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Guidance on the classification of severity of scientific procedures involving
fish
Report of a Working Group appointed by the Norwegian Consensus-Platform for the
Replacement, Reduction and Refinement of animal experiments (Norecopa)
Penny Hawkins (convenor), Kathy Ryder, Ngaire Dennison, Gidona Goodman, Stuart
Hetherington, Sharon Llywelyn-Jones & Adrian Smith
This document aims to facilitate the reliable prediction and classification of the severity of
scientific procedures involving fish.
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Background
Prediction and classification of the severity of procedures when using living animals is
important for several reasons:
1. An evaluation of the effects of a research protocol on the animals concerned helps
ensure that any pain, suffering or distress they may experience will be effectively
anticipated, recognised and alleviated. This is essential not only for reasons of
animal welfare but also for scientific validity, because physiological and behavioural
responses to suffering can significantly affect data quality.
2. Predictions of severity are fundamental to harm-benefit assessments undertaken by
bodies such as regulatory authorities and ethical committees when deciding whether
or not a project should be licensed or funded.
3. Implementation of the 3Rs (Replacement, Reduction and Refinement) of Russell &
Burch (1) is now an integral part of legislation on animal research in many countries.
Severity classification is an important tool in the application of the 3Rs.
4. Information on predicted severity may be required to monitor progress with
refinement or in the name of transparency and accountability.
There may also be a legal requirement to predict and classify severity. For example, the
proposed text of the revised version of the Directive regulating animal use within the
European Union requires severity classification based on the estimated level of pain,
suffering or distress experienced by the animals in each project, with the aim of enhancing
transparency, facilitating the project authorisation process and providing tools for monitoring
compliance (2). Member States will have to ensure that all procedures are classified as 'nonrecovery', ‘mild', 'moderate' or 'severe' on a case-by-case basis, using the assignment
criteria set out in a European Commission (EC) Working Group report on severity
classification (3).
The EC Working Group Report focuses heavily on procedures that are relevant to the
traditional terrestrial laboratory animal species. The aim of the present document is to
complement the EC Report by giving examples of procedures that are especially relevant to
the care and use of fish in research. Where possible, relevant examples from the EC report
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have been incorporated (in italics at the end of each section) in these fish guidelines, to aid
comparison.
Norecopa (www.norecopa.no) is Norway’s Consensus-Platform for the Replacement,
Reduction and Refinement of animal experiments. One of the activities of the organisation is
to arrange international consensus meetings on harmonisation of the care and use of
animals in research. At a meeting in September 2009, the participants produced a
consensus statement describing actions which should be taken to advance the welfare of
fish in research (4). This was followed up by the production by Norecopa of a list of tasks
needed to increase implementation of the 3Rs in fish research (5). One of these tasks was
to produce guidelines on the categorisation of severity in fish experiments.
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Special considerations for fish species
Classification of the severity of procedures when using fish can be problematic for a number
of reasons:
2.1
The fact that fish live in water can affect severity
Many protocols involve catching and handling fish, which in itself is difficult, and some
protocols involve exposing fish to air. All of these interventions can cause stress, adding to
the overall severity of the procedure (6, 7, 8, 9) There are often scientific as well as animal
welfare implications. For example, the results of toxicological trials have been affected by
the degree of handling stress and disturbance experienced by fish (10). Handling can be
refined by using sedation or anaesthesia, preferably while the fish is still in the water, and
avoiding exposure to air wherever possible. However, it is necessary to remove fish from
the water for many procedures, and this may cause physiological complications such as gill
collapse, or the risk of internal injury when large fish are removed from the medium that
supports them.
Fish can habituate to predictable events, such as feeding. It may be possible to reduce the
negative impact of stressors, such as netting, by associating them with feeding. Rodents
respond favourably to repeated handling and show reduced fear to novel situations (11).
Whether it is possible to increase fish welfare by doing the same has not yet been
evaluated.
2.2
The severity of many procedures is very species-specific within fish
Criteria for categorisation of severity in fish should address species-specific and intraspecies variations in the stress associated with capture, handling and immobilisation,
particularly when procedures are performed out of water. There are over 25,000 species of
fish, living in a wide range of habitats. In addition, many species undergo large physiological
changes as a natural part of their life cycle. Therefore it is likely that the impact of a given
procedure will vary between species and age groups. This report gives general guidance
and should be interpreted by specialists on the individual species.
2.3
Indicators of pain, suffering and distress in fish are currently poorly defined
A recent report on the research needs within the welfare of fish used in research highlighted
the need for better indicators of pain, suffering and distress (12). These include indirect
indicators such as water quality parameters, clinical signs in individual fish (e.g. respiratory
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rate, food consumption and health status) and signs of stress in groups of fish (e.g. social
behaviour and activity level).
2.4
Perceptions of fish and their ability to suffer can be inconsistent
There has traditionally been more tolerance of stress, disease and mortality as an endpoint
in fish research, compared to research using mammals, reflecting general attitudes to fish in
society. Assessment of the severity of a procedure in fish is further complicated by the fact
that high mortality rates are a natural part of the survival strategy of many fish species,
making it sometimes difficult to distinguish deaths caused by the experiment from natural
mortality.
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Examples of severity classification for procedures using fish
The examples listed below follow the classification used in the EC report. The examples in
italics, which have been grouped together at the end of each section, are those that are
identical to (or closely resemble) examples given in the EC report.
When considering these examples, it is important to note that:
 Assessment of severity should include an overall assessment of the total harm or
distress produced by a procedure or study, i.e. cumulative suffering caused by all
elements of the procedure.
 In each of the cases below it is assumed that all procedures, including capture and
handling, are performed optimally by competent persons.
 Changes in the definition and application of an endpoint may move a procedure from one
category to another.
 Some of the examples may not be regulated procedures per se, depending on the
purpose for which they are conducted. For example, the European Directive does not
apply to practices undertaken for the primary purpose of identification of an animal or for
the purposes of recognised animal husbandry. However, the guidance in this document
is based upon the animal’s likely total experience of the procedure, so methods such as
marking and tagging are included and classified because they contribute to the
cumulative severity of the procedure, regardless of their purpose.
 Procedures that per se are not particularly invasive may, over a period of time, result in
considerable distress for the animal concerned. For example, Carlin tags may be fouled
by seaweed and shellfish, leading to increased mortality rates (13).
3.1
Sub-threshold
The lower threshold is exceeded if the animals may experience a level of pain, suffering or
distress equivalent to, or higher than that caused by the introduction of a needle. The
administration of anaesthesia for scientific purposes (excluding euthanasia) will bring a
procedure above the lower threshold.
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Non-invasive observation of normal behaviour without disturbing the animal. Behavioural
studies that do not involve any other regulated procedures, e.g. observation of choice of
shelter in an imitation stream.
Open field testing.
Exposure to an artificial predator where escape into a refuge is immediately possible.
Feeding studies where there is no reduction in quantity or quality of the diet compared
with normal feed. Such studies may include weighing and measuring under anaesthesia
at a frequency equivalent to that done for normal husbandry purposes.
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3.2
Feeding studies where food restriction is at a level where weight loss, or reduced weight
gain, is less than 15% of age and sex matched non-deprived fish, or where fish are to be
maintained above 85% of body weight for age and sex matched controls. It should be
noted that this is a more complex issue for fish than it is for most mammals. It is very
species and life stage specific, as some species will naturally stop feeding at some
stages of their life cycle (e.g. spawning) and may experience extreme weight loss and
poor body condition. Likewise, fish may naturally exhibit periods of extreme food intake
and subsequent weight gain. Food restriction is likely to be more stressful to farmed fish
that have been selected for rapid growth rates.
Marking using non-toxic and non-aversive dyes in the water.
Adding inert markers in the diet to follow passage of digesta.
Feeding an experimental diet that meets the full nutritional needs of the animals.
Withdrawal of food for a short interval relative to normal food intake at that stage of the
life cycle, e.g. food withdrawal in adult salmonids for up to 48 hours.
Manipulations of temperature within temperature ranges experienced by the species in
its natural habitat where the speed of change is not more than 1 degree Celsius in a 24
hour period.
Manipulations of photoperiod, for example to delay or accelerate maturation.
Breeding genetically altered animals expected to have no adverse phenotype.
Mild
Procedures on animals as a result of which the animals are likely to experience short term
mild pain, suffering or distress. Procedures with no significant impairment of the wellbeing
or general condition of the animals.
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Maintenance of external parasites on host fish where parasite numbers are at low levels
and clinical or behavioural signs are not seen.
Behavioural studies involving short-term exposure to an artificial predator and where
escape is not possible.
Removal of a small part of one fin of fish where rapid healing is expected. The effect of
fin clipping will, however, depend, among other things, upon the functional importance of
the fin in question. Further studies into the effects of fin clipping are needed.
Removal of a small number of scales for genotyping or age determination.
Induction and maintenance of anaesthesia using a route and agent appropriate to the
species and life stage, for example for the purpose of weighing and measuring fish.
Blood sampling under anaesthesia where volumes are limited to those recommended by
published guidelines (cardiac puncture must, under Norwegian legislation, be performed
as a terminal procedure under general anaesthesia, unless specific dispensation has
been given by the authorities).
Research into some diseases, where humane endpoints are applied at the first clinical
sign of disease or earlier. Note that the severity of the clinical signs will vary with the
severity of the disease.
Toxicological studies where animals are humanely killed at or before onset of clinical
signs. As with applying humane endpoints in disease studies, some agents may cause
severe adverse effects so this may not apply in the case of highly toxic agents.
Pharmacokinetic studies where a single dose is administered and a limited number of
blood samples taken (totalling <10% of circulating volume) and the substance is not
expected to cause any detectable adverse effect.
Non invasive imaging with appropriate sedation or anaesthesia.
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3.3
Superficial procedures, e.g. biopsies and non-surgical implantation of small
transponders.
Application of external telemetry devices that cause only minor impairment to the
animals or minor interference with normal activity and behaviour.
Administration of substances by subcutaneous, intramuscular or intraperitoneal routes,
gavage and intravenously via superficial blood vessels, where the substance has no
more than mild impact on the animal, and the volumes are within appropriate limits for
the size and species.
Breeding of genetically altered animals which is expected to result in a phenotype with
mild effects.
Feeding of modified diets, that do not meet all of the animal’s nutritional needs and are
expected to cause mild clinical abnormality within the time-scale of the study.
Short-term restriction of movement.
Studies involving short-term deprivation of social partners, short-term solitary housing of
sociable species.
Models which expose animals to noxious stimuli which are briefly associated with mild
pain, suffering or distress, and which the animals can successfully avoid.
Moderate
Procedures on animals as a result of which the animals are likely to experience short term
moderate pain, suffering or distress, or long-lasting mild pain, suffering or distress.
Procedures that are likely to cause moderate impairment of the wellbeing or general
condition of the animals.
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Removal of fish from water, for example to induce stress. The impact on the fish is likely
to vary between species, depending upon a number of factors such as their tolerance for
low oxygen levels.
“Shaking” of fish in a net out of water to cause a stress response.
Introduction of a Coded Wire Tag (CWT) into the nose, dorsal fin or other cartilagenous
area.
Fin clipping in conditions where infection may follow e.g. warmer water, or removal of
substantial parts of a fin, or removal of part of a functionally important fin.
Removal of scales in order to promote fungal growth.
Subcutaneous, intramuscular or intraperitoneal implantation of telemetry devices by
surgical procedures or insertion in the stomach by oral gavage (all under general
anaesthesia).
External attachment of telemetry devices where there is a risk of interference with normal
activity and behaviour.
Urine collection by insertion of a catheter into the bladder and attachment with
appropriate suture material around the cloaca.
Gastric lavage.
Cannulation of blood vessels followed by successive blood sampling within acceptable
limits for blood removal.
Blood sampling via the caudal vein at frequent intervals under anaesthesia.
Intraperitoneal injection of substances known to cause adhesions.
Endoscopy.
Disease studies where the disease in question is known to cause death, but where the
study can be controlled so that mortality does not occur but where there is moderate
departure from normal health.
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3.4
Frequent exposure to test substances which produce moderate clinical effects, and
withdrawal of blood samples (>10% of blood volume) in a conscious animal within a few
days without volume replacement.
Acute dose-range finding studies, chronic toxicity / carcinogenetic tests, with non-lethal
endpoints.
Surgery under general anaesthesia and appropriate analgesia, associated with postsurgical pain, suffering or impaired general condition.
Breeding of genetically altered animals which are expected to result in a phenotype with
moderate effects.
Moderate restriction of movement over a prolonged period.
Studies with modified diets that do not meet all of the animal’s nutritional needs and are
expected to cause moderate clinical abnormality within the time-scale of the study.
Evoking escape and avoidance reactions where the animal is unable to escape or avoid
the stimulus, and are expected to result in moderate stress.
Severe
Procedures on animals as a result of which the animals are likely to experience severe pain,
suffering or distress, or long-lasting moderate pain, suffering or distress. Procedures that
are likely to cause severe impairment of the wellbeing or general condition of the animals.
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3.5
Prolonged restraint without anaesthesia or extreme stocking densities.
Methods of marking fish that cause increased mortality or significant interference with
normal behaviour.
Infections with a prolonged disease course, in which extreme loss of condition or other
overt clinical signs, which cause a significant and prolonged departure from normal
health are required for the purposes of the study.
Saltwater/freshwater challenge. Saltwater challenge as a routine test for smoltification is
prohibited in Norway.
Toxicity testing where death is the endpoint, or fatalities are to be expected and severe
pathophysiological states are induced, e.g. single dose acute toxicity testing.
Vaccine potency testing characterised by persistent impairment of the animal’s condition,
progressive disease leading to the animal’s death, associated with long-lasting moderate
pain, distress or suffering.
Surgical and other interventions in animals under general anaesthesia which are
expected to result in severe or persistent moderate post-operative pain, suffering or
distress, or severe and persistent impairment of the general condition of the animal.
Breeding animals with genetic disorders that are expected to experience severe or
persistent impairment of general condition.
Severe restriction of movement over a prolonged period.
Inescapable electric shock, e.g. to produce learned helplessness.
Complete isolation for prolonged periods of social species.
Immobilisation stress to induce pathological conditions.
Forced swimming or exercise tests with exhaustion as the endpoint.
Upper threshold
The upper threshold is exceeded if the animals may experience severe pain, suffering or
distress which is likely to be long-lasting and cannot be ameliorated. Article 50 permits this
upper threshold to be exceeded if there is scientific justification.
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Death as an endpoint.
Pathophysiological studies of disease in which late characterisation of the host-pathogen
interaction is required, such that animals will die.
Description of survival curves after infection with a pathogen.
Closing remarks
Many of the procedures that are commonly performed on the traditional laboratory animals
have very different welfare implications when applied to fish, partly because of the inherent
difficulties in the capture and handling of aquatic species. This, coupled with the biological
variation exhibited in the large number of fish species involved and our limited understanding
of their welfare requirements, makes it difficult to offer detailed guidelines for classifying the
severity of procedures. Many common procedures are undoubtedly a greater challenge in
fish than in terrestrial mammals: these include (but are not limited to) marking, blood
sampling, anaesthesia and analgesia.
The examples in this document are intended to facilitate severity classification, be an aid to
discussion and to stimulate further research in this area. It will be up to the individual
regulator, researcher and animal welfare officer to ensure that the 3Rs are fully
implemented, including humane endpoints, regardless of the category in which a procedure
has been placed.
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Acknowledgements
The authors are very grateful for comments that have been received from participants at
Norecopa’s international consensus meetings and other fish experts during the development
of this document.
We welcome further comments, which may be sent to Penny Hawkins
(phawkins@rspca.org.uk) or Adrian Smith (adrian.smith@vetinst.no).
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Literature references
1. Russell WMS, Burch RL (1959): The Principles of Humane Experimental Technique.
Wheathampstead:
Universities
Federation
for
Animal
Welfare.
http://altweb.jhsph.edu/publications/humane_exp/het-toc.htm
2. Council of the European Union (2010): Proposal for a Directive of the European
Parliament and of the Council on the protection of animals used for scientific
purposes. (Directive 8869/10) Council of the European Union, Brussels
http://ec.europa.eu/environment/chemicals/lab_animals/proposal_en.htm
3. Expert Working Group on Severity Classification Criteria (2009): Expert Working
Group on Severity Classification of Scientific Procedures Performed on Animals:
Final Report. European Commission, Brussels.
http://ec.europa.eu/environment/chemicals/lab_animals/pdf/report_ewg.pdf
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4. Consensus statement issued by the participants at the international consensus
meeting “Harmonisation of the Care and Use of Fish in Research”, Gardermoen, 22 24 September 2009.
http://www.norecopa.no/norecopa/vedlegg/Consensus-sep09.pdf
5. Tasks needed to increase implementation of the 3Rs in fish research. Based upon
discussions at the international consensus meeting “Harmonisation of the Care and
Use of Fish in Research”, Gardermoen, 22 - 24 September 2009.
http://www.norecopa.no/norecopa/vedlegg/Fish-tasks-prioritert.pdf
6. Pickering AD (1992): Rainbow trout husbandry: management of the stress response.
Aquaculture 100: 125-139
7. Wendelaar Bonga SE (1997): The stress response on fish. Physiol. Rev. 77: 591-625
8. Brydges NM, Boulcott P, Ellis T & Braithwaite VA (2009): Assessment of stress
responses induced by different handling methods in three species of fish. Appl. Anim.
Behav. Sci. 118: 137-143
9. Young, P. S; Cech, J. J. (1993): Physiological stress responses to serial sampling
and confinement in young-of-the-year striped bass, Morone saxatilis. Comparative
Biochemistry and Physiology. A, Comparative Physiology. 1993. 105: 2, 239-244.
10. Pottinger TG & Calder GM (1995): Physiological stress in fish during toxicological
procedures: a potentially confounding factor. Environ. Toxicol. Water Qual. 10: 135146
11. Hirsjarvi P & Valiaho T (1995): Effects of gentling on open-field behaviour of Wistar
rats in fear-evoking test situation. Laboratory Animals, 29, 380-384.
http://la.rsmjournals.com/cgi/reprint/29/4/380.pdf
12. Report of a working group appointed by Norecopa and the Norwegian Research
Council (2009). Fish in research - environmental requirements and welfare
indicators. A review of research needs.
http://www.forskningsradet.no/servlet/Satellite?blobcol=urldata&blobheader=applicati
on%2Fpdf&blobheadername1=ContentDisposition%3A&blobheadervalue1=+attachment%3B+filename%3DHAVBRUKFiskif
orskningeng.pdf&blobkey=id&blobtable=MungoBlobs&blobwhere=1253965656551&s
sbinary=true
13. Strand R, Finstad B, Lamberg A & Heggberget TG (2002): The Effect of Carlin Tags
on Survival and Growth of Anadromous Arctic Charr, Salvelinus Alpinus.
Environmental Biology of Fishes, 64, 275-280.
http://www.springerlink.com/content/n66j74t878536603
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