.)

social interactions• direct attack, domination of choice tank locations, schooling

• social isolation, i.e. fish either socially isolated or choosing to isolate them-selves from the group

• not responsive to external stimulation

hyperactivity/hypoactivity (Juell, 1995; Holm et al., 1998)• movement (abnormal movements such as flashing or scraping the body)

(Furevik et al., 1993)

• unexpected jumping or escape behavior

Provoked Behavior feeding activity

threat response

avoidance reaction to mechanical prod

avoidance reaction to light beam

Table 1: Evaluation of Clinical Signs for Fishes Involved in Researchor Testing

Guideline 105:

In any study where there is expected morbid-ity and mortality, the criteria for earlyeuthanasia should be clearly defined.

The use of fish in toxicological research andtoxicity testing is well established. Lethal end-point tests may be required by regulatory agen-cies, for example in the assay of environmentaltoxicant mixtures and in fish vaccine efficacytests. Such tests have the potential to cause painand/or distress in fishes; therefore where feasi-ble, the development of pre-lethal endpoints insuch tests is encouraged.

Regulatory agencies should be contacted duringthe development of the study protocol to agreeon endpoint parameters. In general where lethal endpoints are required, studies shouldmake provision for the humane killing of ani-mals expected to die before the next scheduledobservation.

Fishes should not be held indefinitely in a facili-ty without a protocol describing their future use,as well as endpoints for aging fish and fish inpoor condition (see Section J. Disposition of FishAfter Study).

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9. Monitoring

Guideline 106:

Depending on the study and the time of mor-bidity, monitoring should be done at leastdaily. Frequency of monitoring should allowfor the timely removal of fish before severemorbidity occurs. Frequency of monitoringshould be increased where mortality is expect-ed to be high.

If the timeframe for morbidity is unknown, apilot study should be conducted under veteri-nary and ACC oversight to determine the mostcritical period for observation of the fish duringthe study.

10.Negative ReinforcementModalities

Guideline 107:

Pilot studies and literature searches shouldbe used to establish the least invasivemethod of obtaining a consistent responsewhen using negative reinforcement modali-ties in fishes.

Certain studies may entail the application of nox-ious substances, alarm substance addition totank water, or the application of electrical shockto induce swimming or fear/avoidance respons-es in fishes. The guiding principle of ethicalresearch in this instance is to avoid excessive orunavoidable stress where possible, and to useprocedures that minimize distress. For instance,when designing an experiment, a preference testwith an avoidable negative stimulus should besubstituted for an unavoidable negative rein-forcement schedule.

11.Exercise to Exhaustion

Guideline 108:

Studies involving the forced swimming of fish-es to the point of exhaustion, often in con-junction with negative reinforcement, shouldbe conducted with strict adherence to guidingprinciples of minimization of distress of ani-mals. Fishes used in exercise to exhaustionstudies should be monitored continuously.

Recovery of fishes after exercise to exhaustion mayentail special holding and handling arrangements,such as segregation from normal conspecifics andprovision of low current environments.

12.Environmental ExtremesGuideline 109:

Studies involving the exposure of fishes toenvironmental extremes should select theearliest endpoint possible.

In some instances, fish may be subjected toextreme heat or cold in their holding environ-ment as part of an experimental design. This typeof study would be regarded as highly invasive inmost vertebrate species.

When proposing studies involving exposure toextreme environments, the definition of end-points becomes very important and should becarefully defined to reduce: 1) the use of animalsin future studies; and 2) distress for fish involvedin ongoing studies.

If behavioral endpoints are proposed, it is im-portant that the behavior be observed andrecorded in a repeatable fashion using rigorousmethodology.

Animals undergoing environmental extremesare likely to experience a range of after-effects,including immunological and physiologicalchanges, which may preclude their use as normalanimal models in subsequent studies.

13.Genetically Modified Fish

The local ACC should ensure that investigatorshave complied with applicable regulatoryrequirements before approval of a protocolinvolving an aquatic organism that is an animateproduct of biotechnology (e.g., genetically modi-fied fish). Like many other countries, the federalgovernment of Canada has developed regula-tions, policies and guidelines applicable toresearch involving animate products of biotech-nology in order that the potential for adverseeffects on the environment and human healthmay be assessed. Schedule XIX of the NewSubstances Notification Regulations (NSNR)under the Canadian Environmental Protection

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Act, 1999, specifies the information that must beprovided 120 days in advance of the import ormanufacture of an aquatic organism that is ananimate product of biotechnology. These regula-tory requirements apply to research and devel-opment organisms unless specified containmentcriteria are met (i.e. no release into the environ-ment of the organism, genetic material of theorganism or material from the organisminvolved in toxicity). Organisms determined tohave, or suspected of having, potential adverseeffects on the environment or human health maybe controlled as necessary, including by prohibit-ing or imposing conditions on their import ormanufacture.

Investigators involved in importation, creationor use of aquatic organisms with novel traitsshould contact the biotechnology office of DFOfor more information on the NSNR require-ments. The New Substances Notification Branchof Environment Canada can provide more infor-mation on the NSNR requirements related toother aquatic new substances (telephone: 1-800-567-1999 [toll-free in Canada] or 819-953-7156[outside Canada]; facsimile: 819-953-7155; [email protected]).

Guideline 110:

Genetically modified fishes may have chan-ges in physiology and anatomy as the resultof their genetic alteration, and should beclosely monitored.

A review of transgenic techniques is provided inChen et al. (1996), Jowett (1999), Linney et al.(1999), Fan & Collodi (2002), Lu et al. (2002),Maclean et al. (2002), and Rocha et al. (2004). Inaddition, the CCAC guidelines on: transgenic ani-mals (CCAC, 1997b), and future revisions, shouldbe consulted.

GM fish may have different metabolic and envi-ronmental requirements compared to non-GMfish. The normative tables generated for non-transgenic fish cannot be automatically appliedto transgenic fish (Stevens et al., 1998).

Guideline 111:

Genetically modified fishes must not be per-mitted to enter the food or feed chain unlessthey have undergone a thorough safetyassessment and have received authorizationfor sale, manufacture and/or import as a foodor feed by Health Canada and the CanadianFood Inspection Agency.

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Guideline 112:

Where feasible, the euthanasia of fishesshould consist of a two-step process, withinitial anesthesia to the point of loss of equi-librium, followed by a physical or chemicalmethod to cause brain death.

Physical techniques such as percussive stunningand gill-cut methods, commonly used in com-mercial aquaculture, should be used secondaryto anesthesia; the exception being when animalsare in extreme distress and the time taken inpreparation of anesthesia would result in pro-longed distress.

Use of lethal levels of central nervous systemdepressants, such as buffered TMS, are the pre-ferred method of euthanasia. Alternatively, astunning blow to the head performed by anexperienced person is also acceptable if followedby pithing or cervical dislocation. Use of carbondioxide is not an acceptable method of euthana-sia, nor is suffocation by draining the tank orremoving the fish from water.

Kreiberg (2000) provides discussion on accept-able methods of euthanasia in fishes. The onusremains on the fish handler to be well informedabout the pharmacology and physiologicalimpacts of a proposed method of euthanasia.Robb & Kestin (2002) and Lines et al. (2003) pro-vide further information on humane methods ofeuthanasia for fishes.

Guideline 113:

If a physical technique of euthanasia is usedwhen killing fishes, it should entail the physi-

cal destruction of brain tissue by pithing orcrushing the brain.

Use of hypothermia (including putting fish onice) before euthanasia should be avoided.Because many species of fishes continue to havebrain activity in the face of advanced cerebraland systemic hypoxia, physical euthanasia tech-niques such as decapitation alone should beavoided (Flight & Verheijen, 1993). It is thereforedesirable to physically destroy, freeze, or pith thebrain in fishes that have been euthanised using aprimary physical technique such as blow to thehead.

The American Veterinary Medical Association(AVMA), in their recommendations on methodsof euthanasia for lower vertebrates such as rep-tiles, no longer support the use of hypothermia/freezing as a technique because of concernsabout the induction of pain during ice crystal for-mation (AVMA, 2001).

Exsanguination under anesthesia is also anacceptable method of euthanasia.

Various forms of electrocution have been usedcommercially, including prolonged exposure toAC and DC voltage; however, these methodsmay be associated with spinal fractures and mus-cle damage.

When large numbers of fish need to be eutha-nized, the use of immersion anesthetic at lethaldose in the holding tank is acceptable.

I. EUTHANASIA

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1. Consumption of Fish

Guideline 114:

Fishes destined for food and subjected tosedation or anesthesia should be held for thedesignated withdrawal time before beingkilled.

In studies involving aquaculture fish species, itmay be acceptable to release such fishes forhuman food, providing the fishes have not beentreated with any unlicensed compounds and theadvice of a veterinarian has been sought.

2. Release of Fish to Wild

Guideline 115:

In general, research fishes that have beenkept in captive environments must not bereleased into the wild. Release into the wild isonly permissible under appropriate licenceunder the Fisheries (General) Regulations orsimilar provincial/territorial regulations.

3. Fish as Pets

Some institutions release healthy research fishes(not GM fishes) that are commonly accepted petor companion species to individuals with the

knowledge and ability to provide adequate care.No GM fish may be removed from research facil-ities to private premises.

If fishes are to be released to the care of an indi-vidual as companion animals, the institutionshould develop an appropriate policy describingthe conditions that need to be fulfilled beforetheir release.

4. Transfer of FishBetween Facilities

Guideline 116:

Fishes should undergo health assessmentbefore being transported between facilities.Appropriate regulatory approval and permitsmust be in place before any transfer.

The transfer of unhealthy fishes between facili-ties should be avoided, other than when request-ed by a veterinarian for the purposes of clinicalinvestigation and diagnosis.

5. Disposal of Dead Fish

Fish must be disposed of according to accept-able federal, provincial/territorial and munici-pal regulations for the disposal of biologicalmaterials.

J. DISPOSITION OF FISH AFTER STUDY

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Acclimation — a persisting physiological, bio-chemical or morphological change within anindividual animal during its life as a result of aprolonged exposure to an environmental condi-tion such as a high or low temperature; general-ly, the changes are reversible

Adaptation — the observation that the physiolo-gy, biochemistry and morphology of any animalis usually very well matched to the environmentthat the animal lives in; these features have beenshaped through evolution by natural selectionover many generations, and involve irreversiblechanges in the genetic material

Analgesia — decrease in response to noxiousstimuli

Anesthesia — a state caused by an externalagent, resulting in depression of the nervous system, leading to loss of sensation and motorfunction

Asepsis — absence of living germs, free fromseptic and poisonous putrefactive products

Atraumatic needle — a needle with a suture per-manently attached

Distress — a state of excessive stress in whichthe animal is unable to make the necessary adap-tations to stressor(s)

Ecosystem — a complex of the plant and animalcommunities within an area, along with the non-living components of the environment and theinteractions among these

Ectothermic — an animal that assumes the tem-perature of its surroundings

Euthanasia — literally, a good death; rapid lossof consciousness and death, with no pain or dis-tress accompanying the procedure

Exsanguination — a procedure causing exten-sive loss of blood due to internal or externalhemorrhage

Fish — one or more individuals of one species

Fishes — individuals of more than one species

Fomites — non-living objects that can carry dis-ease organisms (e.g., feeders, mops, etc.)

Hygroscopic — readily absorbs water

Hypothermia — lower than normal body temperature

Integument — the natural outer covering of ananimal; the skin

Lamellae — area of the gills where the exchangeof gases and waste products occurs

Morbidity — visible manifestation of a diseasedstate

Mortality — loss of life; death

Myopathy — muscle damage resulting fromanaerobic muscle function; predisposition maybe due to improper capture procedures

Noxious stimuli — those stimuli that are damag-ing or potentially damaging to normal tissue

Pain (in fish) — fish pain is a response to a nox-ious stimulus that results in a change in behaviouror physiology and the same noxious stimuluswould be painful to humans (a working definition)

Progeny — offspring

Protocol — a written description of a study oractivity that includes details of the goals, the useof animals, the procedures that are to be followedand the personnel involved; the purpose of theprotocol is to ensure the quality and integrity ofthe study or activity

Quarantine — the segregation or isolation of animals from all others to prevent the spread ofdisease

L. GLOSSARY

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Regurgitation — passive return of food or fluidto the mouth from the stomach

Salmonidae — the family of fishes that includessalmon (Oncorhynchus spp. and Salmo salar), trout(Salvelinus spp., Salmo spp.) and char (Salvelinusalpinus); whitefish (Coregonus spp., Prosopium spp.,Stenodus leucichthys); and grayling (Thymallusarcticus). Also used informally as the name of thesubfamily which includes salmon, trout and char

SOP — Standard Operating Procedure; writtendocuments specifying procedures for routineactivities that must be followed to ensure thequality and integrity of the study

Standard Environmental Temperature — thetemperature for optimum growth of a species offish

Supersaturation — a condition where the totalgas pressure in a body of water exceeds the baro-metric pressure in the overlying atmosphere

Telemetry — the use of devices to transmit infor-mation to a distant station where it is recorded;commonly used in field studies to monitor ani-mals in order to answer questions about theirphysiology, behavior, habitat use, survival andmovements

Welfare — a term used to describe the quality oflife that an animal is experiencing

Well-being — a state or condition of physicaland psychological harmony between the organ-ism and its surroundings. Good health and man-ifestation of normal behavioral repertoire are themost commonly used indicators of an animal'swell-being

Withdrawal time — the length of time betweenwhen an animal is given a drug and the pre-scribed time period for clearance of residues ofthat product

Zoonotic — relating to the transmission of a dis-ease from a non-human species to humans

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ACC — Animal Care Committee

CAEAL — Canadian Association forEnvironmental AnalyticalLaboratories

CALAM — Canadian Association ofLaboratory Animal Medicine

CCFAM — Canadian Council for Fisheriesand Aquaculture Ministries

CFIA — Canadian Food InspectionAgency

CITES — Convention on InternationalTrade in Endangered Species ofWild Fauna and Flora

COSEWIC — Committee on the Status ofEndangered Wildlife in Canada

CSA — Canadian StandardsAssociation

CSZ — Canadian Society of Zoologists

CWS — Canadian Wildlife Service

DFO — Fisheries and Oceans Canada

FAWC — Farm Animal Welfare Council

FHPR — Fish Health ProtectionRegulations

FRP — Fiberglass reinforced plastic

FTA — Free Trade Agreement

GATT — General Agreement on Tariffsand Trade

GFI — Ground fault interrupter

HVAC — Heat, ventilation and air conditioning

ICES — International Council for theExploration of the Seas

IV — Intravenous

IP — Intraperitoneal

NAC — North American Commission

NAFTA — North American Free TradeAgreement

NASCO — North Atlantic SalmonConservation Organization

OECD — Organization for EconomicCooperation and Development

OIE — Office International desEpizooties

SOPs — Standard Operating Procedures

SPS — Sanitary/Phytosanitary

TMS — Tricaine methane sulphonate

WAPPRIITA — Wild Animal and PlantProtection Regulations ofInternational andInterprovincial Trade Act

WTO — World Trade Organization

M. ABBREVIATIONS

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American Fisheries Society (AFS) www.fisheries.org

American Society of Ichthyologists andHerpetologists (ASIH) www.asih.org

AquaNet www.aquanet.ca

Canadian Aquaculture Institute, PEIwww.upei.ca/~cai/

Canadian Association of AquacultureVeterinarians

Canadian Association for EnvironmentalAnalytical Laboratories (CAEAL) www.caeal.ca

Canadian Council for Fisheries andAquaculture Ministries (CCFAM) www.aquaculture.ca/English/CAIA_CCFAM.html

Canadian Food Inspection Agency (CFIA)www.inspection.gc.ca

Committee on the Status of EndangeredWildlife in Canada (COSEWIC)www.cosewic.gc.ca

Canadian Society of Zoologists (CSZ)www.csz-scz.ca/jpellerin/csz/

Canadian Veterinary Medical Association(CVMA) canadianveterinarians.net

Convention on International Trade inEndangered Species of Wild Fauna and Flora(CITES) www.cites.org

Environment Canada (EC) (and Intergovern-mental Environmental Toxicology Group) www.ec.gc.ca

European Convention for the Protection ofVertebrate Animals Used for Experimentaland Other Scientific Purposes (ETS 123) -Appendix A: Guidelines for accomodation and care ofanimals — Species-specific provisions for fishwww.coe.int/T/E/Legal_affairs/Legal_co-operation/Biological_safety%2C_use_of_animals/Laboratory_animals/A_texts_docs.asp#TopOfPage

Farm Animal Welfare Council (FAWC)www.fawc.org.uk

Fish Health Protection Regulations (FHPR)laws.justice.gc.ca/en/F-14/C.R.C.-c.812

Fisheries and Oceans Canada (DFO) www.dfo-mpo.gc.ca

Fisheries Society of the British Isles (FSBI)www.le.ac.uk/biology/fsbi

Health Canada (HC) www.hc-sc.gc.ca

Office International des Epizooties (OIE)www.oie.int

APPENDIX ARELEVANT GUIDELINES AND ORGANIZATIONS

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Zoonoses, also called zoonotic diseases, are ani-mal diseases that may be transmitted to humans.Piscine zoonotic diseases have been extensivelyreviewed elsewhere (Nemetz & Shotts, 1993) andwill only be briefly summarized here. In general,transmission of fishborn zoonoses is relativelyrare, and the vast majority of these organismsproduce self-limited bouts of gastroenteritis,usually secondary to consumption of under-cooked fishes, or localized wound infections,usually due to contamination of cuts or abrasionswhile handling either live fishes or fish tissues.However, a few of the more virulent organismshave the ability to produce systemic infections inhumans and, in very rare instances, death(Nemetz & Shotts, 1993). Although zoonotic dis-eases are rare in healthy individuals, the risk ismarkedly increased in people with depressedimmunity (e.g., patients with AIDS, organ trans-plant recipients receiving immunosuppressivedrugs).

Diseases of fishes can be induced by a variety ofbacteria, rickettsiae, roundworms (nematodes),cestodes (tapeworms), flukes (trematodes), pro-tozoa, viruses, and fungi (Nemetz & Shotts, 1993;Fryer & Bartholomew, 1996). The type of infesta-tion is dependent upon a myriad of factors,including the species, the supplier, the geograph-ic origin, and the diet of the fish; other importantissues are water quality and salinity.

The majority of important zoonotic pathogens offishes are gram negative bacteria. In NorthAmerica, members of the genus Vibrio are proba-bly the most important bacterial pathogens ofmarine and estuarine fishes (some species alsoinfect freshwater fishes); members of Vibriospecies (spp.) are also important potential sourcesof zoonotic disease. Other gram negative bacteriacausing fishborn zoonoses include Plesiomonasshigelloides, Aeromonas spp., Escherichia coli,Salmonella spp., Klebsiella spp., Edwardsiella spp.,Yersinia ruckeri, and Leptospira icterohaemorrhagica(Nemetz & Shotts, 1993). Efficacious fish vac-cines are currently available to protect fishes

against some of these gram negative organisms(Fryer & Bartholomew, 1996).

Pathogenic gram positive bacterial infection infishes is less common. Gram positive bacteriathat can cause zoonoses include Streptococcusspp., Erysipelothrix rhusiopathiae, Mycobacteriumspp., Clostridium spp., and Staphylococcus spp.Several cases of cellulitis caused by accidentalinoculation with Streptococcus iniae, a fishpathogen associated with menigoencephalitis intilapia, have been reported in patients in Canadathat had injured their hands while handlingtilapia (Weinstein et al., 1997). Erysipelothrix andMycobacterium also usually cause a localized skininfection but, under some circumstances, cancause a disseminated infection (i.e. septicemia).In contrast, skin contact with Clostridium andStaphylococcus does not usually cause an infec-tion in humans, but rather produce disease whencontaminated fishes containing their bacterialtoxins are consumed.

A number of viruses and fungi have been identi-fied in fishes (Fryer & Bartholomew, 1996).However, no documented case of human infec-tion by either fish viruses or fish fungi have beenreported (Nemetz & Shotts, 1993). Although anumber of fish parasites including both wormsand protozoa can infect humans, such infectionsare almost invariably due to consumption ofundercooked fish and are exceedingly rare inNorth America (Nemetz & Shotts, 1993).

Finally, some marine organisms produce toxinsthat can cause illness and death in humans.Ciguatera and scombroid are examples of foodpoisonings that can result from eating carnivo-rous tropical marine fishes. Ciguatoxin isbelieved to be produced by a marine dinoflagel-late that adheres to reef plants and then is con-sumed by herbivorous fishes; these fishes aresubsequently consumed by carnivorous fishes,thus concentrating the ciguatoxin in their tissues.Patients with ciguatera poisoning usually dis-

APPENDIX BZOONOTIC DISEASE – TRANSMISSION OF FISH

DISEASES TO MAN

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play gastrointestinal and neurological symp-toms. The mechanism of scombroid toxicity isunclear, but is associated with human consump-tion of marine fishes from the familyScombroidae (e.g., tuna, bonito, mackerel, skip-jack, etc.). Scombroid often produces allergicsymptoms in patients (Nemetz & Shotts, 1993).

Although all of these fishborn zoonoses are rare,prompt and accurate diagnosis expedites appro-priate treatment. Because they are so rare, mostphysicians, even those specializing in the treat-ment of infectious diseases, have little or noexperience diagnosing them. Thus, it is impor-tant for people working with fishes to also beaware of the existence of fishborn zoonoses and,if being evaluated by a physician, to mention thatthey have occupational exposure to fishes. Withthe advent of the use of fishes as a source ofimplantable biomaterials, in xenotransplanta-tion, or as bioreactors for large scale productionof human proteins, the possibility of emergingxenozoonoses transmitted from these sources

should be considered. At the present time, dis-ease agents recognized as a hazard in other xeno-transplantation models, such as mammalianendogenous retroviruses, are not a recognizedthreat from fish tissues; however, the knowledgeof this area is in its infancy.

References:

Fryer J.L. & Bartholomew J.L. (1996) Establishedand emerging infectious diseases of fish. ASMNews 62: 592-594.

Nemetz T.G. & Shotts E.B. Jr. (1993) Zoonoticdiseases. In: Fish Medicine (ed. M.K. Stoskopf),pp. 214-220. New York NY: WB Saunders.

Weinstein M.R., Litt M., Kertesz D.A., Wyper P.,Rose D., Coulter M., McGeer A., Facklam R.,Ostach C., Willey B.M., Borczyk A., & Low D.E.(1997) Invasive infections due to a fish pathogen,Streptococcus iniae. New England Journal ofMedicine 337: 589-594.

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Although there are currently no national stan-dards developed and approved by federal agen-cies that are specific for aquatic biocontainmentsystems, the Canadian Food Inspection Agency(CFIA) and Fisheries and Oceans Canada (DFO)are working on developing guidelines that coverboth veterinary biologics and aquatic animallive-holding, in view of meeting several objec-tives, including genetically modified (GM)organisms and fish with novel traits concerns,and introductions and transfers concerns (e.g.,escapee impacts from ecological, spawning andtrophic competition). The standards will bringmuch needed transparency, clarity, consistencyand objectivity for both the handlers of aquaticanimals and their pathogens for experimental orcommercial development purposes, and theinspectors. For aquatic animal pathogen labora-tories conducting in vitro work, such as diag-nostic laboratories, all physical containment and operational practices for the appropriatecontainment level must be followed as per theexisting Containment Standards for VeterinaryFacilities (Agriculture and Agri-Food Canada,1996). These Standards can be accessed at: www.inspection.gc.ca/engl ish/sci/lab/convet/convete.shtml. This Appendix is there-fore intended to provide additional guidance toinvestigators and Animal Care Committees(ACCs) working with aquatic animals and/ortheir pathogens until federal agency guidelinesare developed. The approach taken in theseCCAC guidelines reflects current best practices.Facilities engaged in studies requiring biocon-tainment are subject to inspection by provincialand federal authorities. CFIA's Biohazard Contain-ment and Safety division (www.inspection.gc.ca/english/sci/bio/bioe.shtml) is the point of con-tact for any queries or construction plans regard-ing laboratories conducting in vitro work and forany containment questions related to in vitro useof imported veterinary biologics. DFO's AquaticAnimal Health Office is the point of contact forany containment questions related to live aquat-ic animal transfers or in vivo infection trials.

There are some prescriptive standards providedin the Fish Health Protection Regulations: Manual ofCompliance (Fisheries and Oceans Canada, 1984)concerning importation and movement ofsalmonid fish, their eggs and tissues.

Principles of containment described within thisAppendix also extend to research involving GMfish. DFO is the Canadian lead agency develop-ing requirements and containment standards forlive-holding of aquatic organisms with noveltraits (e.g., GM fish). DFO's biotechnology officeis the point of contact for any containment ques-tions related to aquatic organisms with noveltraits, and DFO's Aquaculture ManagementDirectorate for containment questions related tolive aquatic animal transfers.

1. Aquatic BiocontainmentLaboratory Physical Plant

The categorization of aquatic pathogens toAnimal Biosafety Levels I to IV (established forterrestrial systems) is problematic. Most agentsare currently considered as Biosafety Level I or II (See Laboratory Biosafety Guidelines [HealthCanada, 2004] at www.phac-aspc.gc.ca/ols-bsl/lbg-ldmbl/). The criteria used to assess the levelof risk are subjective, e.g., the lack of viral enve-lope in the case of some viral pathogens.

In aquatic systems, surface contamination, con-tamination of fomites such as nets and tanks,and aerosol-born transmission are all possiblesources of accidental transfer of aquaticpathogens. Biocontainment in aquatic systemsrelies to a large extent on common sensehygiene measures such as fomite control anddisinfection, use of tank-specific hardware,external clothing dedicated to biocontainmentfacilities, hand washing and the use of disinfec-tant footbaths.

APPENDIX CGUIDELINES FOR CONTAINMENT FACILITIES

(FOR PATHOGEN STUDIES)

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Guideline A:

Facilities conducting research, teaching ortesting on fish using aquatic animal patho-gens must do so with the knowledge and per-mission of local DFO, CFIA and appropriateprovincial/territorial authorities.

Guideline B:

Facilities used for aquatic animal pathogenresearch must be properly contained andphysically separate from other holding roomsand facility functions, such as the holdingand rearing of production fish or holding ofbroodstock fish. Effluent must be renderednoninfectious before being returned to theenvironment.

The location and physical attributes of a contain-ment facility must prevent accidental releaseduring flooding, storms or other natural disas-ters. In order to ensure adequate containment,facilities should be built on dry land and sitedabove historic flood levels. If these conditionscannot be met, then projects should be smallenough to permit all animals to be moved safelyto an alternate site, or destroyed, within a realis-tic time-frame permitted by natural disasterwarnings. Containment facilities are particularlysensitive to entry of predators and pests, and thedesign of the entire facility must address thisrisk. In particular, drain systems, portals for per-sonnel and equipment access, and heating, venti-lation and air conditioning (HVAC) systems arevulnerable.

Consideration must be given to maintenance andother required access to mechanical and accesso-ry systems. Where possible, these systemsshould be accessible without entry to the con-tainment area from outside of the facility.

Guideline C:

Materials and surfaces used for facility con-struction should be durable, nonporous andeasily sanitized using surface cleaning andpotent surface disinfectants that have provenefficacy. Wood, porous materials andunsealed concrete should not be used forbiocontainment facilities.

Materials and surfaces used in any fish facilityshould be easily sanitized. However, in contain-ment facilities, surfaces may be subjected to more

rigorous decontamination procedures, and there-fore should be tested to ensure that they canwithstand stronger or more repetitious chemicaltreatments.

Guideline D:

Containment rooms should be ventilated topermit drying conditions and even mixing ofair, but prevent aerosol-born pathogens fromescaping via air movement or condensationon surfaces or clothing.

Temperatures should ensure that wall, floor andceiling surfaces dry rapidly, as most aquaticpathogens survive longer in a wet environment.The use of tank covers ensures that humidity lev-els are maintained at a reasonable level withinthe room and also reduce the risk of splash trans-fer of pathogens between tanks or onto the floor.

Guideline E:

All pathogen control processes must havefail-safe backup.

In the event of a failure in any automated efflu-ent disinfection system, there should be ade-quate containment to prevent untreated waterfrom leaving the facility, as well as an emergencyalert to ensure that the responsible authoritiesrespond as quickly as possible to rectify the situ-ation. Automated systems should be pro-grammed to measure residual disinfectant con-centrations and ensure these fall within pre-setparameters for pathogen inactivation. Theseparameters should be tested prior to bringing inhigh risk research animals and/or theirpathogens, by bacteriological or virological testsof 'spiked' effluent. All effluent from contain-ment systems should be routed through hardplumbing to effluent treatment tanks whichensure adequate contact time for decontamina-tion of effluent. In the event of a system failure,inflow systems should be engineered to shut off,preventing overflow of the system.

Any release of effluent to the environment mustmeet local regulations, and ensure that viablepathogens are not released. In general, this willrequire local municipal sewage authorityapproval and/or an environmental impactassessment if the effluent is to be released to thelocal waterways. Disinfected products, whethersolid or liquid, should be neutralized prior to

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release from the containment facility becausethey can be toxic to fishes and other aquaticresources.

1.1 Facility entranceGuideline F:

Facilities should have secured entry systems,be clearly signed and have means of loggingwho enters and at what time.

Traffic within the facility should be minimized,with entry permitted on an ‘as needed’ basisonly. The entry should be locked and accessiblesolely to authorized personnel. There should be aclothing transfer area immediately adjacent tothe entry area where outside clothing can beexchanged or covered by containment facility-specific outerwear (coveralls, footwear andgloves).

Guideline G:

Foot baths and hand wash stations should beavailable at the entry and exit points of thecontainment facility.

Personnel must use footbaths and hand washstations each time they enter or exit the contain-ment facility.

1.2 RoomsGuideline H:

Individual rooms should have door and wallseals.

Individual rooms should have raised damsacross the doors and floors, with waterproofseals running along the walls to a depth suffi-cient to hold all water within the containmentroom should it leak or spill from the holdingtanks or water supply reservoirs.

Guideline I:

Room surfaces, piping, tanks and watertransfer systems in rooms should bedesigned for complete access and steriliza-tion between studies, and must be designedto prevent back flow from animal holdingtanks and effluent handling systems.

All pipes should be hard-plumbed with remov-able access points for cleaning and quality con-

trol culture following studies. Fail-safe plumbingsystems should be used that prevent tanks fromself-draining in the event of loss of water supply.

Guideline J:

Rooms should be engineered so that in theevent of catastrophic failure, such as tankleakage, all untreated effluent is preventedfrom escape.

Floor drains should be routed to a holding reser-voir that can process all water held within thefacility.

Guideline K:

Room surfaces should be smooth, impervi-ous and able to be disinfected readily.

Floor surfaces should be smooth, sealed andnonporous, and corners should be coved. Wallsand bare fixtures should be disinfectable, as mustall materials used for handling or likely to comein contact with the experimental animals.

Guideline L:

Electrical fixtures should be ground faultinterrupted, gasketed, sanitizable and provid-ed with emergency back-up power.

Wall switches should be sealed and waterproofto allow disinfection. Ceiling lamp fixturesshould be gasketed, waterproof and sanitizable.Ceilings should be smooth, impervious and san-itizable. Electrical outlets should, ideally, be posi-tioned well above floor level, and above watersupply lines.

Guideline M:

Exposed piping should be secured to wall andfloor surfaces with smooth sanitizable brack-ets, and should be accessible for cleaning.

Piping should have access points for cleaning.Valves should be accessible from within and out-side individual rooms to regulate flows in theevent of emergencies. Where walls are penetrat-ed for pipe runs, they should be caulked orsecurely sealed and gasketed to prevent harbour-ing pathogens. Any drain holes that go direct tomunicipal drainage (not recommended) shouldbe protected with sealed standing pipes to pre-

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vent untreated effluent escape. Methods toreduce condensation should be employed.

Guideline N:

Equipment for cleaning and sanitation, dry-moist feed storage bins and equipment usedin the room (such as nets) should be room-specific.

Nets and other equipment should be tank-specif-ic, in order to minimize transfer of pathogensbetween tanks. There should be immersion disin-fection buckets for the regular sanitation ofroom-specific equipment. The regular changingof disinfection solutions used in such roomsshould be scheduled by an SOP, and the efficacyof such methods should be assessed with a regu-lar validation procedure. Appropriate concentra-tion and type of disinfectant to achieve 100% killof pathogens in the environment should be used.

1.3 Water flow and tanksGuideline O:

All tanks must be enclosed with tightly fitting,removable covers to prevent water, aquaticanimal or pathogen escape.

Tank lid perforations for airstones, standpipes,etc. should be gasketed. Tank stands should beconstructed of rigid, smooth, impervious materi-al such as aluminum, stainless steel or fiberglass.For saltwater systems, the tanks and tank standsshould be salt water corrosion proof. The use ofporous or organic materials, such as concrete isinadvisable. Any porous materials, such as air-stones, should be disposed of and replaced bynew airstones between experiments, or shouldbe disinfected between use.

Guideline P:

All effluent water must be collected and heldin treatment tanks for a recommended disin-fectant contact time, and the effluent fromthese tanks must be regularly monitored foreffective level of disinfection.

The disinfection tank should undergo regulartesting for integrity, as should the disinfectantinjection system. The tank should have an auto-mated disinfectant system which is monitored byan alarm system in case of failure. In the event ofany failure in the containment system, automat-

ed alarms to pagers or another means of immedi-ately contacting emergency personnel should bein place. Recirculation systems in aquatic biocon-tainment laboratories represent a special chal-lenge because of the potential to contain and har-bour pathogens, particularly in concentrationcomponents of the system such as biofilters. Suchsystems should be designed with the capacity toisolate, remove and disinfect all system compo-nents including tanks, pipes, biofilters and otherancillary equipment without disrupting animalsin the system.

Guideline Q:

Spill kits to contain and disinfect spills ofpathogen-contaminated water should be inplace in all rooms and areas where spills are a possibility. Designation and training ofpersonnel in the use of spill-kits must bemaintained.

When a spill occurs, it should be physically con-tained using absorbent material, and the infec-tious agent destroyed using an effective disinfec-tant for a recognized contact period; after which,the spill can be cleaned up.

1.4 Facility wearGuideline R:

Facility-specific clothing and footwear wornin the containment area must be kept in thearea.

Street clothing should be kept outside the facilityin separate locker areas away from the contain-ment area. Facility-specific footwear should bedonned to enter the outer areas of containmentfacilities such as hallways. At the room level, acomplete outerwear change with footwearchange is needed. Within individual rooms, per-sonnel should wash their hands before leavingthe room and when leaving the outer area of thecontainment facility.

Facility clothing, tools, etc. should be regularlydisinfected.

1.5 Disposal of materialsGuideline S:

Any contaminated material, including cloth-ing, fomites and carcasses, should be secure-

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ly bagged and autoclaved or incineratedbefore cleaning or disposal.

Clothing which requires cleaning should be dis-infected prior to removal from the facility forcleaning, unless laundering facilities are avail-able within the containment zone and are proveneffective against the pathogens.

Alternative means of sterile disposal of carcassesand other contaminated biological wastes includesincineration or autoclaving and rendering.

2. Operation of an AquaticBiocontainment Facility

The following aspects must be followed in orderto ensure the proper functioning of a biocontain-ment facility:

• only authorized personnel should be permit-ted entry;

• there must be entrance procedures for main-tenance staff;

• if aquatic zoonotic agents are in use (e.g.,Streptococcus iniae or Mycobacterium marinum),a health and medical surveillance programmust be in place to protect personnel frominfection;

• personnel must be trained in all infectious,chemical and physical hazards likely to beencountered, and must demonstrate compe-tence; such training must be documented;

• facility-specific SOPs must be developed andfollowed to ensure consistent practices in all

areas, such as entry and exit procedures, traf-fic flow from clean to dirty, disinfection andgarment change on entry and exit;

• staff must understand the physical structure,plumbing and air handling systems in thefacility and how these work, and adhere tostrict containment protocols (i.e. not wedgedoors open, etc.);

• emergency procedures and equipment to dealwith loss of containment, fire, etc. must be inplace, posted and understood by all workers;and

• all spills, loss of containment and accidentsmust be reported and investigated.

References:

Agriculture and Agri-Food Canada (AAFC)(1996) Containment Standards for VeterinaryFacilities. 71pp. Ottawa ON: Agriculture andAgri-Food Canada. Available at www.inspection.gc.ca/english/sci/lab/convet/convete.shtml

Fisheries and Oceans Canada (DFO) (1984)(revised 2004) Fish Health Protection Regulations:Manual of Compliance. Publication 31 (Revised)50pp. Ottawa ON: DFO. Available at www.dfo-mpo.gc.ca/science/aquaculture/aah/manual_of_compliance_e. htm

Health Canada (2004) Laboratory BiosafetyGuidelines, 3rd ed. 113pp. Ottawa ON: HealthCanada. Available at www.phac-aspc.gc.ca/ols-bsl/lbg-ldmbl/

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85

86

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