a guide to acceptable procedures and practicesfor

A Guide to Acceptable Procedures and Practicesfor Aquaculture and Fisheries Research

by

Barker, D., Allan, G. L., Rowland, S. J. and Pickles, J. M.

NSW Fisheries

for

NSW Fisheries Animal Care and Ethics Committee

Port Stephens Fisheries CentrePrivate Bag 1

Nelson Bay NSW 2315Australia

2nd Edition

ISBN 0 7310 9423 9 March 2002

NSW Fisheries ACEC

A Guide to Acceptable Procedures and Practices 1

TABLE OF CONTENTS

1. SUMMARY ................................................................................................................... 3

2. ANIMAL RESEARCH AUTHORITIES....................................................................... 4

2.1 What is animal research?.................................................................................... 42.2 Why is an animal research authority required? .................................................. 42.3 Animal Care and Ethics committees .................................................................. 5

2.3.1 ACEC membership ................................................................................ 52.3.2 Procedures of ACECs............................................................................. 6

2.4 Where an animal research authority application form can be obtained ............. 62.5 Animal research authority renewals ................................................................... 72.6 Inspection obligations......................................................................................... 7

2.6.1 NSW Fisheries ACEC............................................................................ 72.6.2 NSW Agriculture, Animal Welfare Unit................................................ 8

3. ANIMAL HUSBANDRY .............................................................................................. 9

3.1 Basic husbandry.................................................................................................. 93.1.1 Water quality variables......................................................................... 133.1.2 Water quality management................................................................... 13

3.2 Disease recognition and treatment ................................................................... 143.2.1 Bacterial infections............................................................................... 143.2.2 Ectoparasitic infestations...................................................................... 173.2.3 Fungal infections .................................................................................. 173.2.4 Recommended formalin treatments...................................................... 173.2.5 Recommended neguvon bath treatment ............................................... 18

3.3 Transport protocols .......................................................................................... 183.4 Recognition and relief of distress..................................................................... 203.5 Hatchery requirements...................................................................................... 203.6 Carers .............................................................................................................. 21

4. ANAESTHETICS ........................................................................................................ 22

4.1 Indications for anaesthesia ............................................................................... 224.2 Levels of anaesthesia........................................................................................ 224.3 Available anaesthetics ...................................................................................... 23

4.3.1 Benzocaine (Ethyl-p-amino benzoate) ................................................. 234.3.2 Tricaine methanesulphonate (MS222) ................................................. 234.3.3 Aqui-S ............................................................................................... 23

4.4 Concentrations required for handling, surgery and euthanasia ........................ 244.5 Methods of euthanasia...................................................................................... 24

5. FIELD SAMPLING AND COLLECTION................................................................ 226

5.1 Acceptable methods of fishing for sampling and collection ............................ 265.2 Electrofishing summary and effects on captured fish ...................................... 26

NSW Fisheries ACEC

2 A Guide to Acceptable Procedures and Practices

5.3 Handling techniques for sampling and returning animals................................ 275.4 Techniques for collection of animals for laboratory analysis........................... 28

6. ENVIRONMENTAL CONSIDERATIONS................................................................ 29

6.1 Disposal of chemicals....................................................................................... 296.2 Disposal of animal carcasses............................................................................ 296.3 Translocation and quarantine ........................................................................... 29

7. REFERENCES............................................................................................................. 30

8. ACKNOWLEDGMENTS............................................................................................ 31

9. APPENDICES.............................................................................................................. 32

9.1 NSW Fisheries ACEC members - 2001........................................................... 329.2 Example of a new application for an animal research authority ...................... 339.3 Example of an annual renewal for an animal research authority ..................... 449.4 Example of an animal research authority certificate ........................................ 489.5 Example of an animal research authority final report ...................................... 499.6 NSW Fisheries ACEC Terms of Reference ..................................................... 509.7 NSW Fisheries ACEC Grievance Procedures.................................................. 519.8 NSW Fisheries ACEC Terms of Appointment ................................................ 52

NSW Fisheries ACEC


1. SUMMARY

Research with vertebrates in New South Wales, Australia, is governed by the AnimalResearch Act, 1985. Under this act, all research must be covered by a current AnimalResearch Authority, issued by an accredited Animal Care and Ethics Committee.

The NSW Animal Research Act 1985 was introduced to protect the welfare of animals, byensuring their use in research is always humane, considerate, responsible and justified. TheAnimal Research Regulation 1995 incorporated the Australian Code of Practice for the Use ofAnimal for Scientific Purposes into the legislation.

Any organisation that uses or supplies vertebrate animals for research or teaching in NSW isaffected by the Animal Research Act. The Act applies to all individuals, groups, institutions,organisations, schools and companies which use animals.

Organisations which conduct research with vertebrates must either (under the AnimalResearch Act, administered by the Animal Welfare Unit of NSW Agriculture) become anaccredited research establishment or obtain an Animal Research Licence.

In addition, all research must be covered by a current Animal Research Authority. These areissued by approved animal care and ethics committees (ACECs) which in turn areadministered by the Animal Welfare Unit and the Animal Research Review Panel of NSWAgriculture.

This document includes a guide to procedures and practices for maintaining and collectingfish for the purpose of scientific research that are consistent with the objectives of theAustralian Code of Practice for the Care and Use of Animals for Scientific Purposes. Itprovides information to assist scientists complete the application form necessary to obtain anAnimal Research Authority.

Basic information for most topics is presented and more detailed information provided fortopics that are most frequently raised by researchers or members of Animal Care and EthicsCommittees who must approve applications for Animal Research Authorities. Emphasis hasbeen placed on describing basic husbandry requirements, maintaining water quality,recognising and treating some of the most common diseases and transporting live fish.Methods for collecting and sampling fish in the wild and practical details of methods foranaesthesia and euthanasia are also described.

This document does not attempt to cover all aspects or methods that may be available to theresearcher in any given circumstance. Full details of methods, even if they are included in thisguide, must be provided in the application for an Animal Research Authority.

Examples of a completed Application Form for an Animal Research Authority, and for anAnnual Renewal are attached. Also attached are copies of an Animal Research Authority andan example of a final report that must be provided to the NSW Fisheries ACEC on completionof research. Finally, the NSW Fisheries ACEC Terms of Reference, Grievance Proceduresand Terms of Appointment are provided.

NSW Fisheries ACEC


2. ANIMAL RESEARCH AUTHORITIES

2.1 What is animal research?

Animal research includes the use of animals in:

Experimental researchSurgical, medical, psychological, biological, chemical or physical treatmentAbnormal husbandry or dietary conditionsElectric shock or radiation studiesCollection of blood, tissue or other body samplesProduct TestingTeachingDiagnosisField surveysProduction of biological materialFeeding trials

An ‘animal’ includes any vertebrate, other than a human being, that is any:

- mammal- bird- reptile- amphibian- fish

2.2 Why is an animal research authority required?

The use of animals for scientific purposes is regulated by the NSW Animal Research Act1985. The following extracts from the Australian Code of Practice for the Care and Use ofAnimals for Scientific Purposes (NHMRC, 1997) outline the responsibilities and justificationsfor conducting animal research: i Scientific and teaching activities using animals may be performed only when they are

essential to:- obtain and establish significant information relevant to the understanding of

humans or animals;- the maintenance and improvement of human or animal health and welfare;- the improvement of animal management or production; or- the achievement of educational objectives.

ii Studies using animals may be performed only after a decision has been made that they

are justified, weighing the scientific or educational value of the study against thepotential effects on the welfare of the animals.

iii People who use animals for scientific purposes have an obligation to treat them withrespect and consider their welfare as an essential factor when planning and conductingstudies.

NSW Fisheries ACEC


iv The acquisition, care and use of animals for all scientific purposes in Australia must bein accord with the Australian Code of Practice (ACP) and with Commonwealth, Stateand Territory legislation.

v Investigators have direct responsibility for all matters relating to the welfare of theanimals they use.

vi Institutions such as NSW Fisheries are required to establish an Animal Care & EthicsCommittee (ACEC) to ensure that all animal use conforms to the standards of theACP.

vii Investigators must submit written proposals for all animal studies to the ACEC whichmust take into account the expected value of the knowledge to be gained, thejustification for the study, and all ethical and animal welfare aspects.

viii Scientific and teaching activities must not commence until written approval has beenobtained by the ACEC.

2.3 Animal Care and Ethics committees

Under the NSW, Animal Research Act 1985, all experiments with vertebrates, need to beapproved by an accredited Animal Care and Ethics Committee. The NSW Fisheries AnimalCare and Ethics Committee must approve all applications for research using fish by NSWFisheries scientists before they commence. This Committee also assesses applications forresearch on fish from Australian Water Technologies (Water Board) and on occasion from otheragencies.

NSWF ACEC approves research with fish conducted by NSW Fisheries or by AustralianWater Technologies. The NSWF ACEC’s role is to advise, monitor, discipline and controlanimal research. The Committee ensures that all research conducted complies with thelegislation and Code of Practice. The Committee is responsible for monitoring researchwithin the department, including inspections of animals and facilities.

The Committee must consider and evaluate requests to conduct research, on the basis of theresearchers’ responses to a comprehensive set of questions, including the justification for theresearch; its likely impact on the animals; and procedures for preventing or alleviating pain ordistress. The NSWF ACEC has the power to stop inappropriate research and to disciplineresearchers by withdrawing their research approvals. They can require that adequate care,including emergency care, is provided. They also provide guidance and support to researcherson matters relevant to animal welfare, through preparation of guidelines and dissemination ofrelevant scientific literature.

2.3.1 ACEC Membership

The membership and duties of ACECs are laid down in the Animal Research Regulation1995. The requirements are based on the Australian Code of Practice, which also provides thebenchmark against which the committee judge research, when the research is proposed andcarried out. The categories are:

NSW Fisheries ACEC


a A veterinarian or person with similar expertise relevant to the species being studiedb An animal researcherc An animal welfare representative who is not involved with the establishment or with

animal research or supplyd An independent person who is not a researcher and preferably is not employed by the

establishment

More than one person may be appointed to each category. The Committee also has aChairperson and Executive Officer. See Appendix 9.1 for the list of NSW Fisheries ACECmembers for 2001.

2.3.2 Procedures of ACECs

The NSWF ACEC conducts quarterly meetings to consider issues associated with animalresearch or teaching. The Committee must:- Review, discuss, approve, reject or revoke proposals to perform animal research or

teaching- Document their consideration of proposals- Have guidelines on procedures for animal research or teaching- Inspect animals and facilities used in research or teaching

Investigators (researchers) are ultimately responsible for the care, management and well beingof their research animals. People working within NSW Fisheries in any role, including as: anemployee, a consultant, a post graduate student or an animal attendant; who wish to useanimals in research must hold an animal research authority issued by NSW Fisheries.

All proposals to undertake animal research must be submitted in writing to the NSWF ACECand approved before the research is commenced. Full information should be provided of:- The justification for the use of animals- The justification for the number and species of animals used- The procedures to be used- The expected impact on the welfare of the animals and the methods which will be used

to avoid and alleviate these

Investigators must keep detailed records of the health and experimental use of animals in theircharge. The records may be examined at any time by the ACEC or inspectors of the AnimalWelfare Unit, NSW Agriculture.

Animal Care and Ethics Committees must meet at least 4 times per year and at each meetingat least one member of each category must be present. Only in exceptional circumstances willapplications be considered at other than a scheduled, physical meeting.

2.4 Where an animal research authority application form can be obtained Applications are available from the Executive Officer, NSW Fisheries ACEC, NSW Fisheries,Port Stephens Fisheries Centre, Private Bag 1, Nelson Bay, NSW, 2315, (facsimile [02] 49821107, telephone [02] 4982 1232), or any NSW Fisheries officer currently serving on the NSWFisheries ACEC. An example of a completed form is attached at Appendix 9.2.

NSW Fisheries ACEC


2.5 Animal research authority renewals Animal Research Authorities are valid for a period of twelve months. Any scientist orinvestigator running a project for longer than one year is required to have their Authorityrenewed each year by submitting an ‘Application for Annual Renewal of Proposal to UseAnimals’ (see example of completed renewal form at Appendix 9.3). Some projects run forlonger than three years. After three years, in addition to completing an ‘Application for AnnualRenewal of Animal Research Authority’, a major revision of the project and methods isrequired. As the ‘New Application for an Animal Research Authority’ is constantly changing(to ensure all information required is provided and to reduce the possibility of confusion),scientists or investigators are required to transcribe information for their project to a new formand indicate major changes from the original application. The Executive Officer will notify thePrincipal Investigator when the Authority for a project is due to expire and, if a renewal issought, whether a ‘New Application for Approval to Use Animals’ must be completed (ie. afterthree years). The Principal Investigator will be asked to either apply for a renewal or notify theExecutive Officer that a renewal is not required. In that case a final report will need to besubmitted. Final reports need to include details of relevant aspects, including the number andfate of animals used and the condition of animals remaining after the project. A brief summaryof the outcomes or findings of the project is also required (see Appendix 9.5 for an example of acompleted final report).

2.6 Inspection obligations

2.6.1 NSW Fisheries ACEC

Inspections of all animal housing and laboratory areas must be conducted regularly by membersof the ACEC and appropriate records maintained to ensure compliance with the Australian Codeof Practice for the Care and Use of Animals for Scientific Purposes.

Policy of NSW Agriculture states that inspections by ACECs may be used to:- Ensure the Code of Practice and ACEC directions are being complied with- Familiarise ACEC members with what is happening ‘in practice’ as opposed to ‘on

paper’- Promote interaction between the ACEC members, researchers and animal care staff

All ACEC members should participate in inspections, though it is not necessary for everymember to attend every inspection. NSW Fisheries ACEC inspects each research locationannually and includes a calendar on every agenda and minutes stating when they were inspectedor when they are due to be inspected.

Inspections should include examination of:

- Animal holding facilities- Animals (health and welfare)- Research procedures (on occasion)- Animal identification and care records- Research records (on occasion)

NSW Fisheries ACEC


Inspections are held to ensure animals are held in current and approved protocols and that theprocedures being conducted are in accord with the approved protocol. Discussion with animalcarers and researchers during inspections is encouraged as a means of obtaining information andproviding an opportunity for contact between these people and ACEC members. Researchprocedures should be viewed by ACEC members on occasion. ACEC members are encouragedto view procedures as often as possible as an additional means of assessing the impact ofprojects on animals. Procedures to be viewed may be chosen on the basis of criteria including:

- Contentious research project or procedure- Convenience (procedure coinciding with inspection)- Commonly occurring procedure- Operator has not been seen performing procedure by the ACEC- ACEC member(s) have not previously seen procedure performed

A written report is submitted to the Executive Officer after each inspection, discussed at the nextavailable meeting and attached to the minutes.

2.6.2 NSW Agriculture (Animal Welfare Unit/Animal Research Review Panel)

The Animal Welfare Unit of NSW Agriculture conducts its inspections under the AnimalResearch Act (usually by a veterinary officer of the Animal Welfare Unit and a member of theAnimal Research Review Panel). There are three different types of visits:

i Accreditation site visitsii Inspection of animal research authority and animal supply licence holders’ facilitiesiii Investigation of complaints

These visits are to ensure that animal research and supply complies with the legislation andCode of Practice. Accrediation, authorities and licences are issued subject to a satisfactoryinspection and can be suspended or cancelled. The Code of Practice provides criteria againstwhich institutions are assessed.

This includes assessing the membership, procedures and activities of the ACEC, animal careprocedures, animal research procedures and the physical facilities for housing and using animals.Central to this is an evaluation of the well-being of the experimental or breeding animals.

Assessment commences with an examination of written material provided by the institution orindividual. This includes a list of the protocols considered by the ACEC and the people issuedwith animal research authorities, ACEC minutes, annual report and records of inspectionsconducted, information about the procedures of the Committee’s operation and decisions.

A detailed report is prepared as soon as possible after the inspection. The report covers anevaluation of the ACEC, assessment of the well-being of the animals, housing and holdingfacilities and animal care and monitoring, including emergency procedures. Once the Panel hasconsidered the report, recommendations may arise which will alter the terms of accreditation orlicence. Conditions of an earlier accreditation may have been met or the Panel may feeladditional conditions should be imposed.

NSW Fisheries ACEC


3. ANIMAL HUSBANDRY

3.1 Basic husbandry Facilities

‘Facilities’ includes the ponds, raceways, tanks, cages and aquaria in which animals are kept.Scientists and the investigators, ACEC's and Fisheries aquaculture and research institutionsare responsible for ensuring that facilities are appropriately staffed, designed, constructed,equipped, operated and maintained to achieve a high standard of animal care and to fulfillscientific requirements. The overall condition and management of facilities must permiteffective maintenance and servicing and be compatible with maintaining the animals in goodhealth (NHMRC, 1989, p.30).

Operation of facilities

All fish holding facilities must be operated in a manner that optimises conditions for fish.Appropriate stocking densities, aeration and water management must be used.

All facilities should be aerated; tanks and aquaria continuously with diffused air and pondsnightly for around 8 h/day with mechanical aerators such as paddlewheels. At very hightemperatures and feeding rates, or on overcast, still days, ponds may need to be aerated for 24h/day. Cages should be located in aerated ponds.

In circular, self-cleaning tanks, a constant flow of water must be used to facilitate the removalof solids and dissolved wastes (e.g. ammonia) and to supplement aeration. If tanks need to bestatic, e.g. during chemical treatment, fish should not be fed and water (10-30 %) should beexchanged daily. Tanks should be placed under cover or in a building out of direct sunlight toprovide an environment with relatively low light intensity. It is beneficial to some species,e.g. Murray cod, to partially cover tanks to reduce stress.

Static ponds should be managed (stocking densities, aeration, water quality, diseases, feedingetc.) according to guidelines for particular species, e.g. techniques for the culture of Silverperch have been published (Rowland and Bryant, 1995).

Stocking densities(dependent on prevailing water quality, size of fish, temperature, oxygen supply, etc)

Housing

Optimum

Upper density1

Tanks

10 kg/m3

100 kg/m3

Cages Ponds

20 kg/m3

5 t/ha 100 kg/m3

20 t/ha

1 Requires greater control of water quality, more experience and entails higher risk.

NSW Fisheries ACEC


Monitoring requirements

All fish holding facilities and support systems must be inspected every 24 hours. Thingsto observe include changes to the fishes’ external body, especially any signs of diseases,abnormal swimming behavior, abnormal feeding behavior, as well as unexpected changes inthe appearance of the water. Water quality variables and diseases need to be monitoredregularly (see following).

Nutrition and feeding

Commercial diets are available from a number of feed manufacturers for marine and freshwaterfish including diets for larvae, juveniles and adults.

Wherever possible, manufactured fish diets should be stored for as short a time as possiblebefore use. If the diets are to be stored for longer than a month or two they should be kept incool (<15oC), dry conditions, or frozen. At all times, manufactured diets should be kept cooland dry. If there is any sign of fungal contamination, diets should be discarded.

The manufactured diet should be designed for the target species, life-stage and size. Thenutritional requirements and development of diets for silver perch have been observed (Allanand Rowland, 2001). Fresh or frozen bait fish or other aquatic plant or animal material are oftenused as a food source. They usually need to be stored frozen and care must be taken to ensurethey are not contaminated and do not deteriorate.

Fish should usually be fed to optimise survival and growth. Appropriate feeding strategiesshould be followed for each species, where available. Guidelines for feeding silver perch havebeen published (Rowland et al., 2001). At times, excess feeding can adversely affect waterquality, particularly if fish are not feeding vigorously due to low temperatures or if water qualityis poor or restricted. At such times feeding should be reduced or suspended until conditionsimprove.

3.1.1 Water quality variables

The following variables help ‘characterise’ the water in which fish are held and grown. Eachspecies will have an optimum range for each variable as well as lethal limits. The mostimportant variables are: dissolved oxygen, temperature, salinity, pH, alkalinity, ammonia, nitrite,nitrogen, hardness, hydrogen sulphide, turbidity. Details of each of these variables and theirimportance for holding and growing fish can be found in Rowland (1998) and the followingsummaries are taken from that document.

Temperature

Water temperature influences chemical and biological procedures. As fish are cold-blooded(poikilothermic), the water temperature affects their metabolism, digestion, growth, sexualmaturity and reproduction. Rates of chemical and biological reactions roughly double for every10oC increase in temperature. As water temperature increases, fish become more active,consume more food, use more oxygen and grow faster. However, when the temperature exceedsthe critical level for a particular species, fish become stressed, more vulnerable to disease, maystop growing and can die.

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Salinity

Salinity refers to the total concentrate of all dissolved ions. As salinity rises, the ability of waterto conduct electricity also increases and conductivity is therefore often used to measure orestimate salinity. In general, freshwater is 10-500 mg/L salinity, seawater is 35 000 mg/L (or 35g/L; the units are often referred to as ‘parts per thousand’, ppt or ‰). Many Australian nativefish, such as silver perch, golden perch, Murray cod and catfish can tolerate long-term exposureup to 5 g/L while many estuarine species such as mulloway and snapper can tolerate salinitiesdown to as low as 10 g/L. Rainbow trout, Australian bass and barramundi can tolerate salinityof 0-35 g/L. When changing salinity, fish should be allowed to adjust slowly (e.g. 1 g/L/day).

Dissolved oxygen

Dissolved oxygen is the most critical and limiting variable in fish husbandry and culture. Likeall animals, fish cannot live without oxygen and lethal levels vary from just less than 1 mg/L toabout 3 mg/L. Sub-lethal levels (e.g. around 4 mg/L) can stress fish, reduce growth and increasesusceptibility to disease. Oxygen enters water through diffusion at the air-water interface and asa result of photosynthethis when there are plants (e.g. algae) in the water. For aquaria, tanks andraceways, dissolved oxygen is usually supplied through low pressure compressors or blowers(through diffusers like air stones). In ponds, paddle-wheel aerators are among the most efficientmethods of transferring oxygen from the air to the water. Mechanical aeration also assists withmixing water throughout the pond.

pH and carbon dioxide

The pH is the measure of the hydrogen ion concentration and indicates whether water is acidic(pH less than 7), neutral (pH = 7) or alkaline (pH greater than 7). The desirable range for fish isaround 6-9, depending on species. A pH of 4 is lethal for most species while prolongedexposure to pH levels of above 10 is also lethal.

As with many water quality variables, the interaction of pH with other variables can be criticallyimportant. The inter-relationship of pH with ammonia is one of the most obvious examples.Carbon dioxide affects pH as it has an acidic reaction in water. Phytoplankton and other aquaticplants remove carbon dioxide from water during photosynthesis in daylight hours and allorganisms add carbon dioxide during respiration. Typically, in ponds or water bodies with algalblooms (phytoplankton), or other aquatic vegetation, the pH will rise during the day, peakingnear sunset then declining to a minimum around dawn.

Alkalinity

Alkalinity is the total quantity of ‘bases’ present in water (primarily carbonate and bicarbonateions) and is measured as mg/L of equivalent calcium carbonate (CaCO3). The bases ‘buffer’water against changes in pH. Waters of low alkalinity (e.g. <20 mg/L) are poorly buffered andin general are unproductive for fish culture. A desirable range of alkalinity is 50-200 mg/LCaCO3 and waters with alkalinity over about 250 mg/L CaCO3 are generally less productive.

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Hardness

Is the total concentration of metal ions (mainly calcium and magnesium in water and as it is alsoexpressed as mg/L CaCO3 it is often confused with alkalinity. Waters of low hardness (e.g. <20mg/L) are called ‘soft’ while those above 200 mg/L are ‘hard’. Most productive waters arebetween about 20 and 250 mg/L CaCO3. In general, where the alkalinity is derived fromcalcium or magnesium carbonate, hardness and alkalinity values are similar. However, ifalkalinity is derived from sodium bicarbonate (NaHCO3), it is possible to have soft water with ahigh alkalinity.

Ammonia and Nitrite

Ammonia is the major product excreted when fish (and other aquatic animals) cataboliseprotein. It is excreted across the gills and in urine and faeces. Ammonia is also a by-product ofthe decomposition of organic matter by bacteria. In water, ammonia exists in two forms; ahighly toxic unionised form (NH3) and a much less toxic ionised form (NH4

+). The proportionof ammonia in each form depends on pH, temperature and salinity. For example, at a pH of 8.0,1.6% of ammonia is in the toxic unionised form at 8oC and 8.8% at 32oC. Similarly, at 24oC,0.5% of ammonia is in the unionised form at pH of 7.0 compared with 34.4% at pH of 9.0.Fortunately, ammonia is very soluble and is readily used as a nutrient by plants. So, when pHrises in ponds with algal blooms, ammonia concentrations tend to be low.

Ammonia is ‘nitrified’ to nitrite and then to nitrate through a two step process by the ‘nitrifying’bacteria Nitrosomonas and Nitrobacter. These bacteria occur naturally and are the main agentsresponsible for removing ammonia and nitrite in biological filters.

At high levels, nitrite replaces oxygen in the blood to form methemoglobin. Gill filamentsbecome brown giving rise to the name ‘brown blood disease’ from this condition.

Nitrite toxicity is reduced when chloride is present in the water. Adding salt at 5 g/L is veryeffective in reducing nitrate toxicity and explains why marine and estuarine fish are much lesssusceptible than freshwater species.

Sensitivity of fish to nitrite and ammonia vary between species and age/size of fish. Lethallevels (acutely toxic concentrates that are predicted to kill 50% of the population over 4 days)are around 1.0 mg/L unionised ammonia and 160 mg/L nitrite. However, growth of silver perchwas reduced when concentrations were above 0.36 mg/L unionised ammonia or 1.4 mg/L nitriteover three weeks.

Nitrogen is not toxic to fish but if the water is supersaturated with air, the nitrogen can cause gasbubble disease which can kill fish. Bubbles can form under the skin, behind the eyes or in bloodvessels, in the same way bends occur in humans. Supersaturated water can occur when water ispumped under pressure or sometimes, from under-ground sources. It should be avoided orpassed through ‘degassing’ columns before use.

Hydrogen sulphide

This compound is produced by the bacterial decomposition of organic matter under anaerobic(without oxygen) conditions. Hydrogen sulphide smells like rotten-eggs and is often called

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‘rotten-egg gas’. Concentrations of less than 1 mg/L can be lethal to fish. It can be removedfrom the water using vigorous aeration or by adding potassium permanganate.

Turbidity

This refers to the amount of suspended material in water from clay, organic material or plankton(including plants, phytoplankton or zooplankton). Turbidity is not usually harmful to fish andcan help prevent colonisation of the pond bottom by aquatic plants that can interfere with harvestoperations. However, excessive clay turbidity can also interfere with development of beneficialalgal blooms, while excessive organic turbidity can increase biological oxygen demand and leadto problems with dissolved oxygen.

3.1.2 Water quality management Maintenance of good water quality is the most important aspect of fish husbandry, as themajority of problems occurring in a hatchery/aquarium are associated with poor water quality.Water quality in freshwater aquaculture is discussed in detail by Rowland (1998).Maintenance of good water quality requires the regular monitoring of dissolved oxygen,temperature, pH and ammonia, and for marine and brackish water species, salinity. Water exchange

A significant contributing factor to disease is poor water quality. A reduction in water qualitycan result from inadequate water exchange. The three basic systems of waterexchange/circulation are a) static, b) flow-through and c) recirculating systems.

a Static systems

Static systems do not receive continuous water inflow. They rely either on low biomass ornatural processes within the system to maintain water quality with occasional additions of newwater when required. Most ponds are run as static systems. Water exchange is often achievedby partially draining the pond or tank and then replacing lost water. Water exchange is stillused as a primary method to maintain water quality, especially when ‘crises’ occur (e.g. algalbloom crashes followed by low dissolved oxygen).

b Flow-through systems Flow-through systems are ‘single-pass’, i.e. water is only used once, and the tank, raceway orpond usually remains full with water entering and leaving the system at the same time fromdifferent locations. For tanks and ponds, water inflow should be designed to maximisemixing and help concentrate solids near the drain or outflow (e.g. used to generate a circularwater current). Outflowing water leaves via an overflow (e.g. an external standpipe) and isdrawn from the bottom of the tank; such tanks are ‘self-cleaning’.

c Recirculating systems Recirculating systems are used to conserve water or maintain temperature. Sometimes, risksof introducing pathogens with influent water can be avoided by water re-use or recirculatingsystems. Operators of recirculating systems will often blend fresh (new) water with re-use

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water. Recirculating systems typically involve solids removal through sedimentation and/orfiltration (e.g. drums, disc or sand filters) and biological filtration to remove dissolvednitrogenous wastes such as ammonia and nitrite. Depending upon the complexity required,recirculation systems can also include foam fractionation (air or another gas is bubbledthrough a column of water to trap and remove organic particles) and methods to increaseoxygen concentration (e.g. pure oxygen injection). Treatments to remove or reduce potentialpathogens, such as ozonation or UV filtration are sometimes also included in recirculationsystems. NOTE: Biofilters must also be managed as a living, breathing organism. Most importantly, ittakes time for surface bacteria or biofilters to establish. The time depends upon the amount ofnutrients supplied, temperatures of the water and water flow characteristics. Bacterialpreparations and food (nutrients) are available and can be used to reduce this ‘start-up’ timethat typically is between 1-3 months. Care must be taken to ensure the filter is not starved ofnutrients or that chemicals used to treat fish (e.g. formalin) do not affect the biofilter. Carefulmonitoring should be undertaken when changes in the filter loading rates are made or whenany chemicals are used. Turbulence

Turbulence is caused by aeration or water inflow and may keep solids suspended in the watercolumn. Turbulence is required for mixing but needs to be kept as low as possible whenrearing larvae up to ten days old. Larvae must not be prevented from reaching the surfaceduring this period as swimbladder inflation is occurring. After this period, increasedturbulence assists in food distribution, which may help reduce the incidence of cannibalism.For older fish, turbulence should help concentrate waste materials near the drain or wateroutflow.

Skimmers

If rearing larvae, surface skimmers should be in place during swimbladder inflation to reduceoil scum, which may prevent larvae from gulping air on the water surface. Skimmers are notnecessary after this stage but certainly aid in the removal of excess lipids and proteins. Cleaning

Tanks should be cleaned regularly, by siphon or vacuum pump, to reduce problems with theaccumulation of organic matter (uneaten food, faeces) and fouling organisms, bacteria andalgae. Filters need to be backwashed regularly to prevent build up and decomposition ofaccumulating waste material. Floors, drains, etc., associated with tank rooms should becleaned and sterilized on a regular basis. Dilute pool chlorine or sodium hypochlorite (NaOCl20 ppm) or caustic soda (NaOH 1%) are suitable cleaning agents for this purpose.

3.2 Disease recognition and treatment

3.2.1 Bacterial infections Bacterial infections often follow stress caused by handling, exposure to toxic metabolites (e.g.ammonia, nitrites, hydrogen sulfide), exposure to sub-optimal temperatures or low dissolved

NSW Fisheries ACEC


oxygen levels. Bacteria can also be ingested with food and may proliferate in the gut. Noantibiotic residue is allowed in food fishes in Australia, i.e. zero. As the action of antibiotics is often pathogen-specific, it is important to accurately diagnose abacterial disease to ensure that the most effective antibiotic and dosage can be administered(Rowland & Ingram, 1991). Antibiotics should be used with caution as prolonged or repeatedapplication, at dosages insufficient to kill bacteria, may lead to the development of resistantstrains that will subsequently prove difficult to treat (Rowland & Ingram, 1991).

Recognition / Diagnosis

External bacterial infections are easily recognised by hemolytic inflammation (redness andswelling), fin erosion, lesions and ulcers and/or gross colonies of bacteria (e.g. Flexibacter) ofthe epidermis on the torso or fins. Other signs may be exopthalmia (pop-eye), swelling of theabdomen, fluid in the body cavity and lesions in tissue such as spleen, liver and kidney. Thespecific infecting bacteria may be identified by sending fish to a histology/pathologylaboratory for analysis. The best samples are moribund (dying) fish which should be sent liveto the laboratory if possible. Recently-dead or euthanased fish can be placed on ice forpreservation for postmortem analysis. Laboratories include:

i NSW Fisheries, Aquatic Animal Health Unit, Wollongbar, NSW.ii EMAI (Elizabeth Macarthur Agricultural Institute) Menangle, NSW.iii Macquarie Pathology, Sydney, NSW.iv Aquatic Diagnostic Services International, Brisbane, Qld.

For detailed descriptions of symptoms and treatment for specific fish bacterial diseases referto Fish Diseases (University of Sydney, 1988), Diseases of Australian Native FreshwaterFishes (Rowland & Ingram, 1991) and Diseases of Silver Perch (Callinan & Rowland, 1995). Treatment Procedures

Antibiotics such as oxytetracycline (OTC) are utilized in the treatment of bacterial infectionsin fish. OTC is a artificially engineered antibiotic used sparingly to combat bacterial infection,but also as a biological tagging agent in age/growth experiments (skeletal chronology). OTC is available in powder and liquid forms and may be applied to fish externally (by bathingor immersion), orally (by ingestion) or by intraperitoneal injection. It must be used underveterinary prescription. Note: OTC is a known carcinogen. Safety precautions must therefore be taken to ensure thatOTC powder is not inhaled (read safety requirements before use).

Bathing / Immersion Bacterial infections in fish are commonly external. During bathing or immersion the OTC isapplied directly to the site of infection, and is also absorbed through the gill filaments into the

NSW Fisheries ACEC


blood stream. This method has generally been more effective than all other methods ofantibiotic treatment. Preparation of a treatment solution that may be added directly to tanks requires that the OTCpowder be weighed and dissolved in water. The recommended procedure fortreatment/prevention of bacterial infections is to apply OTC at a concentration of 100 mg/L(active ingredient), for five to seven successive days. Baths should be exchanged andrepeated every 1-3 days as necessary. Prior to treatment, tanks should be thoroughly cleaned and the water well aerated. Watercirculation should cease for the first 12 hours after treatment, allowing time for the uptake ofthe OTC. For the following 12 hours, water exchange should then proceed. Overnightflushing is often convenient as it allows for observations and the re-treatment during daytime. To maintain high water quality, fish should not be fed for the duration of the treatment.Addition of higher doses of OTC can lead to dramatic reduction in pH and this needs to bemanaged. Oral / Ingestion Ingestion is another method of treatment for bacterial infections, provided that the drug iscarried to the site of infection. The addition of sulphonamides assists in this transfer. OTCmay be administered orally in pelleted feed. This method is employed as an alternative to theimmersion/bath method and may be used in ponds or cages. A soft pellet can be prepared by combining 2 kg of minced pilchard and 3 kg of pellet crumblewith OTC powder. The recommended pellet concentration of OTC is 50-100mg of OTC/kgof pelleted feed. Pellets can also be directly coated with OTC. Vitamin C (ascorbic acid) is often added at a concentration of 0.5 - 1.0 g/5 kg to pelleted feedto assist in the healing process and to reduce associated stress. Oxalinic acid too can be usedas a treatment itself and is also a good agent for prophylatic purposes. Intraperitoneal injection Treatment with OTC by injection into the intraperitoneal cavity is most commonly used forskeletal chronology (to mark the bones of fishes for aging or tagging purposes) but it may alsobe used therapeutically especially for larger fish or when it isn’t possible to feed or immersethe animal requiring treatment. The liquid forms of OTC available for injection are:i Roscocycline 5 = 50 mg/mlii Engamycin 100 = 100 mg/ml Recommended OTC injection concentration:i 50 to 100 mg/kg body weight. Engamycin is preferable for the treatment of juveniles or smaller animals as the dosagevolume required is half that of Rosocycline (as it is twice as concentrated as Rosocycline).

NSW Fisheries ACEC


3.2.2 Ectoparasitic infestations Ecto-parasites are pathogens that commonly cause diseases in both freshwater and marinefishes. If left untreated, ecto-parasitic diseases can cause very high mortalities. The parasitesand their etiology, diagnosis and treatment in Australian native freshwater fishes are describedin detail by Rowland & Ingram (1991) and Callinan and Rowland (1995). The most common parasites of freshwater fish are the ciliate protozoans Ichthyophthiriusmultifiliis (which causes the disease white spot), Chilodonella hexasticha and Trichodina sp.,the flagellate Ichthyobodo necator, and the monogenean trematode Dactylogyrus sp. Theseparasites mainly infest gill tissue, and can be readily diagnosed using a microscope.Outbreaks can usually be controlled by using formalin in ponds, or salt or formalin in tanks.

3.2.3 Fungal infections

Water molds (Class Oomycetes) are ubiquitous and cause fungal infections of fish. There areseveral common fungal diseases of freshwater and marine fishes; fungus (cotton wool-likegrowths on skin or gills) caused by Saprolegnia sp., and Epizootic Ulcerative Syndrome (EUSor red spot disease) caused by Aphanomyces invadans. Fungi are opportunists that normallyfeed on dead tissue or become ineffective when the epidermis is damaged and/or the immunesystem is suppressed. Fungal infections can usually be avoided by good husbandry, carefulhandling and the maintenance of good water quality. The prophylactic use of 3-5 g/L NaCl intanks will prevent fungal infections, and formalin may reduce spread once an infection hascommenced in ponds or tanks. Prevention of fungal diseases is generally much easier thancontrol. Listed below are two treatment procedures that are well established and commonly used byNSW Fisheries researchers.

Formalin Treatment A formalin bath is employed for marine and freshwater fish both as an antifungal agent and inthe treatment of ectoparasites (parasites located on the skin or gill filaments) of fish, and fisheggs (GESAMP). Formalin removes oxygen from water, and therefore containers must beaerated during treatment (Rowland & Ingram, 1991). Formalin is rapidly degraded in water,and an initial concentration of 50 mg/L is not detectable after 2-4 days at 20oC.

3.2.4 Recommended formalin treatments

Concentration (mg/L)

Time

200

1 hour 100 2 hours

20-30

Repeat each 2-4 days for some diseases

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Repeated treatments are necessary to eliminate parasites that lay eggs, e.g. Dactylogyrus sp. orhave complex life cycles involving cysts, e.g. I. multifiliis. WARNING: Formalin is a potential carcinogen and should be handled carefully as to avoidskin contact, eye irritation and inhalation (Katz, 1989).

Neguvon (Trichlorfon) Treatment

Neguvon is not an approved chemical and requires a prescription from a veterinary surgeonfor use. Treatment with a Neguvon (anthelminthic powder) bath over a twelve to fourteen dayperiod is often a successful treatment for dactylogyrid gill flukes. If fish show signs of stressdue to ectoparasites or if a treatment appears unsuccessful (i.e. the fish continue to displaysymptoms of stress, such as continuous flashing) further diagnosis is necessary. Fish shouldbe examined after all treatments to evaluate effectiveness.

3.2.5 Recommended neguvon bath treatment

Day

Concentration (mg/L)

1

1 2 3 4 5

0 1 0 1

(continue schedule for twelve to fourteen days)

NB: There is no treatment required on days 2, 4 and 6, etc. Neguvon breaks down quickly. During treatment it is also advised to maintain the 10-20 %exchange water rate. It is possible for the treatment concentration to be doubled to 2.0 mg/L ifrequired, however 1.0 mg/L is the preferred concentration. As the required concentration ofNeguvon is quite low, preparing a larger volume of treatment solution will ensure greateraccuracy of treatment concentration. Fish should be closely monitored for signs of distress during treatments. Neguvon canbecome toxic to fish if stored incorrectly or if used past its expired date. NB. Read handling precautions before use.

For alternative treatments and more detailed explanations, refer to Fish Diseases (Proceedings,1988), Diseases of Australian Native Freshwater Fishes (Rowland and Ingram, 1991) andCallinan and Rowland (1995).

3.3 Transport protocols

Changes in environment, noise, movement and confinement all contribute to the stress thatfish experience. Animals are particularly susceptible to transport stress. The ability to cope

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with stress during transport will depend on the fishes’ state of health, species, age, sex,stocking density, period without food, the duration of the trip, the mode of transport, andwater quality, particularly temperature, oxygen, pH and ammonia. It is imperative that all sources of distress are identified, minimized or avoided to ensure thatthe health and well being of transported fish are not unduly compromised. The general principles given below are derived from sections 4.2 and 5.5 of ACP. Stress during transport can be minimized by: i appropriate size, design and construction of transport containers;ii maintenance of good water quality, including use of pure oxygen;iii limiting exposure to extremes of temperature, noise, visual disturbance and vibration;iv providing, if appropriate for the species, an inner shelter within the transport container;v ensuring that animals are separated where there is incompatibility of species, age, size,

sex, or reproductive status;vi preventing unnecessary handling; andvii administering anaesthetics, if appropriate, during handling, by appropriately trained

persons.viii application of NaCl (5 g/L) to reduce stress in freshwater fish. Conditions for transport Containers must be escape and tamper-proof and should be protected from sudden movementsand extremes of climate (NHMRC, 1989, p.30). The water quality should be of the highestpossible standard at the start of the journey. The temperature should be kept constant.Relatively low or high temperatures should be avoided. A constant air or oxygen supply tothe holding tanks should maintain >5 ppm dissolved oxygen in the water. Pure oxygen ispreferable and is often necessary when fish are stocked at high densities. Handling

Injury and stress-induced disease can occur to animals, which are handled or crowded duringtransportation. Therefore, handling should be kept at a minimum. Appropriate handlingtechniques should be used including sedation with anaesthetics if necessary. Whereverpossible, the short and long-term consequences of capture and handling should be recorded(NHMRC, 1997, p39) Inspection stops The frequency of inspection stops throughout a journey will depend on the stocking densityand duration of a journey. Generally, inspections occur every 2-4 hours of the journey, moreheavily stocked tanks may require checking more frequently.

Quarantine / Acclimation Quarantine procedures are used to isolate fish with actual or potential contagious diseases.Consequently, all new broodstock should be quarantined until acclimatized and given a goodhealth status before transfer to the broodstock facilities. In addition, broodstock that show any

NSW Fisheries ACEC


form of infection or behave abnormally should be isolated until treatments or medicationshave been successfully completed. Ideally fish being moved from one pond to another shouldbe quarantined.

3.4 Recognition and relief of distress

There are a number of signs that fish are in distress. The include ‘gulping’ at the surface,erratic swimming and ‘flashing’ (fish turning on their side). The skin of some fish getsdarker, mottled or banded when they are stressed and this can be an indication that conditionsare suboptimal. Distress is often caused by poor water quality (see Section 3.1.1 above).Gulping is often due to low dissolved oxygen and can be ameliorated using aeration or waterexchange. Erratic swimming can indicate neurological problems (e.g. caused by ammoniatoxicity), disease, developmental problems (e.g. failure of swim bladder to inflate) or be aresponse to poor water quality. Irreversible neurological or developmental problems cannotbe relieved, disease problems require diagnosis and treatment (see Section 3.2) and waterquality can be addressed, at least initially through water exchange and aeration (see Section3.1.1).

Colour changes indicating stress can be a response to crowding, excess light, dominancehierarchies, poor water quality or disease. In general, fish prefer low light (too dark to read)although under some circumstances they can be acclimated to higher light intensities.Grading and sorting fish into relatively tight size classes can interrupt dominance hierarchies.

3.5 Hatchery requirements Marine and freshwater hatcheries are facilities that generally house and maintain a variety ofanimals including algae, invertebrates (as live feeds), larvae, fry, fingerlings and broodstock.It is important that hatchery procedures and protocols are developed, recorded and madereadily available to all staff working within the unit. The following are areas that should be addressed by hatchery managers when developing suchregimes. Culture tanks The infrastructure of all tanks should be sound in construction and provide appropriate sizeand shelter to its inhabitants, details of ACEC requirements are listed in 3.1.

Preparation of tanks

Water management, standard hygiene and sterilization procedures.

Stocking of larvae Quarantine and acclimation procedures.

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Record keeping

Date, daily events, feeding, treatments, water quality, mortalities, animal numbers, carer incharge.

Feeding

Live feeds, (rotifers and brine shrimp), weaning, artificial diets, nutrition, food storage andpreparation.

Harvesting / Size grading

Procedures associated with harvesting/grading regarding methods, handling, destination ofanimals harvested etc.

3.6 Carers

Record Carer Details of staff responsible for animals, at time of action, should be recorded. Qualifications / Experience

Of staff caring for animals/operations should be obtained and judged if appropriate.

NSW Fisheries ACEC


4. ANAESTHETICS

4.1 Indications for anaesthesia

Anaesthetics are often used in:

- fish transportation- fish handling- diagnostic procedures and surgery to sedate and calm fish- to immobilize broodstock during artificial breeding procedures such as gamete

sampling, hormone injection, and egg and milt stripping- to euthanase animals.

Anaesthetics are employed at low doses, such that their limited use in coastal aquaculturepresents no significant environmental risk, although there may be many hazards to users(GESAMP).

4.2 Levels of anaesthesia

Level Description Signs displayed

0 Normal behavior Reactive to stimuli. Good muscle tone, normalequilibrium and operculum rate.

1 Sedation Equilibrium maintained. At lighter levels there issome reaction to external stimuli and normal opercularrates. Deeper levels show no reactivity to mild externalstimuli and reduced opercular rates.

2 Light anaesthesia Opercular rate increases initially, then decreases asanesthesia deepens. Progressive loss of equilibrium.Reacts to only deep pressure stimuli. Color changesmay be seen.

3 Surgical anaesthesia No reaction to any stimuli. Slow opercular rate, withoperculum spread. No muscle tone, no equilibriumcontrol.

4 Medullary collapse Cessation of operculum movements, followed sometime later by cardiac arrest.

NSW Fisheries ACEC


4.3 Available anaesthetics

4.3.1 Benzocaine (Ethyl-p-amino benzoate)

Benzocaine is probably the most commonly used anaesthetic in fisheries research applicationsas it is very effective, relatively safe and easy to use, and relatively inexpensive. Benzocainehas been registered by the National Registration Authority for use in aquaculture.

It is available in a powder form, which is not water soluble and requires dissolving in anorganic solvent before use (e.g. 1 g/10 mls alcohol).

Usually applied during egg and milt stripping and for transportation purposes. Clearance ratesare rapid, as is the animal’s recovery to normal behavior or response to stimuli.

Method of preparation for benzocaine

Ethyl-p-amino benzoate is dissolved in 100% ethanol at an advised concentration of 1 g/10ml, to form Benzocaine solution. This solution should be kept refrigerated and not exposed tosunlight.

Administration

Benzocaine solution should be thoroughly mixed into the anaesthetic bath prior to fish beingplaced into the bath, unless impractical. Dosages for light and surgical anaesthesia are around25 and 50 mg/L respectively.

4.3.2 Tricaine methanesulphonate (MS222)

This is a commonly used anaesthetic, however it is relatively expensive. It is available inpowder form. It is very unstable in sunlight and should be stored in sealed, light proofcontainers.

MS222 causes marked brachychardia, associated with low rates of opercular movement.Forced irrigation of the gills is thus useful during prolonged anesthesia.

Used in stripping eggs at low dosages, light anaesthesia and euthanasia but the marginbetween anaesthesia and acutely toxic concentrations is small. It has a low persistence in theaquatic environment.

Administration and preparation of MS222

MS 222 powder is very soluble in water and may be added directly to anaesthetic baths.

4.3.3 Aqui-S

Aqui-S has been registered and is available commercially. The active ingredient is iso-eugenol. This drug has only recently been recognized as a suitable anaesthetic for fish, andmay be particularly useful to fresh water species including eels, that show some resistance tothe other anaesthetics described above.

NSW Fisheries ACEC


Administration and Preparation of Aqui-S

Aqui-S is water soluble, stock solution is prepared by diluting concentrated Aqui-S to100 mg/L in distilled water.

(Note: Clove oil has also been used as an anaesthetic, and its active ingredient is eugenolwhich has similar effect to iso-eugenol on fish. However, Clove oil is not registered for use.)

Stock solution can be added directly to anaesthetic baths.

4.4 Concentrations required for handling, surgery and euthanasia1

Concentration (mg/L)1Anesthetic

Handling Surgery Euthanase

Benzocaine 25-35 50-75 100MS222 Unknown 12.5 100Clove oil2 15 60 Not RecommendedAqui-S1 25 60 Not Recommended

1 These concentrations are for silver perch (see Stone and Tostin, 1991) and thoserequired for other fish may differ. In the absence of other information, researchers areadvised to start with lower concentrations and increase the dose if necessary.

2 Not registered for use.

4.5 Methods of euthanasia

There are several methods available to euthanase marine vertebrates. The preferred method isto use a lethal dose of anaesthetic. Suggested lethal dose rates of several commonanaesthetics have been provided above. However, sometimes it may not be possible orpractical to use an anaesthetic to euthanase animals. This circumstance might arise where: i)samples need to be collected for analyses which cannot be contaminated by anaesthetics (ortheir carriers); ii) where the need to euthanise an animal arises when the researcher is notcarrying anaesthetics (in the field, for example); and iii) during disease diagnosis where theanaesthetic may affect parasites. In such circumstances it is acceptable that other methods ofeuthanasia are used. These methods include:

Ice slurry – An ice slurry is made by adding 50% water to cubed or crushed ice, a fresh waterslurry is 0.0oC, whilst a salt water slurry is -4.0oC. Either of these is acceptable and willquickly euthanase fish.

NSW Fisheries ACEC


Rapid severing of the spinal cord – This can be achieved in several ways.

i Placing the fish in upright position and cutting downward with a sharp, heavy knifejust posterior to the head.

ii Ikijimi – this method is traditionally used by Japanese fisherman. There are severalways of performing this method; however, the most simple guide is to position askewer or knife where the lateral line of the fish meets the head or operculum, thendrive the skewer in to the vertebrate to sever the spinal cord.

NSW Fisheries ACEC


5. FIELD SAMPLING AND COLLECTION

5.1 Acceptable methods of fishing for sampling and collection

A wide range of minor procedures are used in the field. These may involve only capture andrelease, often facilitated by the use of an anaesthetic. Such procedures could include tagging,examination, measurement, and sampling.

The following list shows methods of fish sampling and collection that may be used byinvestigators, subject to ACEC approval and only if the following requirements are met:

i All procedures are conducted by appropriately qualified and experienced persons,using clean equipment;

ii Equipment necessary to provide for health and welfare of the animals and relief ofpain is readily available;

iii Uneventful recovery to full consciousness should occur in an area where animals canbe readily observed, can maintain normal body temperature and are protected frominjury or predation;

iv The potential impact of procedures on dependent young is minimised andv The methods and equipment used are appropriate for the species.

Methods of fishing

i Gill Nettingii Trappingiii Hauling/Trawlingiv Electrofishing (refer to Australian Code of electrofishing practices)v Drop/hand liningvi Fyke Nettingvii Dredging sediments

Studies are often associated with commercial fisheries, therefore commercial harvestingpractices may often be used for catching fish. In these cases, practices which ensure rapid lossof consciousness, such as stunning or anaesthetising should be used wherever possible(NHMRC, 1989, p.39).

5.2 Electrofishing summary and effects on captured fish

Boat and backpack electrofishing have been the main sampling tools for freshwater fishresearch conducted by NSW Fisheries in recent years. With the boat-mounted units, a pulsedDC electrical field is generated in the water around the boat effecting fish within up to fourmeters under ideal conditions. The standard protocol uses a pulsed DC current with voltageset at a low initial level of 400 v, but this may be varied depending on the conductivity of thewater. The effectiveness of the initial settings is assessed by checking whether any fish arecaught and by the behaviour of shrimps. Fish within the electric field are temporarilyimmobilised, captured with dip nets, and placed into a live well on the boat and subsequentlyinspected, measured and weighed, etc depending on the requirements of the project beingconducted. Backpack units produce a similar but much smaller field, usually only affectingfish within 0.5 m of the hand-held anode.

NSW Fisheries ACEC


Fish recover in several minutes, and can be released back into the water. NSW Fisheries haveconducted fish injury studies investigating the effects of electrofishing on several species ofnative fish. These showed that a very small percentage of fish suffered injuries such asbleeding, nerve damage and opercular damage, but the incidence of injury was much less thanthat caused by more conventional survey capture methods. These results are supported byoverseas research that show electrofishing causes fewer injuries to fish if performed correctlythan other techniques such as gill nets. There have been no reports of any injury to platypusor other aquatic vertebrates resulting from electrofishing in NSW.

In addition to surveying fish populations in rivers and dams, NSW fisheries staff also useelectrofishing for capturing broodstock for its freshwater fish breeding program. Thistechnique is preferred for broodstock collection as fish receive fewer injuries than with othercapture methods and the process does not affect their reproductive success.

NSW Fisheries research staff conduct electrofishing following the guidelines outlined in theAustralian Code of Electrofishing Practice1, which states in Section 5.5, Care of Fish:

‘Only the minimum power necessary to attract and stun the fish effectively should be used.Contact of fish with live anodes should be avoided, as the resulting shock will be muchgreater. If threatened species that are not being targeted are affected, appropriate measuresmust be taken to minimise disturbance and stress to these fish. Commercial operations mustalso take action to minimise harm to any species that they are not authorised to catch underthe conditions of their licence.’

Also, Section 5.6 of the Code, Care of Other Fauna, requires that:

i ‘Electrofishing must be halted within 15 m of any animals standing in or about to drinkfrom the water, or in contact with a wire fence line that enters the water.

ii The utmost care possible should be taken to avoid shocking platypus, birds and othernative aquatic animals.’

1Australian Code of Electrofishing Practice, 1997. NSW Fisheries Management PublicationNo. 1

5.3 Handling techniques for sampling and returning animals

The principle aim of handling techniques is to minimize stress experienced by fish as far aspossible and to prevent any further damage. The following handling techniques for samplingand returning animals are taken directly from ACP (NHMRC, 1997).

i The time for which the fish is held should be minimal and consistent with the aims ofthe study.

ii Fish must be held in such a way as to minimize stress and/or injury. Knowledge ofavailable information on the normal behavior of the species and its likely response tocaptivity is essential and must form the basis for management practices.

iii Wherever possible, fish must be sampled whilst still in the water. This is particularlyrelevant when using any trapping or netting sampling methods.

iv Holding areas must be safe, quiet and hygienic.

NSW Fisheries ACEC


v Close confinement devices must:- allow fish to rest comfortably;- minimize the risk of escape or injury;- be adequately aerated;- maintain constant temperature; and- minimize the risk of disease transmission.

vi Release should be at the site of capture, unless an alternative site is justified in theproject proposal.

vii The time of the release should be consistent with the species usual time of movement.Individuals must be released safely, particularly if the time of day for release is lessthan optimal.

At the time of release all reasonable steps must be taken to protect animals from injury andpredation.

5.4 Techniques for collection of animals for laboratory analysis

To minimise the distress to animals being kept for analysis or preservation it is essential thatanimals be euthanased as soon as possible after capture.

Animals should not be euthanased by placing in preservative. All vertebrate animals shouldbe correctly euthanased prior to fixation. (For suitable procedures in euthanasia refer toChapter 4.5)

When sorting the catches of haul nets, traps, etc. it is recommended that the net or trap be keptin the water for as long as possible to reduce the trauma to all aquatic animals captured.Discarded animals to be returned to the wild should be released in suitable places, safe fromadvantaged predators or unsuitable water conditions which may be found close to sortingareas.

NSW Fisheries ACEC


6. ENVIRONMENTAL CONSIDERATIONS

6.1 Disposal of chemicals

For information regarding the disposal of chemicals, please examine the following reference:

(GESAMP, 1997) (IMO/FAO/UNESCO-IOC/WHO/IAEA/UN/UNEP) Joint group of Expertson the Scientific Aspects of Marine Environmental Protection, 1997. Towards safe andeffective use of chemicals in coastal aquaculture. Rep. Stud. GESAMP, 65.

6.2 Disposal of animal carcasses

Appropriate provision must be made for prompt and sanitary disposal of animal carcasses andwaste material in accordance with any commonwealth, state or territory legislation, localcouncil by-laws and community standards (NHMRC, 1997).

6.3 Translocation and quarantine

There are a number of requirements governing the import, capture, handling and transport ofanimals. Some requirements are listed below. It should be noted that this list is notcomprehensive and it is the responsibility of the investigator to consult the relevant state andterritory authorities to ensure compliance with all requirements (NHMRC, 1997).

i Under quarantine and fauna laws and formal agreements, the Commonwealth andindividual States and territories regulate the movement of animals or animal tissuesinto Australia and across State and Territory borders within Australia.

ii A Certificate of Health may be required to accompany animals traveling interstate.This is normally issued by State or Territory Departments of Agriculture or theirequivalent.

iii For native fauna, the appropriate State or Territory Fauna Authority may requirefurther certification that animals will be taken legally.

iv Permits must be obtained from The Biodiversity Group (Environment Australia)formerly ANCA, for the importation of live animals, except those species which arespecifically exempt. The Australian Quarantine and Inspection Service (AQIS) shouldalso be contacted.

v Permits are also required by The Biodiversity Group (Environment Australia) andAQIS for the importation of dead specimens and tissues.

vi Permits must be obtained from The Biodiversity Group (Environment Australia) forthe export of both live and dead specimens of all native Australian fauna. Priorapproval is also required from The Biodiversity Group (Environment Australia) for theexport of some animal species not native to Australia.

NSW Fisheries ACEC


7. REFERENCES

Allan, G. L. and Rowland, S. J., in press. Silver perch, Bidyanus bidyanus. In: NutrientRequirements and Feeding of Finfish for Aquaculture. C. Webster and C. Lim(Editors). CABI Publishers, UK.

Callinan, R. B. and Rowland, S. J., 1995. Diseases of silver perch. In: Silver Perch Culture,Proceedings of Silver Perch Aquaculture Workshops, April 1994. S. J. Rowland and C.Bryant (Editors), pp. 67-75. Austasia Aquaculture, Sandy Bay, Tasmania, Australia.

GESAMP (IMO/FAO/UNESCO-IOC/WHO/IAEA/UN/UNEP), 1997. Joint group of Expertson the Scientific Aspects of Marine Environmental Protection, Towards safe andeffective use of chemicals in coastal aquaculture. Rep. Stud. GESAMP.

Katz, S., 1989. Environmental impact assessment for the use of formalin-F in the control ofexternal protozoa on penaeid shrimp. Unpublished report to US FDA, August 1989,12pp.

NHMRC (National Health and Medical Research Council), 1997. Australian code of practicefor the care and use of animals for scientific purposes. 6th edition.

NSW Fisheries Management (Publication Number 1), NSW Fisheries, Sydney. Australiancode of electrofishing practice.

Reddacliff, G. L., 1985. Diseases of Aquarium Fishes, A practical guide for the veterinarian.Veterinary Review No. 25. (Published by the University of Sydney).

Rowland, S. J., 1998. Water quality in freshwater aquaculture. Fishfact 1, NSW Fisheries.

Rowland, S. J., Read, P., Glendenning, D. and Allan, G. L., 2001. Guidelines for feedingsilver perch. NSW Fisheries.

Rowland, S. J. and Ingram, B. A., 1991. Diseases of Australian native freshwater fishes.Fisheries Bulletin No 4.

Rowland, S. J. and Bryant, C., 1995 (Editors). Silver Perch Culture: Proceedings of SilverPerch Aquaculture Workshops. Austasia Aquaculture, Sandy Bay, Tasmania, Australia.

Stone, D. and Tostin, N., 1999. Clove oil a big yawn for silver perch. Fisheries NSWMagazine, Spring 1999, p. 19.

University of Sydney, Post-Graduate Committee in Veterinary Science, 1988. Fish Diseases-Refresher Course for Veterinarians, In: Proceedings 106, 23-27 May 1998.

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8. ACKNOWLEDGMENTS

For this second edition of the Guide to Acceptable Procedures and Practices for Aquacultureand Fisheries Research, we would like to thank the current members of the Animal Care andEthics Committee; Kevin Burke, John Kennedy, Daniel Large, Joe Pera, Kevin Taplin, EmmaHughes, Tony Gregory and Peter Scanes. As well as past members John Harris and DavidButcher for all their input into the document. Stewart Fielder, Helena Heasman, Bob Creeseand Peter Gehrke also made valuable contributions and Wayne O’Connor and Mark Boothkindly reviewed the document.

NSW Fisheries ACEC


9. APPENDICES

9.1 NSW Fisheries animal care and ethics committee members - 2002

The current members of the 2002, NSW Animal Care and Ethics Committee are:

Name Category

Dr Geoff Allan (NSW Fisheries), Chair Animal ResearchMr David Barker (NSW Fisheries) Animal ResearchMr Kevin Burke Community RepresentativeMr John Kennedy VeterinarianMr Daniel Large Animal WelfareMr Joe Pera Animal ResearchMs Jo Pickles (NSW Fisheries), Executive Officer Not applicableMr Kevin Taplin Community RepresentativeMs Emma Hughes Animal WelfareMr Tony Gregory Animal WelfareMr Peter Scanes Animal Research

NSW Fisheries ACEC


9.2 Example of a completed application for an animal research authority

ACEC Ref: Office Use

Project Title: Aquaculture Nutrition

Principal Investigator: Dr Geoff Allan

Is this application part of a major three yearly renewal: Yes/No (Please strikethrough)

NEW APPLICATION for an Animal Research Authority [Animal Research Act 1985 and Regulation 1990]

Please note

1 Submit this proposal with a copy of your research protocol/preschedule.

2 Append any other information that you think the ACEC should consider with your proposal.

3 To reduce the risk of delayed approvals, send protocols to reach the Executive Officer at leasttwo (2) weeks before the scheduled ACEC meeting at which you want your proposal considered.This will allow distribution to members and discussion of clarifications/modifications with youbefore the meeting. If further clarification is needed you may be asked to attend the meeting.ACEC meetings will normally be held in March, June, September and December.

4 Send to: The Executive OfficerAnimal Care and Ethics CommitteePort Stephens Fisheries CentreTaylors Beach RoadTAYLORS BEACH NSW 2316Tel: (02) 4982 1232Fax: (02) 4982 1107

GUIDELINES FOR APPLICATIONS TO ANIMAL CARE AND ETHICS COMMITTEEFor NEW applications the ACEC requires clear justification that the work needs to be done. The

Committee also requires that the researchers propose to use the minimum number of animals to producea scientifically satisfactory result.

For RENEWALS the ACEC requires evidence that management can adequately service the researchwith adequate housing, feed and staffing.

Office Use:Received: Considered: Amended:Approved: Authority Issued:

NSW Fisheries ACEC


1 Expected Date ofCommencement: 28/9/2001

Expected Date ofCompletion: 28/9/2004

2 Names and Qualifications

2a Principal Investigator: Dr Geoff AllanQualifications: BSc(Hons) PhDAddress: NSW Fisheries

Port Stephens Fisheries CentreTaylors Beach RoadTaylors Beach, NSW 2316

Telephone: 0249 821232Facsimile: 0249 821107

2b Other Investigators and Animal Care Staff: Mr Mark Booth BSc(Hons)Mr Ian RussellMr Ian CampbellMs Helena HeasmanProf Tom LosordoMr Stewart FielderMr Luke CheviotMr Dave Barker

2c Other Research Collaborators(outside NSW Fisheries):

Yes/No (Please strikethrough)

If Yes, please provide details and evidence of their animal research authority for theircomponent of the research if it is not covered by this application:X

3 Section: (eg. Aquaculture) AquacultureSub-Program: Aquaculture Nutrition

Industry Significance:(Recommendation by ‘Principal Scientist’ or ‘Chief Scientist’ in the case of Principals requestingapproval)X

Signature:…………………………….……… Date: X

4 Location: NSW FisheriesPort Stephens Fisheries CentreTaylors Beach RoadTaylors Beach, NSW, 2316

(If research is in the field, indicate location of Principal investigation)

5 Please explain, in simple terms that will be easily understood by people withoutscientific training, the following:

NSW Fisheries ACEC


5a Need for the research (justification for the project) and why live animalsmust be used:

The overall objective of this program is to develop cost -effective diets foraquaculture species in Australia. Fishmeal is the protein source of choice for mostformulated aquaculture diets. Supplies of fish meal and trash fish are becomingmuch harder to obtain and prices are escalating as availability declines. The majorfocus of this project is, therefore, to replace fishmeal or ‘trash fish’ in aquaculturediets. To achieve this, alternative feed ingredients to fishmeal must be identified,analysed and evaluated for use in fish diets. Many ‘new’ ingredients will be inferiorto fishmeal in terms of nutritional composition and digestibility of energy and majornutrients. Cost-effective methods of improving the value of these ingredientsthrough processing or the addition of supplements (e.g. enzymes, minerals, aminoacids) need to be identified and rigorously evaluated. In addition, experiments todetermine optimal feeding strategies for fin fish are required to reduce theincidence of over feeding. Increased feeding efficiency will lead to reductions infeed costs for farmers and ensure that the release of nutrients to the environmentfrom uneaten feeds is minimised.

To ensure results are directly transferable to feed manufacturers and fin fishfarmers, the use of live animals to evaluate feed ingredients, formulated diets andfeed additives is critical. The primary target species for the previous applicationwas the omnivorous silver perch (Bidyanus bidyanus). Emphasis has now shiftedto carnivourous species such as the Australian snapper (Pagrus auratus) andmulloway (Argyrosomus japonicus). Generic technology to improve digestibilityand potential use of alternative protein sources to fishmeal as well as improvedfeeding strategies will have applicability to most aquaculture species.

5b Specific aims of the project:

To thoroughly review available ingredients which have the potential singly, or incombination with other ingredients, to replace fishmeal or ‘trash fish’ in aquaculturediets.Full chemical composition and nutritional profiles will be determined foringredients where this information is unavailable or unreliable. Novel and newingredients for potential use in fin fish diets, especially those available as bi-products from other processes (e.g. poultry offal meal, feather meal, proteinconcentrates) often have variable nutritional composition. Understanding thenutritional advantages or deficiencies for each of these ingredients is criticalwhen evaluating their potential as feed additives.

To determine the digestibility of alternative protein sources to fishmeal.The measurement of digestibility is the first task in evaluating the potential of anyfeed ingredient for inclusion in a diet. This involves measuring the amount of aspecific nutrient (e.g. fat, protein, amino acids) or energy ingested andsubtracting the amount in the faeces. Digestibility of various vegetable andanimal based feed ingredients has been determined and reported for silverperch (Allan et al., 1999; + others). Data on the digestibility of potential proteinand energy sources for snapper is practically non existent. Some information isavailable from studies undertaken with the Japanese red sea bream and closelylinked species such as the gilthead sea bream, however much of the data iscommercial in confidence. Even less is known about the nutritionalrequirements of the mulloway. Initially, the most appropriate methods fordetermining the digestibility of diets and ingredients will be evaluated. Thedigestibility of up to 25 protein and energy source will then be determined.

NSW Fisheries ACEC


These sources will be selected based on, as far as possible, the limitedpublished data on the requirements of each species and published data on othersimilar carnivorous species. Additional digestibility experiments may beundertaken to assess the effects of processing and supplementation.

Determine optimum protein requirements and the potential for ‘sparing’ proteinusing fat or carbohydrateFish, like all animals, require balanced quantities of essential amino acids.Protein which is supplied in excess of these requirements is used for energy.When protein is used for energy, ammonia production increases, increasingproblems with water quality. As protein is expensive, feed costs areunnecessarily increased. Fats are highly digestible sources of energy whichmay be used to spare protein. However excess energy in the diet will lead toexcessive deposition of fat in the fish. This can reduce the marketability of theproduct. Carbohydrate can also be used for energy, but especially forcarnivorous fish, is poorly digested. Determining the optimal balance betweenprotein and energy offers the potential to reduce the total protein and thus theuse of fishmeal in diets. This reduction will also ultimately reduce the cost of thediet.

To formulate, using digestibility data, nutritionally balanced diets using alternativeingredients to fishmeal or ‘trash fish’ and to compare performance of fish on thesediets with that of fish on fishmeal diets.Initially, these studies will be laboratory based, allowing a large number of dietsand ingredients to be screened. Diets will be formulated so increasing inclusioncontents of promising ingredients can be evaluated in terms of weight gain andprotein and energy retention. The most successful ingredients and inclusioncontents will be combined in diets and tested in larger tank or pond based trials.The effects of processing, enzyme supplementation and synthetic amino acidson performance and utilisation may also be evaluated.

To evaluate methods of increasing the digestibility of alternative protein sources tofish.In terms of digestibility, fishmeal is superior to animal meals which in turn aresuperior to Vegetable protein meals. Fish, especially carnivorous species do nothave well developed mechanisms to digest large amounts of carbohydrate orfibre found in vegetable protein sources. However cooking methods such asextrusion or steam conditioning can improve digestibility and these methods oftreatment are widely used in production of pig and poultry diets. The addition ofexogenous enzymes to diets containing high levels of carbohydrate mayincrease digestibility. As such, the efficacy of different enzyme preparationswarrants investigation.

To determine the potential use of supplements in fish dietsMany of the alternative protein sources to fishmeal are deficient in essential aminoacids. Compared to fishmeal most vegetable protein sources are deficient inlysine and sulphur amino acids. Supplementation of amino acid deficient diets withsynthetic amino acids has been successfully employed to obtain desirableessential amino acid balances (Tacon and Jackson, 1985). However leaching andpoor assimilation have been observed. Certain preparations of amino acids mayincrease their efficiency. As such, diets may be designed in which one or moreessential amino acids are replaced with similar synthetic amino acids in order totest their utilisation by fish. Ionic deficiencies in inland saline ground water maybe overcome by the addition of mineral supplements to the diets of speciescultured under these conditions. This would be an efficient way of deliveringdeficient minerals to fish such as snapper and deserves evaluation.

NSW Fisheries ACEC


To determine optimum feeding frequencies for target speciesResearch is needed to optimise the delivery of formulated diets to target species.Poor feeding practices lead to reductions in feed efficiency and lead todeteriorations in water quality. In order to improve feed practices, experimentswill be undertaken to evaluate the effects of increasing the frequency of feedingon growth and performance of target species. Results and recommendationsfrom this research will be made available to farmers.

5c Research procedures (include experimental design and statistical analyses tobe used):

Statistical methodsThe number of treatments and the number of treatment replicates in allexperiments will be designed for statistical analysis using either single or multifactor ANOVA. Experiments in which weight gain and nutrient deposition follow adose-response relationship will be analysed using linear and non-linear regressionanalysis. The number of replicates chosen for each treatment will be based on theexperimental power desired (>0.8) while at the same time minimising use of fish.Replicates are randomly allocated to each treatment via random numbergenerator.

Analytical methodsBased on relationships formed with analytical service providers during the previousapplication period and others, the majority of proximate (moisture, ash, protein, lipid,fibre) energy and amino acid analyses for digestibility and growth studies will beperformed by the Victorian Agriculture, State Chemistry Laboratories at Werribee orby CSIRO Tropical Agriculture’s Analytical Services Laboratory in Indooroopilly,Qld. These laboratories follow internationally accepted standards and proceduresand most analyses are conducted according to AOAC (1995).

Source of feed ingredients and dietsAn extensive list of contacts involved with the supply and manufacture of feeds forthe terrestrial and aquaculture sector were established during the previousapplication period (e.g. Ridley Agriproducts; Select Nutrition; Pivot Aquafeeds;Janos Hoey Pty Ltd; Cyanotech; Hyfeed: Australian Meat Holdings; Lachley Meats;Steggles Pty Ltd; CSIRO). This network of contacts has ensured a wide variety ofAustralian ingredients and diets have been available for evaluation. The ongoingrelationships formed with many of these companies and incorporations will ensurethat this program continues to evaluate high quality ingredients processed undercurrent technologies. Use of these technologies will be critical in improving both thephysical and nutritional qualities of ingredients and diets for aquaculture.

For most small scale experiments, only limited quantities of feed will be required. Inthese circumstances, diets will be formulated and manufactured on site in the feedlaboratory at PSFC. Diets are manufactured using commercial grade grinders,mixers and meat mincer. Dry ingredients are mixed with distilled water, fortified withvitamins and minerals and formed into pellets suitable for the fish being studied (e.g.1-2-3-4mm etc). All diets are stored at <-15°C prior to use. Feed for large scaletrials is manufactured offsite by commercial or government (CSIRO) providers toNSW Fisheries specifications.

Digestibility studiesThe digestibility laboratory consists of a dedicated room housing 27 X 190L cylindro-conical tanks connected to a recirculating system designed to remove bothparticulate and dissolved solids and metabolites of nitrogen.

NSW Fisheries ACEC


This facility allows the environmental control of photo period, temperature andsalinity. Generally, experiments performed in this facility allow for one reference dietand eight test diets (treatments) to be evaluated. Each treatment is allocated threereplicate digestibility tanks. The reference diet used in such studies will be specificto the species in question and will be chosen from a commercially availableselection or manufactured on site at PSFC. Test ingredients are combined with thereference diet such that between 30 and 50% of the reference diet is replaced on aweight for weight basis. An inert indicator (chromic oxide) is incorporated inreference and test diets for later chemical analysis. Experiments will be run forsufficient duration to collect enough faeces for chemical analysis. This will takebetween three and five weeks. Diets will be fed ad-lib over one three hour periodeach day, after which tanks will be thoroughly cleaned. Faeces will be collectedfrom clean tanks via passive settlement for up to 18 hours. Faeces from individualtanks will be dried and pooled before being stored (<-15°C.) for chemical analysis.Spare fish, which will be held in the same laboratory in smaller tanks will be used toreplace any mortalities. Weight gain will be recorded at the beginning and end ofeach experiment. Surviving fish will be returned to larger facilities and kept separatefrom unused fish stocks.

Growth StudiesPromising ingredients, enzymes or ionic supplements identified from digestibilitystudies or small evaluation trials will be evaluated in aquaria or cage trials (70 to200L experimental units) and later in larger scale experiments (10 000L indoortanks or 0.1ha outdoor ponds). Growth and utilisation studies will evaluate the;• Optimal protein and energy ratio’s for specific fish,• Effects of different processing techniques on diets and or ingredients,• Effects of different ingredient inclusion contents,• Effects of enzymes and ionic supplements• Optimal feeding strategies for specific carnivorous speciesSmall growth and utilisation studies will be run for 8 to 12 weeks. Larger, full scaletrials will be run over a grow-out season (e.g. spring summer). Fish for all trials aresedated (benzocain) and the weight range of the population determined.Afterwards, fish are sedated and individually weighed and stocked to experimentalunits. The number of fish per unit is dependent on fish size and the size of the unititself. Fish are fed on experimental diets twice daily (AM and PM) and bulk weighedeach fortnight or each month. The endpoint for each experiment is determined bythe overall purpose of the trial. This can be achieved by evaluating the statisticalequality or inequality of a growth response to different treatments via regular weightchecks as the experiment progresses (e.g. ANOVA), or by running the experimentfor a predetermined period.Retention studies require the sacrifice of fish at the beginning and end of the study.In these cases a small but representative sample of fish are selected from eachreplicate, euthanased and frozen. Growth studies will employ at least three replicateexperimental units per treatment.

ResultsResults will be published in target journals such as Aquaculture, AquacultureNutrition and Aquaculture Research. Non-technical papers will be published andresults will generally be freely available.

5d Numbers and species of animals to be used (even if only rough estimatesare available) (You must answer this question):Australian snapper (Pagrus auratus) Approximately 15 000Mulloway (Argyrosumus japonicus) Approximately 10 000

NSW Fisheries ACEC


5e Justification for the use of these numbers:In order to begin a growth experiment in which treatment replicates have a lowvariance in biomass (small range) and individuals are as uniform in weight aspossible , high numbers of fish are required. These requirements are lessimportant when stocking digestibility studies. Up to twelve separate experimentswill be performed over the next four year period using Australian snapper.Utilisation and growth experiments will account for at least half these studies. Eachstudy will require about 1500-2000 snapper to begin (6x2000=12000). Smallernumbers will be required for digestibility trials (3000) accounting for approximately15000 fish. Two to three trials evaluating the feeding efficiency of juvenilemulloway stocked at commercial densities (15-35 kg m-3) will require up to 10 000fish.

6 Has this work been undertaken previously? Yes/No (Please strikethrough)If so, why is it necessary to repeat it?X

7 Identify procedures that may cause pain or distress:

7a Procedures:Tank cleaning, weight checks, rapid water exchange

7b Justification for these procedures:Must be undertaken regularly in order to maintain tank hygiene and water quality.Weight assessment is needed to evaluate treatment effects during experiments.

7c What steps will be taken to avoid or minimise this pain or distress?Minimise disturbance to fish, use of anaesthetics where necessary.

8 Will any animals be euthanased during or after thisresearch?


8a If Yes, approximately how many animals will be euthanased?Approximately 20% of animals from growth and retention studies will beeuthanased.

8b Drugs or process for euthanasiaEthyl-p-aminobenzoate (i.e. benzocain)

8c Dose rate (if applicable)> 60 mg L-1.

8d Routes of administrationSeawater bath

NSW Fisheries ACEC


8e Is death intended to be the specific endpoint ofthe experimental procedure? (ie. do you intendfor animals to die to provide results eg. LC50experiments)


Generally, the performance of animals will be assessed in terms of weight gain andfeed efficiency ratio’s, which do not require animals to be killed. For experiments inwhich the retention efficiency of specific nutrients is required, a minimumrepresentative number of animals will need to be killed. This would normally require2 to 4 animals from each experimental replicate

8f If death is not the endpoint, what is the planned endpoint?Growth, e.g. weight gain, FRC, PER

9 Are any of the following to be used? If so, please give details.

9a Surgical procedures: Yes/No (Please strikethrough)Details:X

Who will be conducting the surgery?X

9b Anaesthetics: Yes/No (Please strikethrough)Reason for use:To minimise stress which would occur during handling procedures (e.g. movement offish between tanks and between laboratories, tank cleaning procedures, weightchecks, health inspection).

Drugs: Ethyl-p-aminobenzoate(benzocain)

Dose rates: 10-30mgL-1

Routes of adminstration:Seawater bath

Duration:Varies dependent on dose, generally not longer than 15 minutes.

Who will be inducing/monitoring anaesthesia?:All investigators

9c Analgesics: Yes/No (Please strikethrough)Reason for use:X

Drugs: X Dose rates: X

Routes of adminstration:X

Duration:X

NSW Fisheries ACEC


9d Neuromuscular blockers: Yes/No (Please strikethrough)Note: Neuromuscular blockers must not be used without general anaesthesia orsurgical procedure to eliminate sensory awareness.Reason for use:X

Drugs: X Dose rates: X

Routes of adminstration:X

Duration:X

What steps will be taken to avoid and to monitor pain?X

10 Is blood to be collected as part of your proposalresearch? If Yes, please provide the following details:


10a The volume of blood to be collected?X

10b The frequency of blood collectionX

10c The period over which blood will be collectedX

10d The route by which blood will be collectedX

10e The technique by which blood will be collectedX

10f The methods of animal monitoring and frequency with which these methodswill be implementedX

10g The experience of the operator relevant to the species of animal to be usedand the blood collection procedures to be undertakenX

11 Have any of these animals been used previously forresearch?


If so, please give details of the animals, the previous work and why they are being used againX

NSW Fisheries ACEC


12 Source of animals:NSW Fisheries - Grafton Research Centre (GRC)NSW Fisheries - Fisheries Research Institute Cronulla (FRI)NSW Fisheries – Port Stephens Fisheries Centre (PSFC)NSW Fisheries – Inland Fisheries Research Station, NarranderaPisces Marine - Providence Bay, Port StephensJohn Hedison – Silver Beach, Kurnell.

13 Animal housing and nutrition:Fish will be kept in 10 000L holding tanks before use in experiments. Experimental facilitieshouse a variety of different size plastic and fibreglass tanks. These included small 70L aquariaand 190L digestibility tanks. Some experiments will use 200L or 1200L floating cagessuspended inside larger tanks or in ponds. Fish will be fed commercially available diets forother carnivorous species (e.g. barramundi) or diets designed to evaluate alternativeingredients and nutritional requirements.

14 Animal care and monitoring including provision of emergency care:Fish will be monitored daily. Fish will be fed twice daily. Water quality parameters will beassessed and adjusted daily to ensure they fall within the appropriate range for each species.Therapeutic baths of formalin (100-200 ppm) and copper sulphate (2 ppm) will be administeredto control the outbreak of protozoan diseases. Primary and secondary bacterial infections maybe treated with seawater baths of broad spectrum antibiotics such as oxytetracycline (100 mgL-

1) after consultation with NSW Agriculture’s veterinary staff.

15 Method of disposal of animals at completion of the experiment:Fish may be released to the wild, used in other ACEC approved applications at PSFC or soldto permit holders.

16 Are there any aspects of the proposal that raise specialethical considerations? If so, please detail


X

17 Does the proposal present any health risk to otheranimals or people? If so, please detail


X

NSW Fisheries ACEC


Application by each Investigator and Animal Care staff member for an Animal ResearchAuthority

I have read this application, understand the aims of the project and I am aware of the responsibilitiesimposed on me under the Animal Research Act 1985 and the Animal Research Regulation 1990including the Code of Practice.

Name Work Address Signature DateDr Geoff Allan PSFC, Taylors Beach, NSW, 2316Mr Mark Booth PSFC, Taylors Beach, NSW, 2316Mr Ian Campbell PSFC, Taylors Beach, NSW, 2316Ms Helena Heasman PSFC, Taylors Beach, NSW, 2316Mr Ian Campbell PSFC, Taylors Beach, NSW, 2316Prof Tom Losordo PSFC, Taylors Beach, NSW, 2316Mr Stewart Fielder PSFC, Taylors Beach, NSW, 2316Mr Luke Chevoit PSFC, Taylors Beach, NSW, 2316Mr Dave Barker PSFC, Taylors Beach, NSW, 2316

Application by Other Research Collaborators (outside NSW Fisheries)

I have read this application, understand the aims of the project and I am aware of the responsibilitiesimposed on me under the Animal Research Act 1985 and the Animal Research Regulation 1990including the Code of Practice.

Name Work Address Signature Date

Declaration by Principal Investigator

I hereby apply for an Animal Research Authority in respect to the above application. I am aware of theresponsibilities imposed on me under the Animal Research Act 1985 and the Animal ResearchRegulation 1990 including the Code of Practice. I declare that the qualifications and experience ofinvestigators and other operators involved in this project are appropriate to the procedures to beperformed. I undertake to submit annual and final returns stating the number and fate of animals usedand the condition of surviving animals and their fate at completion of the work.

Signed:............................................................................. Date ...........................................................

NSW Fisheries ACEC


9.3 Example of a renewal for an animal research authority

ACEC Ref: Office Use 93/5 Port Stephens

Project Title: Aquaculture Nutrition

Principal Investigator: Dr Geoff Allan

Is this application a major three yearly renewal: Yes/No (Please strikethrough)If Yes, you must also complete a ‘New Application for Approval to Use Animals’

Application for ANNUAL RENEWAL of Animal Research Authority [Animal Research Act 1985 and Regulation 1990]

Please note

1 To reduce the risk of delayed approvals, send protocols to reach the Executive Officer at leasttwo (2) weeks before the scheduled ACEC meeting at which you want your proposal considered.This will allow distribution to members and discussion of clarifications/modifications with youbefore the meeting. If further clarification is needed you may be asked to attend the meeting.ACEC meetings will normally be held in March, June, September and December.

2 Send to: The Executive OfficerAnimal Care and Ethics CommitteePort Stephens Fisheries CentreTaylors Beach RoadTAYLORS BEACH NSW 2316Tel: (02) 4982 1232Fax: (02) 4982 1107

GUIDELINES FOR APPLICATIONS TO ANIMAL CARE AND ETHICS COMMITTEEFor NEW applications the ACEC requires clear justification that the work needs to be done. The

Committee also requires that the researchers propose to use the minimum number of animals to producea scientifically satisfactory result.

For RENEWALS the ACEC requires evidence that management can adequately service the researchwith adequate housing, feed and staffing.

Office Use:Received: Considered: Amended:Approved: Authority Issued:

NSW Fisheries ACEC


Please provide a brief summary of the need for the Project: (maximum 200 words)Should summarise the need described in original application

The overall objective of this program is to develop cost -effective diets for aquaculture species inAustralia. Fishmeal is the protein source of choice for most formulated aquaculture diets. Supplies offish meal and trash fish are becoming much harder to obtain and prices are escalating as availabilitydeclines. The major focus of this project is, therefore, to replace fishmeal or “trash fish” in aquaculturediets. To achieve this, alternative feed ingredients to fishmeal must be identified, analysed andevaluated for use in fish diets. Many “new” ingredients will be inferior to fishmeal in terms of nutritionalcomposition and digestibility of energy and major nutrients. Cost-effective methods of improving thevalue of these ingredients through processing or the addition of supplements (e.g. enzymes, minerals,amino acids) need to be identified and rigorously evaluated. In addition, experiments to determineoptimal feeding strategies for fin fish are required to reduce the incidence of over feeding. Increasedfeeding efficiency will lead to reductions in feed costs for farmers and ensure that the release ofnutrients to the environment from uneaten feeds is minimised.

The primary target species for the previous application was the omnivorous silver perch (Bidyanusbidyanus). Emphasis has now shifted to carnivourous species such as Australian snapper (Pagrusauratus) and mulloway (Argyrosumus japonicus)). Generic technology to improve digestibility andpotential use of alternative protein sources to fishmeal as well as improved feeding strategies will haveapplicability to most aquaculture species.

Please provide a brief summary of results and implications of the research to date:(maximum 100 words)

A growth assay and digestibility study using 30g juvenile snapper (Pagrus auratus) has beencompleted. These experiments were undertaken to compare the weight gain and performance ofsnapper fed one of 8 commercially available diets designed specifically for other carnivorous species.Results have been conveyed to growers where appropriate.

Two digestibility experiments evaluating protein and energy sources for snapper have beencompleted. Results were used to design and stock (in progress) an experiment to investigate theinteraction of digestible protein and energy levels in diets for this species. Protein and energyretention will be evaluated at the conclusion of this experiment following comparative slaughter andchemical analysis.

Please indicate any minor changes to the original methods, eg. differences in collectionmethods, changes in animal care staff etc (You only need to complete this section if this is not amajor three yearly renewal, because if it is, you need to fill in a new application form as well as thisAnnual Renewal form)

Please see major three yearly renewal for changes to animal care staff.

The previous application was primarily focused on the omnivorous silver perch (Bidyanusbidyanus). Research involving this species under the previous animal research authority(Aquaculture Nutrition ACEC REF 93/5 Port Stephens) has now ceased and the major focus hasshifted to nutritional studies involving either Australian snapper (Pagrus auratus) or mulloway(Argyrosumus japonicus). In addition, as focus has now shifted to snapper and mulloway, we willnot be seeking renewal of the research authority known as Silver Perch Digestibility Studies (ACECREF 93/4 Port Stephens; see final report). In the interests of efficiency, we seek approval toincorporate all digestibility studies involving Australian snapper and mulloway within the new threeyearly application (Aquaculture Nutrition). Experimental protocols and the handling procedures fordigestibility experiments involving Snapper or mulloway will remain fundamentally the same as thoseoutlined in the previous application for silver perch and the present re-application for AquacultureNutrition.

NSW Fisheries ACEC


NSW Fisheries has identified certain bodies of inland saline ground water with potential for aquacultureof marine species such as snapper. However chemical analysis of these water bodies has detected adeficiency in potassium ions which will limit this potential. Potassium may be added directly to thewater body, which would prove to costly, or incorporated in the diet. We seek approval to modify theoriginal application to include experiments investigating potassium supplementation of diets for thesespecies.

Poor feeding practices can account for considerable economic losses as well as deterioration in thewater quality of culture units and receiving waters. We seek approval to modify the original applicationto included experiments which will determine the optimal feeding frequencies for snapper and mullowayfed formulated diets.

Animal Use in Proposal

Species Requested2000

Requested2001

Actuallyused

Died1 Euthanased2 Total3

Australiansnapper (Pagrusauratus)

2000-3000 5000 2500 50 400 450

Mulloway(Argyrosumusjaponicus)

0 10000

1 Died as an unintentional result of research2 Deliberately euthanased (eg. for samples or analyses)3 Total of animals that died (ie. 1 plus 2)

NSW Fisheries ACEC


Renewal Application by each Investigator and Animal Care staff member for an AnimalResearch Authority

I have read this renewal application, understand the aims of the project and I am aware of theresponsibilities imposed on me under the Animal Research Act 1985 and the Animal ResearchRegulation 1990 including the Code of Practice.

Name Work Address Signature DateDr Geoff Allan PSFC, Taylors Beach, NSW, 2316Mr Mark Booth PSFC, Taylors Beach, NSW, 2316Mr Ian Russell PSFC, Taylors Beach, NSW, 2316Mr Ian Campbell PSFC, Taylors Beach, NSW, 2316Ms Helena Heasman PSFC, Taylors Beach, NSW, 2316Prof Tom Losordo PSFC, Taylors Beach, NSW, 2316Mr Stewart Fielder PSFC, Taylors Beach, NSW, 2316Mr Luke Cheviot PSFC, Taylors Beach, NSW, 2316Mr Dave Barker PSFC, Taylors Beach, NSW, 2316

Renewal Application by Other Research Collaborators (outside NSW Fisheries)

I have read this renewal application, understand the aims of the project and I am aware of theresponsibilities imposed on me under the Animal Research Act 1985 and the Animal ResearchRegulation 1990 including the Code of Practice.

Name Work Address Signature Date

Declaration by Principal Investigator

I hereby apply for a Renewal for another year of an Animal Research Authority in respect to the aboveapplication. I am aware of the responsibilities imposed on me under the Animal Research Act 1985 andthe Animal Research Regulation 1990 including the Code of Practice. I declare that the qualificationsand experience of investigators and other operators involved in this project are appropriate to theprocedures to be performed. I undertake to submit annual and final returns stating the number and fateof animals used and the condition of surviving animals and their fate at completion of the work. I certifythat the aims and objectives of the project are unchanged, the source, housing, diet and health care ofthe animals is unchanged, the procedures to be undertaken on the animals are unchanged, theultimate fate of the animal is unchanged. I have reported and will continue to report significantdeteriorations in the animals’ health and welfare to the Animal Care and Ethics Committee. Thenumbers and fates of animals used in the past year and proposed for the next year are detailed onPage 3.

Footnote: If you are unable to certify all of the above please delete the relevant clause(s) and attachan explanation.

Signed:............................................................................. Date ...........................................................

NSW Fisheries ACEC


9.4 Example of an animal research authority certificate

A N I M A L R E S E A R C H A U T H O R I T Y

Names of Applicants: Address:

DR GEOFF ALLAN PORT STEPHENS RESEARCH CENTREMR MARK BOOTH TAYLORS BEACH ROADMR IAN RUSSELL TAYLORS BEACH NSW 2316MR IAN CAMPBELL Telephone (02) 4982 1232MS HELENA HEASMAN Facsimile (02) 4982 1107MR TOM LOSORDOMR STEWART FIELDERMR LUKE CHEVIOTMR DAVID BARKER

are authorised by

NSW FISHERIES

to conduct the following type of research

AQUACULTURE NUTRITION ACEC REF 93/5 - PORT STEPHENS

as approved by and in accordance with the establishment'sAnimal Care and Ethics Committee

NSW FISHERIES ANIMAL CARE AND ETHICS COMMITTEE

This authority remains in force from

28 SEPTEMBER 2001 to 28 SEPTEMBER 2002

unless suspended, cancelled or surrendered.(Major three yearly review due in 2004)

__________________________ _______________________________

JO PICKLES GEOFF ALLAN EXECUTIVE OFFICER CHAIR

7 September 2001

NSW Fisheries ACEC


9.5 Example of an animal research authority final report

FINAL REPORT: ACEC Proposal 93/4 – Port StephensPRINCIPAL INVESTIGATOR: Geoff AllanTITLE: Silver perch digestibility studies

Aims of projectTo investigate the potential for increasing marketing opportunities for Australian oilseeds,grain legumes and other agricultural products by using them to partially or totally replace fishmeal and other protein sources in aquaculture diets. Specifically, this will involve themeasurement of digestibility coefficients for oilseeds, grain legumes and other promisingprotein sources. Secondly, the evaluation of methods of increasing the digestibility ofalternative protein sources to fishmeal will be investigated. This data will be used to designand formulate aquaculture diets for silver perch in which some or all of the fishmeal can bereplaced.

Number of animals used – approximately 13 500Digestibility experiments were conducted in a laboratory that housed 27 digestibility and 9spare tanks. Generally, for each experiment between 10 –15 juvenile silver perch werestocked into each tank ( 36 X 15 = 540 fish/exp). Since the inception of this proposal,approximately 25 digestibility experiments have been performed, accounting forapproximately 13 500 animals.

Fate of animals usedThe condition of fish in each experiment was closely monitored. Survival, weight gain andfeed intake were also assessed in addition to digestibility as a measure of a diets nutritionaladequacy. Fish which became moribund during experiments were removed as soon as theywere identified and immediately killed by administering an overdose (>60 mg L-1) of ethyl-p-aminobenzoate (benzocain) in a freshwater bath. A representative sample of 10-15 fishfrom the stocked population was killed at the beginning and end of each experiment forsubsequent whole carcass composition if required.

Condition of surviving animalsSurviving animals were returned to the holding facilities at PSFC and kept separate from theunused population of fish. After a short period of observation, these fish were transportedand released into enclosed freshwater dams (e.g. Tocal Agricultural College).

Outcome of studyThe partial or full replacement of fishmeal in aquaculture diets remains a priority for themajority of aquaculture species in Australia and throughout the world. We have evaluatedthe digestibility of over 60 different ingredients with potential for use in silver perchaquaculture diets. Some of the ingredients with the most potential include lupins, field peasand other grain legumes, oilseeds such as peanut meal, cereal grains such as wheat andseveral rendered animal meals. We have also investigated and developed cost effectivemethods of improving the digestibility of some ingredients through additional processingsuch as dehulling, protein concentration, steam conditioning and extrusion. Thesedigestibility coefficients have been fundamental in formulating diets for silver perch whichcontain little if any fishmeal and which have reduced the costs of feeding. The outcomes ofthis research has been extensively reported in target scientific journals such as Aquacultureand Aquaculture Research as well as in non-scientific publications and newsletters such asNSW Fisheries Magazine, Aquafeed International Magazine and final reports to fundingagencies such as the Fisheries Research & Development Corporation.

NSW Fisheries ACEC


9.6 NSW Fisheries ACEC Terms of Reference

The Australian Code of Practice for the Care and Use of Animals for Scientific Purposesestablishes the duties and responsibilities of an Animal Care and Ethics Committee.

The Terms of Reference of the NSW Fisheries’ ACEC are to:

1 Monitor the acquisition, transport, production, housing, care, use and disposal ofanimals by researchers of NSW Fisheries

2 Recommend to the principal investigator of an animal research authority any measuresneeded to ensure that the standards of the Code are maintained

3 Examine and approve, approve subject to modification, or reject written proposalsrelevant to the use of animals in scientific and teaching activities. Also to approveonly those studies for which animals are essential and which conform to therequirements of the Code, taking into consideration ethical and welfare aspects as wellas scientific or educational value.

4 Formally withdraw approval for any project that does not conform to the requirementsof the Code or authorise the treatment or humane killing of any animal.

5 Recommend issue, removal or restriction of animal research authorities by NSWFisheries, or other disciplinary action.

6 Examine and comment on all plans and policies, which may affect animal welfare.

7 Maintain a register of approved proposals.

8 Report annually to the Director of Fisheries on the ACEC’s activities including:- numbers and types of animal research proposals approved- physical facilities for the care and use of animals operated by authorised

researchers- any administrative or other difficulties in the operation of the committee- minutes of all meetings held for that year- current committee members- inspections- any requirements for staff training

9 Perform all other duties imposed by the Act, Regulations or Code of Practice.

NSW Fisheries ACEC


9.7 NSW Fisheries ACEC Grievance Procedures

The following Grievance Procedures have been prepared to follow the requirements andrecommendations of the Australian Code of Practice for the care and use of animals forscientific purposes and the Animal Research Act.

Procedures have been developed to handle the following:

a) complaints from independent people who are not associated with researchb) complaints by staff within the accredited research establishment (NSW Fisheries)

without jeopardising the person’s employment or entitlementsc) disputes between investigators and the ACECd) serious disagreements between members of the ACECe) disagreements between the ACEC and the NSW Fisheries’ management

Types a) and b) may be referred to the ACEC for investigation. Investigation of a complaintor dispute may take the form of: interviews of the complainant, the subject of the complaint,witnesses; unannounced inspection of the facility or animals in question; seeking expertadvice on technical matters from outside NSW Fisheries.

A report (formal record of all steps taken in the process) is then referred to the Director ofFisheries with recommendations for any action considered appropriate. This may includecancellation or suspension of the Animal Research Authority, formal reprimand, suspensionof duties or dismissal. The complainant will be advised of the outcome.

Any member of staff or the public with a grievance may report it to a member of the ACECwho is obliged to raise the matter with the ACEC as soon as possible to allow the complaint tobe resolved quickly. Confidentiality must be maintained at all times to protect thecomplainant.

Types c), d) and e) may need referral to a higher authority than the ACEC, ie. Director ofFisheries or the Animal Research Review Panel (ARRP)- guidance on policy matters may alsobe sought from the ARRP.

The Director of Fisheries may investigate procedural matters, may request the ACEC to re-examine the matter or refer new information to the ACEC for consideration but may notoverrule a decision of the ACEC made on the basis of correct procedure in compliance withthe legislation.

All complaints must be treated confidentially.

NSW Fisheries ACEC


9.8 NSW Fisheries ACEC Terms of Appointment

The following Terms of Appointment have been prepared for the NSW Fisheries’ ACEC:

1 Each member of the Committee shall be appointed for a term of three years.

2 Nominations for vacant positions will be advertised through NSW Fisheries websiteand in newspapers or newsletters relevant for the category being recruited. When aposition for a Veterinary Surgeon is vacant, a notice will be put in the AustralianVeterinary Association NSW Newsletter. Positions for Welfare representatives will beadvertised in the Australian and New Zealand Association of Animal SocietiesNewsletter (or other appropriate publication). Positions for Communityrepresentatives will be advertised in the local newspaper serving the Cronulla area (StGeorge and Sutherland Shire Leader).

3 Candidates may serve two or more terms but must nominate each time.

4 Animal researcher categories filled by nomination by NSW Fisheries Executive forperiod of three years.

5 Chair position should be for period of three years.

6 Chair position may be filled by representative for any category provided thenomination is endorsed by NSW Fisheries Executive.

Notwithstanding the above, positions for Animal Welfare and Community representation needto be endorsed by Animal Welfare Unit.

Nominations for any position will be assessed by remaining members of the Committee. Aformal vote will be taken if necessary.

a guide to acceptable procedures and practicesfor

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