Guidelines for the Care and Welfare of Cephalopods in Research -A consensus based on an initiative by CephRes, FELASA and the Boyd Group

Abstract

This paper is the result of an international initiative and is a first attempt to develop guidelines for the care and welfare of cephalopods (i.e. nautilus, cuttlefish, squid and octopus) following the inclusion of this Class of ∼700 known living invertebrate species in Directive 2010/63/EU. It aims to provide information for investigators, animal care committees, facility managers and animal care staff which will assist in improving both the care given to cephalopods, and the manner in which experimental procedures are carried out. Topics covered include: implications of the Directive for cephalopod research; project application requirements and the authorisation process; the application of the 3Rs principles; the need for harm-benefit assessment and severity classification. Guidelines and species-specific requirements are provided on: i. supply, capture and transport; ii. environmental characteristics and design of facilities (e.g. water quality control, lighting requirements, vibration/noise sensitivity); iii. accommodation and care (including tank design), animal handling, feeding and environmental enrichment; iv. assessment of health and welfare (e.g. monitoring biomarkers, physical and behavioural signs); v. approaches to severity assessment; vi. disease (causes, prevention and treatment); vii. scientific procedures, general anaesthesia and analgesia, methods of humane killing and confirmation of death. Sections covering risk assessment for operators and education and training requirements for carers, researchers and veterinarians are also included. Detailed aspects of care and welfare requirements for the main laboratory species currently used are summarised in Appendices. Knowledge gaps are highlighted to prompt research to enhance the evidence base for future revision of these guidelines.

Full text

aboratory
limited
lanimals
Guidelines
Guidelines for the Care and Welfare of
Cephalopods in Research –A consensus
based on an initiative by CephRes, FELASA
and the Boyd Group
Graziano Fiorito
1,2
, Andrea Affuso
1,3
, Jennifer Basil
4
,
Alison Cole
2
, Paolo de Girolamo
5,6
, Livia D’Angelo
5,6
,
Ludovic Dickel
7
, Camino Gestal
8
, Frank Grasso
9
, Michael Kuba
10
,
Felix Mark
11
, Daniela Melillo
1
, Daniel Osorio
12
, Kerry Perkins
12
,
Giovanna Ponte
2
, Nadav Shashar
13
, David Smith
14
, Jane Smith
15
and Paul LR Andrews
16,2
Abstract
This paper is the result of an international initiative and is a first attempt to develop guidelines for the care
and welfare of cephalopods (i.e. nautilus, cuttlefish, squid and octopus) following the inclusion of this Class of
700 known living invertebrate species in Directive 2010/63/EU. It aims to provide information for investiga-
tors, animal care committees, facility managers and animal care staff which will assist in improving both the
care given to cephalopods, and the manner in which experimental procedures are carried out. Topics covered
include: implications of the Directive for cephalopod research; project application requirements and the
authorisation process; the application of the 3Rs principles; the need for harm-benefit assessment and
severity classification. Guidelines and species-specific requirements are provided on: i. supply, capture and
transport; ii. environmental characteristics and design of facilities (e.g. water quality control, lighting require-
ments, vibration/noise sensitivity); iii. accommodation and care (including tank design), animal handling,
feeding and environmental enrichment; iv. assessment of health and welfare (e.g. monitoring biomarkers,
physical and behavioural signs); v. approaches to severity assessment; vi. disease (causes, prevention and
treatment); vii. scientific procedures, general anaesthesia and analgesia, methods of humane killing and
confirmation of death. Sections covering risk assessment for operators and education and training require-
ments for carers, researchers and veterinarians are also included. Detailed aspects of care and welfare
requirements for the main laboratory species currently used are summarised in Appendices. Knowledge
gaps are highlighted to prompt research to enhance the evidence base for future revision of these guidelines.
1
Stazione Zoologica Anton Dohrn, Villa Comunale, Napoli, Italy
2
Association for Cephalopod Research ‘CephRes’, Italy
3
Animal Model Facility - BIOGEM S.C.A.R.L., Ariano Irpino (AV),
Italy
4
Biology Department, Brooklyn College - CUNY Graduate Center,
Brooklyn, NY, USA
5
Department of Veterinary Medicine and Animal Productions -
University of Naples Federico II, Napoli, Italy
6
AISAL - Associazione Italiana per le Scienze degli Animali da
Laboratorio, Milano, Italy
7
Groupe me
´moire et Plasticite
´comportementale, University of
Caen Basse-Normandy, Caen, France
8
Instituto de Investigaciones Marinas (IIM-CSIC), Vigo, Spain
9
BioMimetic and Cognitive Robotics, Department of Psychology,
Brooklyn College - CUNY, Brooklyn, NY, USA
10
Max Planck Institute for Brain Research, Frankfurt, Germany
11
Integrative Ecophysiology, Alfred Wegener Institute for Polar and
Marine Research, Bremerhaven, Germany
12
School of Life Sciences, University of Sussex, Sussex, UK
13
Department of Life Sciences, Eilat Campus, Ben-Gurion
University of the Negev, Beer, Sheva, Israel
14
FELASA, Federation for Laboratory Animal Science Associations
15
The Boyd Group, Hereford, UK
16
Division of Biomedical Sciences, St George’s University of
London, London, UK
Corresponding author:
Graziano Fiorito, Research – CephRes, via dei Fiorentini 21, 80133
Napoli Italy.
Email: cephres@cephalopodresearch.org
Laboratory Animals
2015, Vol. 49(S2) 1–90
!The Author(s) 2015
Reprints and permissions:
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DOI: 10.1177/0023677215580006
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Keywords
Cephalopods, Directive 2010/63/EU, animal welfare, 3Rs, invertebrates
Contributors
People listed here provided data, information and com-
ments, and contributed to different extents during the
preparation of this work.
The following list is arranged by country in alpha-
betical order; different contributors are merged by
Institution.
France
Christelle Alves, Cecile Bellanger, Anne-Sophie
Darmaillacq, Ce
´line Gaudin
Groupe me
´moire et Plasticite
´comportementale,
EA4259, University of Caen Basse-Normandy,
Caen, France
Joe
¨l Henry
Physiologie de la reproduction des Mollusques,
University of Caen Basse-Normandy, Caen, France
Germany
Tamar Gutnick
Max Planck Institute for Brain Research, Frankfurt,
Germany
Italy
Anna Di Cosmo
Department of Biology - University of Naples
Federico II, Napoli, Italy
Carlo Di Cristo
Department of Biological and Environmental
Sciences - University of Sannio, Benevento, Italy
Viola Galligioni
CIBio - Centre for Integrative Biology, Trento, Italy
& Association for Cephalopod Research ‘CephRes’,
Italy
Anna Palumbo
Stazione Zoologica Anton Dohrn, Napoli, Italy
Perla Tedesco
Department of Biological and Environmental
Science and Technologies - University of Salento,
Lecce, Italy
Letizia Zullo
Istituto Italiano di Tecnologia, Department of
Neuroscience and Brain Technologies, Genoa, Italy
Portugal
Anto
´nio Sykes
C. Mar – Centre of Marine Sciences, Universidade
do Algarve, Faro, Portugal
Spain
Roger Villanueva Lo
´pez
Renewable Marine Resources Department - Institut
de Cie
`ncies del Mar, Barcelona, Spain
United Kingdom
Ngaire Dennison
Home Office, Animals in Science Regulation Unit,
Dundee, Scotland, UK
Penny Hawkins
RSPCA Research Animals Department,
Southwater, West Sussex, UK
United States of America
Gregory J. Barord, Heike Neumeister, Janice Simmons,
Roxanna Smallowitz
Biology Department, Brooklyn College - CUNY
Graduate Center, Brooklyn, NY, USA
Jean Geary Boal
Biology Department, Millersville University,
Millersville, PA, USA
Roger Hanlon, William Mebane
Marine Resources Center, Marine Biological
Laboratory, Woods Hole, MA, USA
Judit R Pungor
Hopkins Marine Station of Stanford University,
Pacific Grove, CA, USA
James B. Wood
Waikiki Aquarium, University of Hawaii-Manoa,
Honolulu, HI, USA
1. Introduction
Cephalopods (i.e. nautilus, cuttlefish, squid and
octopus) have been used for diverse scientific pur-
poses across Europe for over 100 years.
1,2
However,
until recently, scientific procedures involving cephalo-
pods have not been covered by EU regulations, with
2Laboratory Animals 49(S2)

the exception of procedures using Octopus
vulgaris in the United Kingdom (see discussion in
Smith et al.
3
).
The inclusion of ‘live cephalopods’ (Article 1, 3b) in
EU Directive 2010/63/EU on the ‘protection of animals
for scientific purposes’ represents a landmark. It is the
first time that an entire class of invertebrates, covering
approximately 700 known species,
4,5
has been included
in laboratory animal legislation throughout the EU.
The decision was largely based upon a review of the evi-
dence for sentience and capacity to experience pain,
suffering, distress and lasting harm (PSDLH) in ceph-
alopods
6
(see also Table 1) which is now supported by
more recent circumstantial (for reviews see
7,8
) and
objective evidence
9–11
for the existence of nociceptors
in cephalopods. Annexes III and IV to the EU
Directive provide general guidance on care and accom-
modation requirements and methods of humane killing
for all species covered by the Directive, but specific
guidance is restricted to vertebrates, and there are no
specific details for cephalopods.
Prompted by the need for guidelines on these and
other matters covered by the Directive, members of
the international cephalopod research community
have met on several occasions over the past 3 years
and have produced publications aimed at cephalopod
researchers, on: i. requirements of the EU Directive,
implementation, ethics and project review;
3
ii. PSDLH, anaesthesia and humane killing;
8
and
iii. implications for neuroscience research and the
Three Rs, i.e. Replacement, Reduction, Refinement.
2
This work has led to the development of a set of
consensus Guidelines for the Care and Welfare of
Cephalopods in Research which aim to assist research-
ers in complying with the Directive, and are the subject
of this paper. These guidelines have been developed as a
joint initiative between CephRes (www.cephalopodre-
search.org), FELASA (www.felasa.eu) and the Boyd
Group UK (http://www.boyd-group.demon.co.uk/).
The Guidelines for the Care and Welfare of
Cephalopods in Research, which should be regarded as
a starting point for future developments, begin with a set
of general principles of good practice, representing the
present state of knowledge that may reasonably be
applied to all cephalopods. These are followed by a tabu-
lated set of specific guidelines (see Appendices) for typical
cephalopod species, currently used in EU laboratories,
which also reflect well-established principles.
1.1 What is a cephalopod?
For the purpose of these guidelines, cephalopods are
defined as all living species that are members of the
molluscan class Cephalopoda.
4,5,12
The term ‘live ceph-
alopod’ is not defined in the Directive, but guidance
indicates that these animals are covered by the
Directive from ‘when they hatch’.
13,14
Cephalopods are characterised by bilateral body
symmetry, a prominent head and a set of arms, includ-
ing tentacles in Decapods, which are considered as mus-
cular hydrostats and derived from the primitive
molluscan foot.
15–21
The class contains two, only dis-
tantly related, living subclasses: Nautiloidea (repre-
sented by Nautilus and Allonautilus) and Coleoidea,
which includes cuttlefish, squid and octopuses.
20,22
In the Nautiloidea, the external shell, common to the
molluscan Bauplan, still exists, whereas in the
Coleoidea it has been internalised or is absent. The var-
iety of species that compose the taxon is reflected in the
diversified habitats they have adapted to: oceans, ben-
thic and pelagic zones, intertidal areas and deep sea,
polar regions and the tropics.
23–26
Understanding the requirements of a particularspecies
in relation to its natural habitat is fundamental in main-
taining healthy laboratory populations of cephalopods.
Assumptions for housing, care and use of these animals
based on fish, whilst appropriate in some circumstances,
should be made with great caution as the evolutionary
convergence between fish and cephalopods
24,27
does not
reflect the actual requirements of different species.
Generally, cephalopods have a high metabolic rate,
grow rapidly and are short-lived.
28,29
These animals are
exothermic, highly adapted to the marine aquatic envir-
onment and are therefore unlikely to tolerate rapid or
significant changes in the quality or temperature of the
water they are housed in. They react rapidly to environ-
mental changes/external stimuli with immediate physio-
logical consequences that can be relatively long lasting.
Such changes, as well as having potential welfare impli-
cations, will also impact upon experimental results.
Cephalopods are considered among the most
‘advanced’ invertebrates, having evolved many charac-
teristic features such as relatively large, highly differen-
tiated multi-lobular brains, a sophisticated set of
sensory organs, fast jet-propelled locomotion, and com-
plex and rich behavioural repertoires.
25,30–37
1.2 What the Directive 2010/63/EU means
for cephalopod research
The entry into force of the Directive 2010/63/EU (here-
after referred to as ‘the Directive’)
38,39
means that, from
1st January 2013, scientific research and testing invol-
ving ‘live cephalopods’ is regulated by a legal frame-
work at both EU and Member State levels, and as a
consequence all scientific projects that cross the thresh-
old set for regulation (i.e. involve procedures that may
cause PSDLH equivalent to, or higher than that caused
by the insertion of a hypodermic needle in line with
good veterinary practice) will require authorisation by
Fiorito et al. 3

Table 1. Summary of evidence for the capacity of cephalopods to experience pain based upon the criteria used by the EFSA 2005 panel (as the basis for recom-
mending the inclusion of cephalopods in revision of Directive 86/609/EEC), and here updated with more recent studies. See also Andrews et al. for review
8
and
additional references.
Criterion used by EFSA 2005 panel Judgement Comment and references
Presence of receptors sensitive to noxious stimuli,
located in functionally useful positions on (or) in the
body, and
YES Circumstantial evidence, e.g. cutaneous free nerve endings
491
available at time of EFSA
report. Recent neurophysiological afferent recording studies
10,11
have provided direct
evidence for presence of mechano-nociceptors in both squid and octopus.
Connected by nervous pathways to the ‘lower’ parts of
the nervous system
LIKELY
(but not proven)
Evidence that peripheral afferent axons project to brain from the arms and
mantle,
492,493
but modality not identified although likely to include nociceptors if
present.
Possession of higher brain centres [in the sense of
integration of brain processing], especially a structure
analogous to the human cerebral cortex
YES Most complex brain structure amongst invertebrates and clear hierarchical
organisation.
34,35
Vertical lobe approximates to the hippocampus in mammals and is unique in inverte-
brates in having gyri.
26,142
Studies in progress investigating self-awareness and consciousness, as discussed in
Edelman and coworkers.
494,495
Possession of nervous pathways connecting the noci-
ceptive system to the higher brain centres
LIKELY
(but not proven)
Evidence for ascending afferent projections from ‘lower’ to ‘higher’ brain regions
including the vertical lobe, but no neurophysiological studies showing projection of
signals from nociceptors.
492,493
Indirect evidence from behavioural studies for projection of signals from nociceptors to
higher brain regions (see below).
Receptors for opioid substances found in the central
nervous system especially the brain
LIKELY
(but not proven)
Not studied directly. Opioid system is highly conserved in evolution.
496
Limited evi-
dence for presence of enkephalins
497,498
and opioid receptors.
497–502
Analgesics modify the animal’s response to stimuli
that would be painful for a human
Not studied Investigation of candidate substances is required using a combination of neurophysio-
logical recording from nociceptive afferents and behavioural studies.
An animal’s response to stimuli that would be painful
for a human is functionally similar to the human
response (that is, the animal responds so as to avoid
or minimise damage to its body)
YES Good evidence of learned avoidance of punishment (e.g. electric shock) but assumes
that this stimulus activates nociceptors and not some other afferent modality that
evokes an aversive but non-painful sensation (e.g.
26,503
). Limited supportive evidence
from behavioural studies of predatory behaviour.
26,36,72,504–507
Equivocal evidence for wound-directed behaviours.
9,10,189
An animal’s behavioural response persists and it
shows an unwillingness to resubmit to a painful pro-
cedure; the animal can learn to associate apparently
non-painful with apparently painful events.
YES Evidence for: peripheral mechano-nociceptor sensitisation (at least 48 h) following
injury to either an arm or fin; contralateral afferent sensitisation (but see Alupay
et al.
10
); hyper-responsiveness to visual stimuli following arm injury.
9,11
4Laboratory Animals 49(S2)

Figure 1. Schematic overview summarising the major components of a project application and stages of project approval under Directive 2010/63/EU. Note that the
details of the project approval process may differ across member states. For details see text and review in Smith et al
3
.
Fiorito et al. 5

the National Competent Authority (see the list avail-
able at: http://ec.europa.eu/environment/chemicals/
lab_animals/ms_en.htm).
3
Three key aspects of the project that will need to
be considered by researchers and those responsible for
animal care and welfare are outlined below (see also
Figure 1). Specific topics for inclusion and consideration
are listed in Appendix 1, and a more detailed overview of
issues relating to implementation of the Directive for
cephalopods can be found in Smith et al.,
3
which also
includes some hypothetical worked examples of project
review, particularly in relation to opportunities for
implementing the 3Rs (see 2.2.1 below).
2. Project application requirements and
authorisation process
2.1 Application requirements
The key requirements of project authorisation are out-
lined here to provide a background to the technical
sections, which aim to show how these requirements
can be fulfilled specifically for cephalopods (Figure 1).
Before they can begin, all projects involving live
cephalopods must be authorised by a competent
authority appointed by the Member State in which
the project is to take place (see Text Box 1 and Text
Box 2 for definitions of key terms in the Directive). This
will involve ‘comprehensive project evaluation’, ‘taking
into account ethical considerations’ and ‘implementa-
tion of principles of reduction, refinement and replace-
ment’ of the use of animals, the 3Rs (Recital 38 and 39).
An application for project authorisation must
include, as a minimum, the project proposal and the
items listed in Appendix 1. A non-technical project
summary will be required unless waived by the
National Competent Authority.
Applications must also include specific scientific
justification for any requests for exemptions from
certain requirements of the Directive (where permitted
– see also Appendix 1). This will include requests for
permission to: i. use an endangered cephalopod species
Text Box 1. Definition of key terms utilised in Directive 2010/63/EU.
Aproject is a programme of work with a defined scientific objective involving one or more procedures, which can run for a
term of up to 5 years, after which authorisation must be renewed.
Aprocedure is any use of an animal covered by the Directive for experimental or other scientific or educational purposes,
which ‘may cause the animal pain, suffering, distress or lasting harm equivalent to or higher than that caused by the
introduction of a needle in accordance with good veterinary practice’. This can include procedures that do not involve any
‘invasive’ technical acts such as administration of substances or surgery, but which cause psychological distress (such as
anxiety) above the threshold level of suffering defined above. Unless specifically justified as part of the authorisation
process, procedures may only be carried out at authorised user establishments.
Authorisation is limited to the procedures and purposes described in the application. If, during the life of the project, there
is need for any amendments to the project plans that may have a negative impact on animal welfare, these must also be
authorised.
ACompetent Authority is a body responsible for implementing a specific task (or tasks), laid down by the Directive, within
a Member State; for example, project evaluation and/or project authorisation. Member States must designate one or more
competent authorities to fulfil these tasks.
Text Box 2. Definitions of the levels of severity of procedures according to Directive 2010/63/EU, Annex VIII; see also Lindl
et al.
490
Mild:Procedures on animals as a result of which the animals are likely to experience short-term mild pain, suffering or
distress, as well as procedures with no significant impairment of the well-being or general condition of the animals shall
be classified as ‘mild’.
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 as well as procedures that are likely to cause
moderate impairment of the well-being or general condition of the animals shall be classified as ‘moderate’.
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 as well as procedures, that are likely to cause severe impairment of
the well-being or general condition of the animals shall be classified as ‘severe’. Note that the Competent Authority will
require retrospective assessment of projects involving ‘severe’ procedures.
Non-recovery:Procedures which are performed entirely under general anaesthesia from which the animal shall not
recover consciousness.
6Laboratory Animals 49(S2)

where it falls within the criteria laid out in Article 7.1 of
the Directive; ii. use cephalopods taken from the wild
(Article 9, see also section 3.3 below); iii. carry out pro-
cedures in a place that is not an authorised users’ estab-
lishment (Article 12); iv. re-use (in a different
procedure) animals that have already undergone a pro-
cedure (Article 16, see also section 8.10 below); v. use
drugs, such as neuromuscular blocking agents, that
could stop or restrict an animal’s ability to show
pain, without an adequate level of anaesthesia or anal-
gesia (Article 14§3); vi. depart from any of the general
standards of animal care and accommodation outlined
in Section A of Annex III of the Directive (Article 33).
For species other than cephalopods, specific justifi-
cation is also required for killing animals by a method
not listed in Annex IV of the Directive, or for departing
from species-specific standards of animal care and
accommodation outlined in Section B of Directive
Annex III. However, at present, cephalopods are not
included in either of these Annexes.
Once a project is authorised and underway, it should
continue to be critically evaluated by the Principal
Investigator and all members of the project team,
using the factors listed in Appendix 1, so as to ensure
that ethical considerations and opportunities for imple-
menting the 3Rs are identified and addressed in an
ongoing process for the entire duration of the project,
not only at the start.
2.2 Factors to be evaluated in project
authorisation and project operation
The Directive sets out the factors that must be evalu-
ated during project authorisation and throughout the
lifetime of authorised projects. These factors are listed
below as a series of action points and associated ques-
tions for consideration.
2.2.1 Implement the 3Rs to minimise the harms
caused to the animals. The Three Rs (3Rs) principles
were first described by Russell and Burch
40
and are now
internationally accepted as an essential requirement for
the ethical and humane conduct of scientific studies
involving animals (Recital 11; Articles 4 and 38 §2b).
In addition, it is widely recognised that implementa-
tion of the principles can enhance scientific
quality.
41–44,45
The following are examples of questions that will
need to be addressed:
Replacement
.What on-going efforts will you make to identify ‘sci-
entifically satisfactory’ alternative methods that could
replace the use of some or all animals? (Article 4§1)
.Could you avoid the use of animals by asking differ-
ent type of question, or making better use of existing
data or literature to address the scientific objectives?
.Could in vitro studies or in silico-modelling be used
to replace some or all of the animals?
Reduction
.How will you ensure that the number of animals
used in the project, and in individual studies within
the project, is ‘reduced to a minimum without com-
promising the scientific objectives’? (Article 4§2)
.Could any further reductions be made, e.g. by taking
expert statistical advice to help optimise experimen-
tal and statistical design?
Refinement
.How have you refined the ‘breeding, accommoda-
tion and care of the animals’ and the ‘methods
used in procedures’, so as to ‘reduce to the minimum
any possible pain, suffering, distress or lasting harm
to the animals’ throughout their lives? (Article 4§3)
.Have you considered and implemented all the possi-
bilities for refinement described elsewhere in these
guidelines?
.How will you ensure that all relevant personnel
working on the project are adequately educated
and trained, and are supervised until they have
demonstrated their competence in the procedures?
2.2.2 Assess and assign the severity classification of
the procedures used in the project. Each procedure
outlined in a project application must be classified
according to the severity of its adverse effects on the ani-
mals (Article 38§2c). This prospective severity classifica-
tion sets an upper limit on the level of suffering that an
individual animal undergoing the procedure is
allowed to experience.
46,47
The categories are: ‘non-
recovery’ (for procedures carried out entirely under
general anaesthesia from which the animal does not
recover consciousness), ‘mild’, ‘moderate’ and
‘severe’ (Article 15§1 and Annex VIII; see also section
8 below, and Text Box 2).
The following points must be considered for all pro-
jects, in order to fully address the 3Rs and meet the
requirements of the project evaluation process:
.Have you tried to identify and predict all possible
adverse effects that could be caused to the animals in
the project? Include any pain, distress, lasting harm
and other forms of suffering, such as hunger, anx-
iety, boredom and osmotic or thermal stress caused
to the animals, which may occur at any time during
Fiorito et al. 7

the animals’ lifetime; for example, a result of capture
and transport to the laboratory, routine handling,
housing and husbandry, or method of killing, as
well as the effects of the procedures themselves.
.Have you taken steps to minimise each of these
adverse effects as far as possible, by: i. applying the
3Rs in the design of procedures (see above), ii. using
these guidelines, other relevant literature, advice
from colleagues and from the institution’s Animal
Welfare Body to assist you?
.Based on the above, have you set prospective sever-
ity classifications (see section 8) and clear humane
end-points for all procedures?
.How will you monitor the welfare of the animals
used in the project, and when and how will you inter-
vene to ensure that the animals do not suffer beyond
the upper limit of severity needed to achieve the
objectives of any particular procedure?
Member States must also collect retrospective infor-
mation on the actual severity of procedures, after they
have ended, and must make this publicly available on
an annual basis (Article 54§2). The assessment of actual
severity is based on day-to-day observations of the ani-
mals, and the ‘most severe’ severity experienced by the
animal is reported to the National Competent
Authority. For example, if records of observations indi-
cate that suffering is moderate at the beginning of a
procedure and then mild for the remainder, actual
severity for reporting purposes is ‘moderate’.
Annex VIII of the Directive further explains the
severity categories (see also section 8), and the
European Commission has put together a series of
examples* to illustrate the process of prospective sever-
ity classification, day-to-day observation and monitor-
ing of animals and actual severity assessment.
2.2.3 Weigh the harms and benefits of the project and
the individual studies within it. Taking into account
all the points listed above:
.What is the basis for your overall assessment that
‘the harm to the animals in terms of suffering, pain
and distress is justified by the expected outcome,
taking into account ethical considerations, and
may ultimately benefit human beings, animals or
the environment’? (Article 38§2d)
.Have you explained this evaluation in your project
application and, where relevant, your non-technical
project summary (Article 43) – the latter using lan-
guage suitable for the general public?
.How will you ensure that the ‘weighing of harms and
benefits’ is an on-going process throughout the pro-
ject – i.e. part of the day-to-day practice of ‘ethical
science’, and not just a one-off event at the time of
authorisation?
A detailed examination of procedures for harm-ben-
efit analysis in animal research and testing is available
in a document from the Animal Procedures Committee
in the UK.
48
3. Supply, capture and transport
3.1 Source of animals
Article 9§1 of the Directive requires that animals must
not be taken from the wild for use in procedures, unless
an exemption has been granted by the relevant
National Competent Authority, based on ‘scientific jus-
tification to the effect that the purpose of the procedure
cannot be achieved by the use of an animal that has
been [purpose-] bred for use in procedures’.
This means that, in principle, cephalopods used for
experimental or other scientific purposes should be bred
and reared in captivity. However, there are significant
difficulties in captive-breeding most cephalopod species
(for exceptions, see
2,49,50
) and, therefore, this may not
be feasible at the time of writing.
Development of more successful, standardised breed-
ing procedures is urgently required. Article 38§1c indi-
cates that projects must be designed ‘to enable
procedures to be carried out in the most humane and
environmentally sensitive manner possible’.
Where there is scientific justification for using animals
taken from the wild, animals may be captured ‘only by
competent persons using methods which do not cause the
animals avoidable pain, suffering, distress or lasting
harm’ (Article 9§3).
2
All those involved must observe a
strict ethic of respectful treatment of animals, take into
account their conservation status (section 3.2 below), and
minimise the impact on the local ecosystem (section 3.3).
Care should be taken to prevent physical injury and
stress to cephalopods at all stages in the supply chain,
including capture (section 3.3), transportation (section
3.4), acclimatisation to laboratory conditions (3.5) and
quarantine where required (3.6). It is also important to
check local requirements for transport of animals in all
countries along the route.
3.2 Cephalopod species commonly used in
research and conservation status
An analysis of cephalopod species used in EU labora-
tories and the types of research undertaken can be
found in Smith et al.
3
Species from the main taxa of
*
http://ec.europa.eu/environment/chemicals/lab_animals/pdf/
examples.pdf
8Laboratory Animals 49(S2)

cephalopods used in research are shown in Table 2.
Note that in some cases, e.g. Nautilus sp. or Idiosepius
sp., animals are mainly available by importation and
this requires permits and documentation from both
exporting and importing countries
y
.
At the time of writing, species of the Class
Cephalopoda have not yet been assessed for possible
inclusion in the IUCN Red List of Threatened
Species
z
and hence none is listed as endangered.
However, concerns are being raised for some rare
species, based on local evidence and experience.
Examples of locally protected species are: Euprymna
scolopes in Hawaii; Octopus cyanea, Sepia elongata,
Sepia pharaonis and Sepia prashadi in Israel
(N. Shashar, pers. comm.); and some
Mastigoteuthidae species in New Zealand.
51
Indeed,
assessments are now underway for cephalopods
§
under the Sampled Red List Index (SRLI) initiative**,
which indicates the relative rate at which the conserva-
tion status of certain species groups changes over time,
and aims to broaden the taxonomic coverage of the
IUCN Red List.
3.3 Capture methods
Commonly used cephalopod species are listed in
Table 2 together with information on currently avail-
able sources (e.g. wild, captive bred, eggs), and Table 3
indicates possible capture methods.
Several reviews describe commercial capture meth-
ods for cephalopod species.
29,52
Current methods
include, but are not limited to, nets, traps and pots
(see Table 3).
53,54
Environmentally destructive methods
(e.g. trawling) should be avoided wherever possible.
Hand-jigging is considered the ‘best’ method for cap-
turing squid, but may not be appropriate for all squid
species.
51,55
Whichever method is used for capturing animals for
research, it must not cause ‘avoidable pain, suffering,
distress or lasting harm’ (Article 9). As noted above,
animals may be captured only by competent persons.
Moreover, researchers (and institutions) should only
accept animals from suppliers who use appropriate
capture and transportation methods; and the compe-
tence of third party providers should be evaluated
based on the condition and survival of the animals sup-
plied (see also section 3.4).
3.4 Transport (local, national and
international)
Transport of cephalopods should always be in well-
oxygenated seawater.
56
Whenever possible and applicable to the research
project, transport of eggs is the simplest, most success-
ful, and, hence, preferable approach. Details of meth-
ods for egg transport are available for some cephalopod
species.
56,57
When juvenile and adult cephalopods are trans-
ported, high survival rates should be achieved through
careful selection of container type, maintenance of sea-
water quality in appropriate volumes and consideration
of other measures to support animal welfare, such as
food deprivation and cooling. The following discussion
outlines general principles for transport of cephalo-
pods, along with variations according to the duration
of transport.
In the following sections, short-duration (i.e. short
distance) transport is defined as requiring less than 2
hours; and long-duration (i.e. long distance) transport is
for any longer period. These working definitions are
based upon the consumption of available oxygen and
detrimental changes in water chemistry (e.g. accumula-
tion of ammonia and carbon dioxide and depletion of
oxygen) as the duration of confinement for transport
increases.
Since no specific systematic studies of transport
methods for cephalopods are currently available, it is
recommended that transport requirements should be
based on the FAO guidelines for fish,
58–60
paying par-
ticular attention to oxygen-sensitive species as they are
considered to be comparable to cephalopods in their
metabolic rate. Transport should also comply with
the European Convention on the Protection of
Animals during International Transport (ETS no 65,
ETS no 193).
Table 2. Summary of major species of cephalopod used in
research together with their source. Eggs may either wild
caught or captive bred (e.g. for S. officinalis). See also
reviews in Smith et al. (2013)
3
and Fiorito et al. (2014).
2
The
possibility of obtaining animals through a given source is
indicated by (ˇ), and (X) indicates this source is considered
possible, but not currently available. For details and com-
ments on welfare issues see also text.
Source
Wild caught Eggs
Captive
bred animals
Nautiloid ˇ
Cuttlefish ˇˇX
Sepiolid ˇXX
Squid ˇXX
Octopus ˇXX
y
http://ec.europa.eu/environment/cites/info_permits_en.htm
z
www.iucnredlist.org
§
http://www.zsl.org/blogs/wild-science/animals-in-the-red
**
http://www.zsl.org/science/indicators-and-assessments-unit/the-
sampled-red-list-index
Fiorito et al. 9

3.4.1 Transport containers. Since seawater is crucial
for the survival of the animals, steps must be taken to
ensure that water will not leak at any time (e.g. by
containing animals in double bags and placing
bags within a sealed container). It should be noted
that several cephalopod species (e.g. octopuses) are
occasionally reported to cut and bite through thin plas-
tic bags (M. Kuba, pers. comm.), hence stronger trans-
port containers should be used for them. In any
case, the external container should be able to contain
the entire water volume even if all inside containers/
bags rupture.
Short-duration transport does not necessarily require
animals to be placed into plastic bags, and other appro-
priate containers (large plastic bucket or box with a lid)
may be preferred containing sufficient pre-oxygenated
seawater to allow the animal to be completely
immersed. Animals should be kept in dim or dark
conditions, and movement and vibration of containers
should be minimised.
For long-duration transport, similar to the protocol
for transporting fishes
58
and depending on body size,
one animal should be placed with 1/3 pre-oxygenated
seawater and 2/3 oxygen-enriched air in double
common aquarium bags (see also description in
61–63
).
Once the bag is aerated, and the animal placed within
it, it should be properly sealed (e.g. twisted at the top
and folded over) and doubly secured (e.g. two rubber
bands or cable grips). For transport periods over
12 hours, aeration and oxygenation may be necessary
during transport, but care must be taken to use meth-
ods which do not add adverse conditions (i.e. bubbling)
that may cause distress the animals.
Sealed holding bags (transparent to facilitate inspec-
tion if required) containing oxygenated seawater should
be placed into insulated boxes (e.g. Styrofoam) to
ensure that a temperature appropriate to the species is
maintained during transport. The transport box should
be darkened with a secure lid to keep the animals in
darkness during transport, which reduces their stress.
Bags should be packed with cushioning material
(e.g. paper, Styrofoam pellets) to ensure they do not
move during transport.
The use of specialised aerating and insulated trans-
port containers is not yet common for cephalopods.
However, the development of specialised transport
containers should be encouraged (for reviews on fish
transport containers see
58,60
).
3.4.2 Other factors for consideration. Food depriv-
ation before shipping may be desirable, where appro-
priate, dependent on i. shipping distance, ii. the species
and iii. size of animal. It is common practice among
cephalopod researchers to withdraw food before ship-
ping. This is to help prevent fouling of the water and
ammonia build-up during transport. The duration of
food deprivation should be based upon a consideration
of normal feeding frequency, oro-anal transit time and
renal ammonium ion excretion. However, food depriv-
ation may require authorisation by the National
Competent Authority.
Food deprivation before transport, alongside
lowered water temperature, can also help to avoid
build-up of toxic ammonia and carbon dioxide.
64,65
With the aim of lowering metabolic rate before
shipping it has been suggested that pre-cooled seawater
at 2–3C above the thermal tolerance minimum of an
individual species should be used, ensuring that the
temperature stays at that level for the duration of trans-
port (see e.g. for the giant octopus
66
). However, the
impacts of this method on animal welfare are not yet
known.
Sedation is not essential and is not recommended for
transport of most cephalopods. However, sedation
methods (i.e. cold water
67
or ‘chemical’ methods
68
)
has been used during transport in some instances with
cephalopods.
It is interesting to note that in 1928 Grimpe
suggested that very long duration transportation, i.e.
requiring more than 2 days, should be achieved in
Table 3. Schematic overview of the most common methods utilised for capture of cephalopod species for research
purposes. See text for comments about welfare and environmental issues assocated with each method.
Capture methods
Collection of eggs Hand net Traps Pots Hand jigging SCUBA Trawling
Nautiloid ˇˇ
Cuttlefish ˇˇˇ ˇˇ
Sepiolid ˇ
Squid ˇˇˇˇˇˇ
Octopus ˇˇˇˇ ˇˇ
10 Laboratory Animals 49(S2)

steps allowing ‘resting’ periods in appropriate
locations.
56
3.5 Acclimatisation after transport
Transport inevitably causes animals stress. Therefore it
is important to allow time for them to recover from
transport-related effects, to acclimatise to the new
conditions including possible differences in water qual-
ity, temperature, illumination, diet, and the shape and
arrangement of the environment (i.e. the tank). Allowing
time for acclimatisation is vital for both animal welfare
and science, as stress can confound scientific results.
Almost all cephalopods are highly stenohaline and
stenotherm, and care should be provided to avoid any
difference in salinity and water temperature of the
container utilised for the transport and the tank
where the animals will be placed. In the case of signifi-
cant difference in water temperature, an adjustment of
the different ‘media’ after transport (e.g. placing the
container inside the final tank for slow adaptation of
the temperature to the final one) should be considered.
Cephalopods arriving in a facility should be examined
for injuries or other health issues, and treated and/or
quarantined (see section 3.6) or humanely killed (see sec-
tion 8.11) where necessary. It is also recommended that
NH
4
and CO
2
levels in the transport water are measured.
Together, these observations can help in assessing the
quality of transport methods and suppliers.
Based upon species-specific requirements (i.e. individ-
ual or group living; see also Appendix 2) animals should
be placed in a holding tank until they are habituated to it.
The requirements and duration of this practice are spe-
cies-specific (generally from 1 to 5 days) and for experi-
mental reasons may be reduced to a minimum (e.g. when
studying individual preferences towards a prey item or a
stimulus), because evidence is available for contextual
learning to occur in most cephalopods.
26,69–71
To facilitate the required ‘habituation’ to the captive
situation, it is best if the holding tanks are designed
according to the same principles as the maintenance/
experimental tanks for the species. Where food has
been withdrawn during transport, a slow reintroduc-
tion is also recommended.
In the classic literature on cuttlefish and octopus, an
adequate predatory performance is considered a sign
of acclimatisation to a holding tank.
26,36,72
In addition,
while excessive inking upon introduction to a new tank
is a sign of stress, low swimming rates, reduced likeli-
hood of inking in response to a small disturbance near a
tank, can serve as indications of successful acclimatisa-
tion (N. Shashar, pers. comm.).
When moving animals from one tank to another
within the laboratory a standardised, minimally
stressful protocol should be applied. In these cases,
only a brief acclimatisation time may be needed (in
the order of minutes to few hours). However, the
experimenter should be aware of potential handling
and relocation stress and its physiological consequences
that may impact on the research.
3.6 Quarantine
The purpose of quarantine after reception of animals is
to isolate cephalopods from the main population
accommodated in the facility to allow observation
and testing until animals are assessed as healthy and
free from potential infectious diseases. Individuals iden-
tified as ill should either be separated for treatment, if
the cause can be identified and treatment is available
(see section 7), or humanely killed (see section 8.11) and
autopsied (see section 6.3).
Quarantine is also useful to isolate individuals that
become sick while being maintained in the facility,
allowing time for sanitary measures to be put in place
and ensure appropriate containment of organisms and
waters.
Currently, quarantine is not the general practice in
the cephalopod research community. However, further
studies are required based on recent research of ceph-
alopod diseases and diffusion of parasites.
73
The duration of quarantine should be sufficient to
assure health of the individual animals. The needs of
individual quarantined animals vary according to the
biology and behaviour of the species (e.g. group hold-
ing maybe appropriate for gregarious species, but
others may require individual accommodation; see
also Appendix 2).
Quarantine should involve complete separation
between animals to be quarantined and the current
laboratory population; this should be achieved either
by using separate rooms or equipping facilities with plas-
tic screens to separate quarantine tanks from others. In
addition, water supply should also be separate, to pre-
vent diffusion of any potential harm to the water circu-
lation and/or the environment. Similarly, equipment
(e.g. nets) should not be shared between tanks.
During the quarantine period, animals should be
monitored closely for unusual clinical signs or behav-
iours (see also sections 6 and 7), and detailed examin-
ations (including autopsy; see section 6.3) made of any
individuals who are considered to be ‘abnormal’. In the
cases of identification of diseased animals present in the
laboratory holding facility, this should be regarded as a
possible indicator of disease in the entire stock/holding
group, and hence particular attention should be pro-
vided, and eventually they should all be treated or
humanely killed.
Fiorito et al. 11

4. Environment and its control
4.1 Seawater supply and quality
Both natural and artificial seawater (see also below) are
suitable for the maintenance of cephalopods. For fish,
Annex III of Directive 2010/63/EU requires that ‘an
adequate water supply of suitable quality [is] provided
at all times’, and that ‘at all times water quality param-
eters’ are ‘within the acceptable range that sustains
normal activity and physiology for a given species
and stage of development’ and such requirements
apply equally well to cephalopods.
4.1.1 Types of seawater circulation system. There are
two principal seawater systems for keeping cephalo-
pods: closed systems which recycle a reservoir of sea-
water, and open systems which either draw a continuous
supply of water from the ocean (flow-through systems),
or pump seawater into a reservoir and regularly replen-
ish it with fresh seawater (semi-closed systems).
Closed systems have the advantage of enabling
control of all parameters of the environment, but are
more costly due to the need for additional environmen-
tal monitoring and control equipment.
Open systems rely on fresh seawater being drawn
from the ocean. While this has some advantages (espe-
cially not needing expensive filters), it limits the facility
to keeping animals that can live within the given water
parameters. For example, non-native species cannot be
kept in this type of system without considerable efforts
purifying and sterilising the reflow. Naturally open sys-
tems are also limited to areas close to the shore.
Closed systems: Efficient and relatively easy-to-main-
tain closed aquarium systems have been developed for
cephalopods.
74–78
Commercially available artificial sea-
water preparations are considered adequate, provided
they contain the necessary substances and trace elem-
ents to meet the physiological needs of the particular
cephalopod species, and for this reason mixtures
designed for marine invertebrates and corals should
be preferred.
Water flow in recirculating systems or filtration
within enclosures should be sufficient to remove sus-
pended waste and to ensure that water quality param-
eters are maintained within acceptable levels. Filtering
systems in recycling/recirculating seawater should be
adequately planned and maintained.
79,80
Appropriate
processes for monitoring water parameters should be
implemented, and alarms in place to ensure flow and
seawater levels are adequate.
Open systems: seawater drawn from the ocean
should be tested for contaminants and pathogens, and
treated to remove them. The water supply should also
be evaluated to ensure that there is sufficient capacity,
including ability to cope with periods of maximum
demand and emergency situations. To protect animals
from potential contaminants, other measures, such as
appropriate filtering or a reverse osmosis system, may
be required.
79,80
Tests to determine the chemical composition and
presence of contaminants/toxins will determine the
treatment necessary to make the water suitable for
use. Seasonal factors such as phyto- or zoo-plankton
blooms, tidal cycles, and seasonal seawater mass turn-
overs can have periodic effects (on a scale of hours,
days or months) for seawater and these should be
anticipated.
All systems: water flow should enable cephalopods to
maintain normal locomotion and behaviour.
Cephalopods can use rapid expulsion of water through
the funnel to power jet propulsion, which results in
swift movement. Cuttlefish and squid have fin-like
structures on the mantle to assist in locomotion.
Squid are in continuous motion due to their pelagic
nature, thus water flow
75,77
needs to be sufficient to
ensure appropriate life-style requirements, and ade-
quate water quality including quick removal of ink
(if released).
4.1.2 Water quality. As for other aquatic species, water
quality is the most important factor in maintaining the
health and well-being of cephalopods. Insufficient water
quality will cause stress and disease. Water-quality par-
ameters should at all times be within the acceptable
range that sustains normal activity and physiology for
a given species and individual (see Appendix 2); and
should remain stable, unless the life style of a given
species requires changes (e.g. because of large vertical
migrations during a day or seasonal changes)
z
.
Optimum conditions vary between species (e.g. deep-
sea benthic octopuses are especially sensitive to
changes), between life-stages (e.g. paralarvae, juveniles,
and adults) and according to physiological status of the
individual (e.g. females preparing to lay eggs).
Most cephalopods show little adaptability to chan-
ging water-quality conditions, and so when animals
need to be moved between tanks or systems, it is
important to ensure that water parameters are mirrored
and maintained. If this is not possible, gradual acclima-
tisation will be needed (see section 3.5), as for other
marine invertebrates and fishes.
Dissolved oxygen, pH, carbon dioxide, nitrogenous
compounds and salinity should be monitored and
maintained according to the appropriate range for
each species. Appendix 2 provides a list of water quality
criteria for optimum health and welfare of cephalo-
pods; for more detailed discussion of monitoring
water quality see below.
z
but this may be difficult to achieve in laboratory facilities.
12 Laboratory Animals 49(S2)

4.2 Monitoring water quality (O
2
, pH, CO
2
,
nitrogenous material, salinity and
metals)
Seawater parameters should be monitored (continu-
ously by specific electrodes or intermittently by chem-
ical methods) and recorded at an appropriate frequency
(at least daily), thus allowing proactive, rather than
reactive, management of water quality. Parameters
that need to be measured and the frequency of meas-
urement vary (see also Appendix 2), depending on
whether the system is open or closed. For example,
while there may be no need to measure nitrites/nitrate
in a high volume flow-through system (depending on
the source of the water), such measurements are critical
with recirculation systems.
At a minimum, environmental monitoring systems
should provide information on water flow, oxygen
saturation and water temperature. Parameters
measured should also be relevant to the health and
welfare of the particular species housed in the facility
(see Appendix 2). In general, recirculation systems
should be monitored for a larger number of param-
eters, including, but not restricted to, dissolved
oxygen, pH, nitrogenous material, salinity, total dis-
solved salts and temperature (see below). As a min-
imum, water quality analysis should be carried out at
times of greatest demand on the system (usually after
feeding) to identify potential problems.
Water and tanks should be kept clean particularly of
faeces and uneaten food. In semi-open and closed sys-
tems, water should be treated to reduce potential
pathologies, for example, using UV light or ozone.
If ozone is used, measurements of ozone concentrations
and/or redox potential of the reflow entering the system
are necessary to avoid toxicity.
Alarm and notification values must be set and their
significance as potential indicators of problems in the
system explained to all relevant personnel. There must
be an agreed, clear protocol for contacting those
responsible for the facility when problems are identified
outside of normal working hours.
The monitored parameters should be recorded and
the information stored for at least 5 years. For all par-
ameters considered below and for techniques of keeping
animals information is also available through a recent
compilation of research on the culture of cephalopods.
50
4.2.1 Oxygen. Cephalopods have high metabolic rates,
so oxygen concentration should generally be kept high
(close to saturation); and where tank inflow is not
sufficient, supplementary aeration of the water must
be provided. However, supersaturating the water is
not advisable as it may cause gas bubbles to become
trapped in the mucus layer of the animals, thus limiting
gas exchange at the gills (for octopus: G. Fiorito, pers.
comm.; for several cephalopod species: J. Rundle, pers.
comm.).
Information on oxygen consumption in some ceph-
alopods is provided by Winterstein
81
(see also
82,83,83–87
)
and reccomended requirements are summarised in
Appendix 2.
It is important to note that monitoring oxygen levels
is not informative of the oxygen available to the ceph-
alopod unless it is combined with measurements of pH
(see next section).
4.2.2 pH. Due to the effects of pH on the carriage of
oxygen by blood pigments, cephalopods tolerate low
pH poorly.
88–96
Regular measurement and careful
maintenance of pH is therefore critical. Acceptable
pH levels depend on many water quality factors, for
example, carbon dioxide (see below), and calcium; as
a consequence, control of soluble gases and water sol-
utes is important. This may be a particular issue for
establishments that use synthetic marine salts rather
than natural seawater and closed filtration sys-
tems, especially where water changes are limited.
Acceptable pH values for keeping cephalopods are
summarised in Appendix 2.
4.2.3 Carbon dioxide. Carbon dioxide is produced
during respiration and dissolves in water to form car-
bonic acid, thus lowering the pH. Since stability of pH
is very important, accumulation of carbon dioxide
should be avoided. Situations that may increase
carbon dioxide levels include high stocking density
and poor aeration.
Care should be taken that water supply systems, par-
ticularly in the case of groundwater-based systems, do
not introduce harmful quantities of carbon dioxide to
the enclosures.
4.2.4 Nitrogenous material. As cephalopods are
carnivores, hence requiring a high protein diet, the accu-
mulation of potentially toxic nitrogenous compounds
can be a problem, particularly in closed systems. It is
also necessary to avoid accumulations of ink (especially
when keeping cuttlefish, that ink in large volumes).
Timely removal of uncomsumed food, use of adequate
protein skimmers and suitable water flow rates, along
with careful water filtering in closed systems, will help
to reduce levels of organic waste, including ammonia,
nitrites and nitrates. It is also beneficial if the flow rate
in the tanks can be adjusted to different situations (e.g.
increased after inking). Additional water changes can
provide a supplementary means of removal of waste
products and substances such as ink.
Build up of nitrogenous compounds may lead to
behavioural changes and/or changes in skin
Fiorito et al. 13

colouration in cephalopods. For example, at nitrate
levels >80 mg/l cuttlefish become very agitated, will
ink profusely and their skin tone may be dark; and
larvae and hatchlings are more vulnerable to bacterial
disease (A. Sykes, pers. comm.).
Levels of nitrogen compounds tolerable by different
species are reviewed in Appendix 2 (see also Iglesias
et al.
50
).
4.2.5 Salinity and metals in seawater. As cephalopods
are marine organisms, maintaining an appropriate salt
concentration is vital. The salinity should match the
natural habitat of the animals.
Commercially available artificial seawater
preparations and especially any mixture designed for
marine invertebrates and corals are considered adequate
as they contain all the necessary substances and trace
elements to keep cephalopods in good health. However,
in accordance with instructions for the different brands of
salt, some trace elements, in particular strontium and cal-
cium should be monitored and added if necessary.
Copper and its alloys are considered to affect the
salinity and ‘poison’ the seawater
56
and therefore
should be avoided in any system holding cephalopods.
Cephalopods are reported to accumulate and be
sensitive to heavy metals so care should be taken to
ensure these are monitored and maintained within
normal ambient ranges for the species, to limit poten-
tial damage (see above and also
2
for relevant
literature).
4.3 Lighting control
Light influences, either directly or indirectly, almost all
physiological and behavioural processes in cephalo-
pods, including growth, development and reproduc-
tion. Lighting requirements vary between cephalopod
species, and both wavelengths and intensity of lighting
should ‘satisfy the biological requirements of the ani-
mals’, where these are known (Directive, Annex III,
section 2.2a). The natural history of the species, in par-
ticular the normal living depth, can provide clues to
help meet the species lightling preferences: for example
there are many cephalopods that prefer very little light.
There is limited specific knowledge on wavelength per-
ception for almost all cephalopod species.
97
However, it
is estimated that simulated sun-light equivalent to that
normally experienced at 3–8 m depth at sea should be
acceptable for the majority of cephalopod species com-
monly used as laboratory animals (but see further
information in Appendix 2).
Photoperiod should also be maintained according to
the natural requirements of the species.
98–103
However,
there is evidence that some cephalopods may easily
adapt to changes in day/night regime.
26,104–106
Where task lighting is needed for people working in
the room, it should be restricted in its dispersion, and/
or be placed below the level of the tank surface, to
reduce disturbance to the animals. Use of automated
dimmer controls that allow light intensity to be grad-
ually increased is important and recommended (G.
Fiorito, pers. comm.; see also
107
). For example, for
decapods and nocturnal octopuses sudden changes in
light level may cause escape reactions, and in some
cases inking, thus a simulated dusk and dawn period
is desirable. Care should also be taken to ensure that
animals are not disturbed by night-time security light-
ing entering through windows in the holding facilities.
The output of fluorescent lights can be diminished by
using dummy bulbs to reduce light levels.
4.4 Temperature control
Water temperature should be controlled within the
natural range for the species; and, where necessary,
appropriate chilling/heating equipment must be used to
ensure the optimal temperature range for the animals.
Cephalopod species vary in their sensitivity to
changes in water temperature. In general, higher
water temperatures create problems for animals from
temperate climates like octopus and the cuttlefish.
Transitions of temperature should not be sudden.
56,108
Where water changes are performed on larger scales,
temperature spikes, which may cause adverse effects,
should be controlled and avoided.
4.5 Noise and vibration control
Background noise, and vibration from housing sys-
tems, such as pumps or ventilation units, should be
minimised as they are likely to impact on cephalopod
welfare.
Several studies suggest that cephalopods can detect
sounds even at low frequencies,
109–115
and other recent
work shows that cephalopods are as likely as other
marine organisms, to suffer from low-frequency noise
traumas.
116–118
In common with other aquatic species, cephalopods
dislike vibrations, such as drilling or banging on tank
sides, and some species, such as cuttlefish, may respond
by inking. Therefore, the most important aspect of
sound reduction is to minimise disruption and avoid
sudden noises, which could startle the animals.
4.6 Aquatic life support systems and
emergencies
Tanks should be built so that complete drainage is
impossible when they are inhabited (although ability
to drain tanks may be required for cleaning purposes).
14 Laboratory Animals 49(S2)

Two independent sources of water movement/
oxygen supply are also recommended, for example,
pumps for water circulation plus extra air sources to
provide additional aeration.
Electronic alarm systems help to ensure that prob-
lems in a system are detected promptly. All facilities
must have an emergency plan in place should problems
arise (including out-of-hours), with clear actions that
are understood by all and effectively communicated to
everyone. There must be a backup system to enable an
appropriate response to the worst case scenario of a
complete system failure, and so avoid circumstances
in which animals would have to be humanely killed
due to suffering from anoxia or a build up of organic
waste.
5. Accommodation and care
5.1 Background and requirements of the
Directive
About 50 species of cephalopod have been kept success-
fully in aquaria (M. Kuba, pers. comm.; see also
108
).
These range from small species such as bobtail squid
(E. scolopes) to larger pelagic squid (e.g. Loligo vulgaris,
Doryteuthis pealeii), octopuses (e.g. O. vulgaris,Eledone
cirrhosa) and cuttlefishes (Sepia officinalis), and the
giant pacific octopus (Enteroctopus dofleini).
At the Stazione Zoologica di Napoli, considered to
be the first large-scale facility for the maintenance of
cephalopods
56
(mostly for cuttlefishes and octopuses,
A. Droesher and G. Fiorito, pers. comm.), outdoor
tanks were preferred to indoor rooms, to allow animals
to be kept in natural light with seasonal daylength.
However, shading was provided to reduce direct
sunlight to the animals. In the following years, indoor
tanks were installed and artificial lighting was intro-
duced to supplement natural illumination
(A. Droesher and G. Fiorito, pers. comm.).
The knowledge accumulated in various laboratories
around the world, with a variety of cephalopod species,
supplemented the original studies at Stazione Zoologica
and facilitated the design of closed systems for
maintenance of species which adapt less readily to
laboratory housing such as squid (for review and
methods see
75
).
Annex III of Directive 2010/63/EU sets out require-
ments for care and accommodation of animals. Section
A lists general requirements pertaining to all species
and section B lists species-specific requirements, for
all vertebrate classes, including brief guidance for fish
(but with no distinction between the different classes of
fish). Some of these Section B requirements for fish
might also apply to cephalopods,
2
but cephalopods
are not specifically mentioned in Annex III.
5.2 Holding facilities for cephalopods
Planning design and maintenance of new accommoda-
tion facilities for cephalopods should take into consid-
eration key points outlined by the Committee for the
Update of the Guide for the Care and Use of
Laboratory Animals.
119
Access to the facility should be allowed only to
people who have received relevant training and have a
legitimate need for access. Movements of personnel
inside the facility should also be monitored and con-
trolled to minimise disturbance to the animals and
ensure biosecurity, which may require measures such
as physical barriers and access restriction/control.
Walls of holding rooms should generally be of dark
neutral and continuous colours. However, very dark
colours may make it difficult to identify dirty areas,
so specific evaluation of the appropriate colour may
be required.
Cephalopods require large volumes of seawater.
All facilities should have an emergency contingency
capacity, capable of maintaining aerated and filtered
seawater should normal systems fail. Monitoring sys-
tems including remote alarm notification should be
designed and used in cephalopod facilities.
Noise should be minimised to avoid disturbing ani-
mals in both housing and experimental rooms. When
applying sound-attenuating material to the ceiling or
walls, always consider that it has to be sanitisable. All
vibration sources (e.g. mechanical equipment, electrical
switches, through ground-borne transmission) should
be identified and vibration isolation methods should
be used to reduce noise (e.g. by placing equipment on
rubber pads). Noise-producing support functions, such
as tank and filter washing, should be separated from
housing and experimental areas, wherever possible.
Fire and environmental-monitoring alarm systems
should be selected and positioned to minimise potential
disturbance to animals.
All procedures and other manipulations of living ani-
mals should be carried out inside the facility to minimise
stress to the animals, unless there is scientific justification
for doing otherwsise. Therefore, a typical cephalopod
facility should have available: i. adult animal housing/
holding room(s) divided by species if possible, and
breeding/’hatching’ room(s); ii. quarantine room(s) (if
needed); iii. an area for acclimatisation of animals; iv.
procedure rooms separated from holding and breeding
rooms, for experimental techniques, including regulated
procedures (e.g. surgery, behavioural experiments, ima-
ging, clinical treatment, humane killing, necropsy, etc.);
v. separate ‘service’ rooms for storage of food, supplies,
chemicals, etc., and for waste (including biological
material) storage before incineration or removal.
Shared facilities, where cephalopods are kept in
water systems and rooms hosting a range of other
Fiorito et al. 15

types of marine organisms, are not recommended.
Additionally prey species should never be accommo-
dated in the same tank as their predators.
In designing holding facilties for cephalopods and
selecting the construction materials, it is recommended
that guidelines developed for fish are followed
(for review see
79
).
Materials used to build aquatic facilities should be
non toxic. Any unavoidable use of material with
the potential to be toxic should be reduced to the min-
imum, recorded and the information made available to
staff, veterinarians and inspectors. In particular, mater-
ials that may release specific ions, chemicals or corro-
sion by-products from their surfaces should be avoided.
The use of metals requires consideration of their inter-
actions with seawater, and the potential effects of that
interaction on the animals.
Special attention should also be given to the behav-
ioural needs of the animals. For example, non-gregarious
animals or animals that might show aggressive inter-
actions (e.g. males during mating season) may require
housing out of sight of others. Attention must also be
paid to species-specific differences in terms of the level
of disturbance that may be acceptable; for example, O.
vulgaris appears to be quite resilient whereas cuttlefish or
squid react more strongly to unfamiliar and sudden
movements.
5.3 Housing
Cephalopods are strictly marine, and all require
high-quality sea-water, but their varying habitats,
social behaviour and especially nature and level of loco-
motion determine how they should be housed.
Aquarium size and stocking density should be based
on the physiological and behavioural needs of the indi-
vidual species, and requirements for their health and
welfare (see Appendix 2).
Section 3§3b of Annex III of the Directive indicates
that all animals, including cephalopods, ‘shall be pro-
vided with space of sufficient complexity to allow expres-
sion of a wide range of normal behaviour’, including
social behaviour, locomotion and feeding, and ‘shall
be given a degree of control and choice over their envir-
onment to reduce stress-induced behaviour’.
Stocking density will vary depending on the animals’
natural history and behaviour, water flow, size, age
and health. Water quality is critical (see section 4.1.2
above).
Most octopuses are solitary and should be kept in
isolation. Nautilus are primarily solitary in the wild but
may be housed together at low densities.
The social structures of many species, including the
European cuttlefish (S. officinalis) are not known, but
in general social animals including many squid, are best
kept in groups. However, social interactions should be
monitored to check for adverse welfare effects; animals
should be grouped according to age to avoid fighting
and possible cannibalism, particularly in the breeding
season or where there could be territorial antagonism.
Such measures should not alter the overall welfare of
the animal, and, in general, should be respectful of the
behavioural needs of each individual species.
Depending on the species, individuals may require
dens, shelters and other devices (mostly for bottom-
living cephalopods).
Enriched environments must be provided, to allow
the animals to express their normal behaviour (see fur-
ther discussion of enrichment in section 5.11 below).
5.3.1 Tanks. Tank requirements and stocking density
vary among cephalopod species and ages (see Appendix
2 for a summary of requirements). For example, for
benthic species, like O. vulgaris and S. officinalis, the
available bottom area is an important requirement,
while for pelagic species this is represented by the
volume of water; the depth of the tank should be con-
sidered for species with known diel (diurnal) vertical
migration (e.g. Nautilus pompilius).
All cephalopods should be kept in opaque tanks of
neutral colour. O. vulgaris and S. officinalis may also be
kept exceptionally in transparent aquaria, as long as the
floor of the tank is opaque (and/or covered by sand).
Tanks can be of rectangular or of any other shape,
but for decapod cephalopods they should have
rounded corners to minimise potential injuries (see
Appendix 2).
In general (and especially for Sepia and other deca-
pods), sharp objects and rough surfaces that can cause
skin damage must be avoided. Jetting can lead to col-
lisions with the walls of the tank if animals are startled,
or there is insufficient space for escape reactions.
Tanks can be made of PVC, fibreglass, glass or any
non-toxic material capable of being adapted to achieve
appropriate shapes and allowing a smooth internal sur-
face, and which is easy to clean and sterilise or decon-
taminate as necessary. As potentially toxic materials
might have been used during initial tank assembly
(e.g. silicone-based adhesives and sealants), it is recom-
mended to wash the tank thoroughly, leave it filled with
water for at least 24 hours, and then rinse with sea-
water, before animals are introduced.
Tanks should be equipped with a covering (e.g. tank
net or rigid transparent covering) that prevents animals
escaping. Lids also serve as a barrier against the acci-
dential introduction of any foreign objects, animals or
chemicals. Tanks lids may be constructed of materials
such as plexiglass or clear acrylic to allow visual inspec-
tion. The distance between the water surface and lid
should be enough to minimise the risk of damage, for
16 Laboratory Animals 49(S2)

example, in the case of squid which are capable of leav-
ing the water using their ‘jet-propulsion’.
5.3.2 Tank labelling. Tanks must be carefully labelled,
to identify and record the histories of individuals or
groups of animals.
Labels should include detailed information for each
individual, including origin, first dates in captivity and
arrival in the laboratory, sex and morphometric meas-
urements if possible (e.g initial body weight, dorsal
mantle length), along with the number of animals in
the tank (which may be an estimate depending on devel-
opmental stage).
For animals undergoing procedures, the label should
identify: i. the procedures being performed (e.g. the label
could refer to a detailed protocol filed for easy access by
all relevant staff); ii. the date when the procedures were
started; iii. the person responsible for the animals (e.g.
the Principal Investigator). Records of any adverse
effects shown by the animals should be also be easily
accessible ‘tankside’ and should be carefully maintained
(see below for further discussion). Taken together, all
these points should make it is easy for animal care
staff and scientists to identify animals showing signs of
welfare compromise, determine the likely cause of the
adverse effects (e.g. whether procedures applied, such
as recent anaesthesia) could explain the abnormalities,
and take action to mitigate them.
5.4 Cleaning of tanks
Water quality should be monitored daily as a minimum
(see also section 4). When water changes are necessary,
the smallest possible amount should be removed.
Tanks should be free of organic waste (e.g. uneaten
food or faeces), otherwise water quality, and thus
animal health will be harmed.
Open systems: tanks should be regularly drained and
cleaned to prevent fouling and reduced water exchange.
There should be no risk of back-flushing, and conse-
quent fouling of enclosure water. The sides and bottom
of enclosures should be cleaned regularly to avoid the
accumulation of detritus.
Closed systems: waste material should be removed as
soon as possible after feeding. Total water replacement
and whole tank cleaning should be avoided, as the bio-
chemistry and flora that develop in a mature system are
essential to well-being, as known in common practice
for acquaria keeping. Depending on the size (i.e. num-
ber of tanks/system) care should be given to facilitate
the most appropriate conditions at equilibrium. Where
complete draining out of a system is required for decon-
tamination reasons, the system must be allowed to re-
mature after the addition of clean seawater, prior to
adding animals.
When cleaning of tanks occupied by animals is
necessary, the process should be designed to minim-
ise disturbance and distress; in most cases animals
will need to be removed from the tank during
cleaning. Capture and transfer methods should con-
form to the principles outlined in these Guidelines,
and the time spent in a holding tank should be
minimised.
Disinfectants should be used with extreme caution
and only in dry tanks, which are then rinsed with clean
water. Detergents should be avoided and substitutes are
preferred.
120
Animals must not be exposed to any
substance used for cleaning of tanks.
5.5 Methods for individual identification and
marking of cephalopods
Depending on stocking density, it can be difficult
to identify individual cephalopods. Marking or tagging,
other than in species with external shells such as
Nautilus, is difficult, owing to vulnerability to tissue
damage. Individual cephalopods may have unique nat-
ural markings, and whenever possible these should be
used for identification.
121,122
Several marking methods
have been successfully applied to different species of
cephalopods (for examples see review in
123
). Methods
used with success, but which require anaesthesia for
their application – and hence scientific justification
and approval from the National Competent
Authority – have included implanted fluorescent elasto-
mer tags in squid and octopus,
124,125
subcutaneous dye
injection into the arm of octopus,
123,126
and external
tagging of cuttlefish, octopus and other species.
127–132
Careful consideration of harms, benefits and justifi-
cation is therefore needed for invasive tagging, and
development of minimally invasive individual marking
methods for cephalopods is an important goal.
31,133
5.6 Food and feeding for adult cephalopods
Most cephalopods are carnivorous and active preda-
tors,
134–136
hunting their prey using a range of strategies
(review in
134
). However, nautiloids are scavengers and
some species of octopus will eat dead food items.
For many cephalopod species at different life-stages,
live prey is the only known method of feeding. This
prey may be fish or invertebrates, such as crustaceans,
which need to be treated ethically and legally,
137,138
and
the feeding regime should suit the lifestyle, natural diet
and developmental stage of the animals.
The duration of digestion (food intake to elimin-
ation) is 6–15 hours in the common laboratory species
of cephalopods and is slower at lower temperatures in a
given species,
139–141
so feeding frequency (and appetite)
may alter with season (temperature) in open systems.
Fiorito et al. 17

Data on the richness of cephalopod diets in their nat-
ural habitats is limited, but known to include, amongst
others, zooplankton, molluscs (including other ceph-
alopods), polychaete worms, crustaceans, chaetog-
naths, sea urchins, fishes and jellyfish.
26,142
An
estimation of the relative breadth of diet has been
attempted for some cephalopod species, including spe-
cies most frequently used as laboratory animals,
26
and
shows that some species’ ‘natural’ diets are restricted to
certain prey items (i.e specialists), such as Spirula spir-
ula, which feeds on detritus and zooplankton,
143
whilst
others are more opportunistic species (i.e. generalists)
such as S. officinalis or O. vulgaris (for review see
26
).
However, estimation of diet variety is substantially
biased by research effort.
In laboratory conditions, animals usually adapt to
prey on several different types of food.
36,108
Nautilus requires food with a high level of calcium
carbonate, such as shrimp with carapace, lobster moult
shells or fish heads. Most cephalopods have a higher
metabolic rate than fish, and their daily food intake
which is rich in protein can be considerable: for exam-
ple, 3–10% body weight per day.
144
The feeding regime,
palatability and method of food presentation should
ensure that animals are adequately fed. Young and/or
wild caught animals need particular attention. In some
cases, enrichement with favoured foods and touching
the animals’ arms with food may trigger feeding.
Refusal to eat can be an early sign of illness (see also
section 6.1.1).
Cuttlefish and squid are especially sensitive to inad-
equate nutrition; the most evident signs include: pro-
truding eyes, poor body condition and floating
(especially in juveniles). Consequently, in general
over-feeding is preferred, as long as excess food is
removed in an appropriate time-frame for the feeding
habits of the species.
145
However, ad libitum feeding of
S. officinalis may cause buoyancy problems, so this is
not advised (K. Perkins, unpublished data).
Artificial diets have been developed for cuttlefish and
other species
108
and are continuing to be explored in
aquaculture research (Table 4; see also
50
). However,
whilst an artificial diet may be ethically preferable
and carries reduced risk of infection, studies to date
indicate that growth and possibly welfare of the ani-
mals is reduced.
146,147
The frequency of feeds is important and depends on
the species and water temperature in the tank. The dur-
ation of digestion also depends on the species, and
other factors including the animals’ size, maturity and
the type of food (for review see
148
). In O. vulgaris, gut
transit times are quite rapid (about 12 hours at
18–19C) suggesting that crop capacity is not great
and so daily feeding should be the norm.
141,149,150
Daily feeding is also common practice for most coleoid
cephalopods. However, other evidence from adult
Table 4. Use of alternatives and/or artificial/synthetic food to natural prey for rearing of some cephalopod species. The
table is based on an overview of recent literature (most representative papers are included) mostly for aquaculture
purposes (unless otherwise stated). For review see also Iglesias et al.
50
Species Artificial food Food item References
Sepia officinalis Yes Pellet
Surimi
508–510
No Shrimps e.g.
511
Yes Lysine diet 4
151
No Natural frozen diet
512
Octopus maya Yes CPSP
a513
Yes Purina @ 51%
514
Yes Shrimp pellet þCPSP
a 515
Octopus vulgaris Yes Moist pellets (fish and prawn mixed with alginate or
gelatin as binders)
b
508,516–518
Yes CPSP
a,b 519
Yes Diet S (50% water, 20% gelatin, 10% egg yolk, 5%
S. aurita, 15% T. sagittatus)
520
No Crustaceans; aquaculture by-products
521–527
a
CPSP: namely ‘Concentre
´s de Prote
´ines Solubles de Poisson’ is a concentrated fish hydrolysate currently trademark of COPALIS (http://
www.copalis.fr/en/home/products-and-applications/animal-nutrition/aquaculture-breeding.html); see also Kristinsson and Rasco
528
b
This study is also aimed to the understanding of nutritional requirements of octopus
18 Laboratory Animals 49(S2)

cephalopods, particularly cuttlefish and octopus, sug-
gests that they may not need to eat every day.
146,147,151
5.7 Food and feeding for larvae and
hatchlings
Evidence is provided that different dietary needs are
required for cephalopod species during the early
stages post-hatching.
28,152,153
For example, hatchlings
of S. officinalis often have a yolk sac which provides
nutrition until they start feeding a few days later; in
contrast, O. vulgaris paralarvae need to feed immedi-
ately in the water column. However, not all taxa of ceph-
alopods have been successfully reared in laboratories, and
so information on the dietary requirements of hatchlings
is limited (for review see
50
). Evidence available for some
species suggests that embryonic development often
requires trace nutrients that are present in natural sea-
water,
154,155
in which case development might be hin-
dered in closed artificial systems. It has also been
suggested that ‘dissolved gases and nutrients may also
contribute to metabolic and nutritional requirements
via absorption through the epidermis’.
144,156
In addition,
a close relationship between the fatty acid profile of the
dietary components and of the individual at early stages
after hatching has been reported.
157
This emphasises the
importance of improving understanding the nutritional
needs of juveniles, especially if artificial diet is being con-
sidered for rearing purposes.
5.8 Handling and moving cephalopods in
the laboratory
Handling procedures should be carried out only by com-
petent, trained personnel using techniques that minimise
the potential for injury and reduce stress to the animals
(see also section 10). It is recommended that laboratory
coats and gloves should not be of white/pale colours, as
the handler can be mistaken for a ‘predator’.
The skin of cephalopods acts as an organ
158
and is
very delicate and so every effort should be made to
minimise handling and removal of animals from the
water. It is especially beneficial to standardise handling
procedures, as anecdotal evidence indicates that ceph-
alopods can habituate to handling.
It is preferable to move the animals in water using
containers where they can be gently restricted before
moving from one tank to another or any other location.
Training animals to enter a container, possibly using
small rewards, may reduce stress and habituate them
to the transfer.
Cuttlefish and squid should be immersed at all times
and a dark net should only be used to coax the animal
into a container.
Nautilus is particularly sensitive to air, and repeated
air exposure is anedoctally reported to have negative
effects on the health of the animals (R. Smallowitz,
unpublished data).
Octopuses can be moved using nets (suggested dark
nylon 2-mm mesh) with a long sleeve to reduce the risk
of escape; exposure to air should be minimised. A con-
tainer method has been developed for O. maya
159
and,
although not currently in use, represents a useful
approach indicative of methods that should be devel-
oped for animal transfer within a facility.
Nets and containers should be clean, disinfected and
rinsed before use. Agitation during moving should be
minimised, as all cephalopods have a sensitive statocyst
system.
160
Handling and other human interactions should be
monitored and recorded, as the frequency and nature
of the interactions can influence behavioural perform-
ance of individual animals.
5.9 Environmental enrichment
Environmental enrichment should not compromise the
need for adequate levels of hygiene and the ability to
observe the animals’ health (section 6 below) without
causing too much disruption. The impact on health and
welfare of environmental enrichment should be evalu-
ated objectively,
161–163
particularly to avoid the appli-
cation of ‘environmental changes’ which may be
detrimental to the animal well-being, and to ensure
health or water quality are not compromised.
Section 3§3b of Annex III of the Directive states that
‘Establishments shall have appropriate enrichment
techniques in place, to extend the range of activities
available to the animals and increase their coping activ-
ities including physical exercise, foraging, manipulative
and cognitive activities, as appropriate to the species.
Environmental enrichment in animal enclosures shall
be adapted to the species and individual needs of the
animals concerned.’ The same section also states that
‘the enrichment strategies in establishments shall be
regularly reviewed and updated’.
These provisions require on-going consideration of
the effects of laboratory housing on animal welfare and
efforts to enhance well-being wherever possible.
Exemptions from these, and other, requirements out-
lined in Annex III have to be approved by the National
Competent Authority, and must be for scientific,
animal welfare or animal health reasons.
Environmental enrichment aims to enhance the well-
being of animals in captive conditions, by identifying
and providing stimuli that enable animals to express as
wide a range of their normal behaviours as pos-
sible.
164,165
Enrichment is proven to be effective for
Fiorito et al. 19

many species, including fishes,
166–168
cephalopods and
other invertebrates.
138,169–175
Enrichment may be accomplished through changes
in the tank environment, for example, by varying fac-
tors, such as the shape of the tank, flow of water,
variety of live prey items (if these are essential), conspe-
cifics and environmental complexity; and also by pro-
viding opportunities for animals to engage in specific
activities and exercise some choice.
Enrichment strategies should be tailored to the needs
of the particular species concerned. For example, open-
water species may require large but less complex
environments. Social animals should be housed in
groups. Benthic cephalopods are better kept in complex
environments with suitable substrates (sand, gravel or
pebbles) and dens.
Nautiluses should have access to vertical space for
movements and attachment at a variety of levels, thus
meeting their natural habit of daily vertical
migrations.
176,177
However, not too many vertical
attachments should be added, as nautiluses naturally
swim up and down whilst circling around the perimeter
of tanks and require space to do so. Adding texture
(artificial coral reef) to at least one wall of the tank
may make it more attractive to the animal (and may
promote egg laying; G. Barord, pers. comm.).
In octopuses, interaction with objects is a common
form of enrichment and is recommended; providing a
den as refuge is not considered to be enrichment, as it is
a basic requirement for octopuses, and for all benthic
species that use refuges in the wild. Artificial shelters
can take the form of many different objects (e.g. bricks,
ceramic pots, plastic jars), but dark and opaque dens
are preferred over clear ones.
Suggestions for the type of objects (artificial and/or
natural) to be added in tanks as enrichment for most
common cephalopod species are provided by Grimpe.
56
Recent systematic studies are missing and data avail-
able are mostly anedoctical.
Caution should be taken to avoid objects added to
holding tanks that could harm or limit full expression
of the behavioural repertoire of the animal.
Mirrored surfaces should be avoided, since may
create agonistic reactions expressed by some individuals
towards the ‘ghost’ reflected image (G. Fiorito, pers.
comm.). Accounts of tank design for coastal and reef
squid species provide also information on environmen-
tal enrichment for these animals.
76,178
6. Assessment of health and welfare
Annex III, Article 3.1 of the Directive requires that
establishments have a strategy to ensure that the state
of health of the animals safeguards animal welfare and
meets scientific requirements. This should include
regular health monitoring and plans for dealing with
health breakdowns (see section 7). The starting point
for fulfilling these requirements is objective monitoring
and recording of the health and welfare of the animals
and recognition of the factors likely to cause deviations
from optimal status.
The primary factors that could cause a decline in
health and welfare and which require monitoring are
effects of:
genvironmental and housing conditions (including:
capture, transport, handling, stocking density,
tank design); nutrition; variations in: water tem-
perature, oxygen levels, pH, salinity and water con-
taminants (sections 4 and 5);
ginfectious disease (section 7); and
gexperimental (regulated) procedures (section 8).
Irrespective of the cause, objective criteria for assess-
ment of the overall health and welfare status of animals
are required to:
(i) ensure that animals arriving in the laboratory are
healthy;
(ii) ensure that housing and care are adequate for the
maintenance of good health and welfare;
(iii) assess the impact of experimental procedures (sec-
tion 7) in terms of severity and identification of
pre-established humane end-points;
(iv) identify and implement measures to rectify health
and welfare problems and enhance the well-being
of animals, and refine procedures so that they
cause less harm to the animals; and
(v) monitor the efficacy of any therapeutic
interventions.
As for other animals, the key parameters used to
assess the health and welfare of cephalopods are
behaviour and appearance, supplemented in some
cases by measurement of a number of physiological
‘biomarkers’.
Animals should be inspected immediately on arrival
in the laboratory and at least daily thereafter; a
consistent method should be used for recording obser-
vations, evaluation and actions to be taken modified
as required for each species. An example of the types
of observation that could be made is shown in Table 5
and provides a starting point for the development of
species specific observation sheets. Oestmann et al.
145
recommend that general behaviour, indicative of
well-being, in cephalopods is assessed 2–4 times per
day; and G. Fiorito (pers. comm.) recommends at
least twice a day.
For animals that are being used in an experimental
(regulated) procedure (section 8), the observation
20 Laboratory Animals 49(S2)

Table 5 A-C. Potential generic indicators of health and welfare in cephalopods (primarily cuttlefish, squid and octopus) that could be used in daily assessment and
adapted for monitoring animals following a procedure (see section 7). For each sign (see section 5 for details and references) a guide is given to show how each can be
graded (indicated from green to red) to indicate an escalation of aspects of PSDLH from mild to moderate and severe. The table makes no assumptions about the
underlying cause or what the animal is actually experiencing. The table utilises the principles of health and welfare assessment developed by Morton and
Griffiths
385,387
and widely adopted for welfare assessment,
485
adapted for cephalopods by Andrews et al.
8
and incorporated into Directive 2010/63/EU severity
assessment framework (Expert Working Group 2012: http://goo.gl/DozPKi). It should be appreciated that for practical implementation this table will need to be
adapted for each species, validated by research in multiple laboratories and revised accordingly. Note that the table is included here to prompt consideration of the
challenges involved in objective assessment of PSDLH in cephalopods and to stimulate research.
Table 5A. Appearance (Physical state) - see text section 6.1.2 for details.
Types of sign
Positive welfare status (‘health
and good welfare’)
Monitor animal with increased
frequency of observation depend-
ing upon parameter; Additional
checks of water quality
Monitor for signs of resolu-
tion or increased severity;
Seek advice and treat where
possible
Requires immediate action
(including euthanasia) when
observed or at the end of a
defined monitoring period
Skin colour (arms, head and
dorsal and ventral surfaces of
mantle and arms [suckers])
Normal skin colour, pattern and
reflectance/iridescence, appro-
priate prompt changes to exter-
nal stimuli (prey, threat,
conspecific).
Occasional inappropriate flash-
ing, wandering clouds, deimantic
display in absence of an overt sti-
mulus; transient pallor such as
that seen during general anaes-
thesia (reversible); unusual skin
markings or colouration should
always be monitored for changes
with time.
Frequent abnormal displays;
uncoordinated colour
changes between arms, head
or mantle; some continuously
pallid areas or areas with an
unchanging colour or pattern
(often associated with a
swelling or skin lesion).
Entire animal pale and fails to
change colour when
challenged.
Skin texture (dorsal and ven-
tral surfaces)
Skin smooth with a thin mucus
layer except when there is a sti-
mulus appropriate display of
papillae; no swellings.
Small swelling (relative to size of
animal), not in a location that
interferes with vision or feeding
and with no breach of the skin.
Occasional behaviourally unre-
lated display of papillae.
Continuous display of papillae
possibly indicative of an
aroused state. Larger swel-
ling suddenly appearing; a
small swelling increasing in
size; a swelling interfering
with vision or ability to feed
(buccal area); swelling with
signs of infection (e.g. fluid
filled cyst); excessive skin
mucus production.
Swelling associated with
breach of the skin; gas filled
swelling likely to interfere
with posture.
Skin integrity Skin intact (no underlying muscle
visible) over entire body (dorsal
and ventral surfaces).
Small, punctate breaches on
arms or mantle (often caudal
regions-’butt burn’) showing a
distinct adherent wound edge
indicative of healing; no overt
signs of infection.
Larger and more numerous
breaches especially with
irregular detached edges;
small breaches that increase
in size or develop a stable
colouration distinct from
adjacent skin.
Full thickness (muscle visible
and possibly penetrated) skin
lesions in multiple parts of
body (arms and mantle)
covering >10% of apparent
surface; wound dehiscence
following a surgical proce-
dure especially cranial or
mantle if liver capsule opened
because of gut herniation risk;
externalised portion of cuttle
bone or gladius.
(continued)
Fiorito et al. 21

Table 5A. Continued
Types of sign
Positive welfare status (‘health
and good welfare’)
Monitor animal with increased
frequency of observation depend-
ing upon parameter; Additional
checks of water quality
Monitor for signs of resolu-
tion or increased severity;
Seek advice and treat where
possible
Requires immediate action
(including euthanasia) when
observed or at the end of a
defined monitoring period
Abnormal body morphology Normal positional relationship
between arms, head and mantle
appropriate to location in tank.
In octopus arms unaligned with
mantle during jetting; in cuttle-
fish pendulous/dangling arms
during jetting.
In octopus an arm with a
permanent acute angle indi-
cative of a muscle trauma.
Dangling pallid arms in
cuttlefish.
Tentacles un-retracted in
cuttlefish and squid; Mantle
deformation in cuttlefish
indicative of damaged cuttle
bone; dorsal ridge on
cuttlefish.
Eyes Normal prominent position, clear
cornea and pupil diameter and
orientation appropriate for light
level and cranial axis.
Unilateral clouding of cornea;
nystagmus.
Eyes sunken indicative of
weight loss; pupils incor-
rectly orientated in relation to
head; exophthalmos.
Bilateral clouding of corneas
(functionally blind-unrespon-
sive to visual stimuli); fixed
constricted or dilated pupils
unresponsive ambient light
change; absence of consen-
sual pupil response but see
section 6.1.2 b.
Number of arms or tentacles All arms, tentacles and suckers
present and intact and no indi-
cation of regeneration.
Part or all of one arm missing
with signs of wound healing.
Loss of one tentacle club
(cuttlefish, squid).
Loss of both tentacle clubs
(cuttlefish, squid) and >3
arms in octopus.
Animal found dead N/A N/A If an animal is found dead in
the tank especially following a
procedure the cause should
be investigated immediately
(including autopsy) An
assessment should be made
of the degree of suffering
prior to death as this will be
required for the report of
‘actual severity’ of the
procedure.
22 Laboratory Animals 49(S2)

Table 5B. Behaviour (psychological state) - see text section 6.1.1 for details.
Types of sign
Positive welfare status (‘health
and good welfare’)
Monitor animal with increased
frequency of observation depend-
ing upon parameter; Additional
checks of water quality
Monitor for signs of resolution or
increased severity; Seek advice
and treat where possible
Requires immediate action
(including euthanasia) when
observed or at the end of a
defined monitoring period
Unprovoked behaviours
Apathetic and/or withdrawn Animals normally explores tank,
is curious about novel objects in
tank.
Reluctance to leave den/refuge
area; rarely seen exploring tank.
Has not been observed to leave
den/refuge on two consecutive
days; adopts defensive posture in
den (Octopus).
Does not leave den/refuge even
when challenged.
Abnormal body position in the
tank or the water column
Animal able to maintain a posi-
tion in the tank/water column
with ease and to move in relation
to a stimulus (e.g. food, light
change, conspecific).
Animal continually swimming
and appears to experience diffi-
culty in maintaining a stable
position in the water.
Squid located near/on floor of
tank for extended periods; cut-
tlefish spending prolonged peri-
ods at/near surface; octopus with
prolonged periods with part/all
of the body out of water.
Squid that do not move from the
floor of tank; cuttlefish that do
not move from near the water
surface; octopus in a fixed loca-
tion with most or all the body out
of water.
Stereotypic behaviour Normal diversity of behaviour
with no indication of repetitive,
overtly purposeless activity.
Occasional. Daily but not continuous. Present continuously (irrespec-
tive of when the animal is
observed)
Abnormal motor or locomotor
coordination
Locomotion and other motor
activity (e.g. prey capture) is
precisely coordinated.
Inability to coordinate arms/ten-
tacles during attack on 2 conse-
cutive occasions; inability to
maintain a straight line; persis-
tent tremor/twitching in limbs.
Further deterioration or resolu-
tion in 48hours; stiff movement;
bradykinesia; ataxia.
Convulsions, seizures or exten-
sive muscle spasms.
Cleaning/grooming Cleaning behaviour is most
obvious in octopus that can reach
all parts of the body with arms
and is a normal period activity.
Sucker rings floating in the water
may be a surrogate marker for
grooming.
Animal spends progressively
more time demonstrating
grooming behaviour or signs that
grooming is reducing leading to a
deterioration in skin condition or
obvious rings hanging from
suckers.
Continues grooming when pre-
sented with food or a distraction;
mucus accumulation; skin infec-
tion or algal deposits may be a
marker of significantly reduced
grooming.
Absent grooming or continuous
grooming as indicated by contin-
uous wiping of mantle by the
arms in octopus.
Wound/lesion directed
behaviour
The existence of this behaviour is
controversial and likely to be
most relevant following an inva-
sive procedure (injection site,
surgery).
In octopus probes wound occa-
sionally in first 24 hours post
procedure; with an arm lesion
may examine arm with mouth; in
cuttlefish attempts to reach
dorsal mantle with an arm.
One or more arms continuously
in contact with wound or
attempting to reach wound in 24
hours post procedure.
One or more arms continuously
in contact with wound in 24 hours
post procedure and wound
shows signs of dehiscence or
infection.
Changes in social interactions
with conspecifics
For social species interaction
(e.g. display) is a normal
behaviour.
Animal becomes withdrawn from
the group on occasions and does
not always respond to signals
from conspecifics.
Animal withdraws for 24 h from
all normal social interactions.
Withdrawal from all normal
social interactions on consecu-
tive days; Inappropriate and per-
sistent aggression.
Autophagy Not a normal behaviour so any
occurrence of autophagy should
be investigated promptly.
Removal of a few suckers or a
skin lesion on the arm may indi-
cate incipient autophagy.
Removal of distal 50% of an arm. Removal of an entire arm.
(continued)
Fiorito et al. 23

Table 5B. Continued
Types of sign
Positive welfare status (‘health
and good welfare’)
Monitor animal with increased
frequency of observation depend-
ing upon parameter; Additional
checks of water quality
Monitor for signs of resolution or
increased severity; Seek advice
and treat where possible
Requires immediate action
(including euthanasia) when
observed or at the end of a
defined monitoring period
Feeding
Changed urge/speed to attack
prey and possibly time to
subdue live prey
Rapidly approaches and cap-
tures/takes food. Attack latency
within 1 SD of the normal range
established for a given lab/spe-
cies/prey type; for live prey
attack should be coordinated and
prey subdued quickly.
Reduced urge; increased time to
attack.
Progressive increase in attack
latency and uncoordinated
attack; misses target.
No desire to attack or unable to
subdue live prey.
Provoked behaviours
Defaecation Defaecation may be triggered by
handling (cf. mammals) but the
significance as an index of
‘stress’ in cephalopods is not
known.
???
Inking Inking is part of a defensive
response but the threshold for
induction in relation to stimuli
likely to cause PSDLH is
unknown. Threshold differs
amongst individuals exposed to
the same stimulus (e.g.
handling).
On consecutive days animal inks
when the tank is opened or a
human is visible (wearing dark
clothing); signs of ink in the tank.
Inking behaviour/signs of ink in
tank continues for a third day;
signs of ink leaking uncontrolla-
bly from ink duct/anal canal (loss
of control).
Persistent inking in the absence
of an overt stimulus; continuous
leakage of ink from ink duct/anal
canal.
Response to humans and non-
food items placed in the tank
(see also withdrawal/apathy
above)
Cephalopods are naturally cur-
ious and will usually interact with
humans without aggression.
Objects placed in the tank are
usually explored and octopus
may use them for den
construction.
Fails to respond to presence of
human or novel non-food object
on consecutive days. Apparent
aggressive behaviour indicated
by directed squirting at a human;
withdraws to den on appearance
of human.
Continuation of behaviours for a
third day.
Behaviours continue for a fourth
day.
24 Laboratory Animals 49(S2)

Table 5C. Clinical signs (physiological/biochemical state) - see text section 6.1.3 for details.
Types of sign
Positive welfare status (‘health
and good welfare’)
Monitor animal with increased
frequency of observation depend-
ing upon parameter; Additional
checks of water quality
Monitor for signs of resolution or
increased severity; Seek advice
and treat where possible
Requires immediate action
(including euthanasia) when
observed or at the end of a
defined monitoring period
Digestive
Food intake (criteria criti-
cally depend upon species,
body weight and age)
Cephalopods are ready feeders
with a relatively high metabolic
rate so any reduction in appetite
should be monitored carefully.
Fails to take food on two conse-
cutive days (assumes a daily or
every two days feeding schedule)
or fails to completely eat a
normal size meal.
Fails to take any food on 3 con-
secutive days including an
attempt with a novel food or live
prey if animals not normally
given live prey.
Fails to take any food on 4
consecutive days including
when pieces of food are
placed in the arms /near
mouth.
Faecal output (consider in
combination with food
intake)
No data on faecal weight/com-
position and normal frequency of
defaecation. Likely to be a large
normal range. Faeces may be
hard to detect/collect especially
fresh.
Reduced; presence of parasites
or cysts; cytological markers of
epithelial damage.
Very reduced. Absent (if animal has fed
recently or is still feeding
may indicate gut obstruction).
Vomiting/regurgitation Not normally present so any
occurrences should be a cause
for concern but controversy over
existence.
Rare. Often occurs following ingestion
of food.
Always occurs following
ingestion of food.
Rates
Ventilation Ventilation is normally regular,
clearly inflates the mantle
(depth), has a steady frequency
and is coordinated with siphon
opening and closing. Frequency
can be measured and depth
estimated in both conscious and
anesthetised animals by an
observer.
Small rate change (increase or
decrease), but remains coordi-
nated; no indication of laboured
breathing.
Sustained rate change; periods
of tachypnoea /apnoea/dys-
pnoea/hyperpnoea; uneven depth
(augmented breaths).
Slow, shallow, poorly coordi-
nated (mantle/siphon); fre-
quent periods of apnoea/
dyspnoea/hyperpnoea.
Heart rate (bradycardia and
tachycardia)
Heart rate appropriate for beha-
viour but may be affected by drug
treatments including anaesthe-
sia. Currently no telemetric
methods available for HR moni-
toring in unrestrained conscious
animals but can be monitored in
anaesthetised animals to check
physiological status.
???
Blood biomarkers
Increased concentration of
catecholamines and phago-
cyte number/type
Utility requires ‘normal’ values to
be established utilising methods
which do not result in changes
and which allow good temporal
resolution.
Transient increase in catechola-
mine (<24hours) indicates expo-
sure to a mild stressor.
Large increase in phagocytes
maintained for 48 h indicates an
infection/illness/sustained
stressor exposure.
Increase in phagocytes unre-
sponsive to treatment and
accompanied by other signs
of an infection/illness.
(continued)
Fiorito et al. 25

frequency may be increased depending upon the nature
of the procedure and its anticipated impacts on animal
welfare. Factors to be considered are described in detail
below, but most require further validatation, including
consideration of their severity (see section 2.2.3 and
8.2).
It should be noted that prompt identification of
problems is essential so that i. action can be taken to
reduce and preferably eliminate any suffering and ii.in
the case of procedures, to ensure that humane
end-points are promptly implemented where appropri-
ate and that severity limits are not exceeded (sections
2.2.3 and 8).
6.1 Objective assessment of health and
welfare
Proposed parameters are described in detail below and
summarised in Table 5. Welfare assessments should be
performed in the animals’ home tank and without
removal of the animal from the water wherever
possible. Each element of the assessment will ideally
require some form of quantification to enable recogni-
tion of points at which particular parameters reach a
pre-set humane end-point and to enable actual severity
of a procedure to be reported.
Observation and evaluation of the following criteria
can help to determine whether ‘something is wrong’
with the animals, and, considering the overall pattern
of observations, can help decide strategies for rectifying
any health and welfare problems.
6.1.1 Observation of spontaneous and provoked
animal behaviour
.
a) Feeding. The common laboratory species of cephalo-
pod have voracious appetites and eat relatively large
amounts of food in relation to body weight, reflect-
ing a relatively high metabolic rate.
Therefore, altered feeding behaviour manifests as a
reluctance to take food or an increase in the time to
attack or subdue live prey, and is usually the first
and most apparent behavioural indicator that there
could be a health or welfare problem. For example,
in octopuses fed on crabs or mussels, the tank
should be checked for empty carapaces and shells
to ensure the animals are ingesting prey and not just
attacking them. In O. vulgaris the willingness to
attack can be tested equally well with an artificial
crab as with a live crab.
179
b) Location in the tank and water column. Each species
normally locates in a characteristic place in the tank
and water column.
Nautiluses spend much of their time attached to the
sides of tanks, and undergo daily vertical migrations
Table 5C. Continued
Types of sign
Positive welfare status (‘health
and good welfare’)
Monitor animal with increased
frequency of observation depend-
ing upon parameter; Additional
checks of water quality
Monitor for signs of resolution or
increased severity; Seek advice
and treat where possible
Requires immediate action
(including euthanasia) when
observed or at the end of a
defined monitoring period
Body weight
Reduction in body weight (or
other external morphologi-
cal indicator) over specific
time periods
Maintenance of normal growth
depending on food availability,
season, species, age, reproduc-
tive status.
Reduced rate of growth. 10% loss of body weight over 1
week?
20% over 1 week?
Free observations
Observations of other
behaviour not anticipated in
the checklist and which may
have a negative impact upon
welfare.
26 Laboratory Animals 49(S2)

(see above), so animals spending large amounts of
time at the bottom of the tank other than when
engaged in feeding are exhibiting abnormal behav-
iour.
Cuttlefish alternate between hovering/swimming in
the water column and resting on the bottom, par-
tially covered by the substrate, and an animal spend-
ing a considerable period of time at or near the
surface of the tank should be inspected closely for
signs of physical damage to the mantle. Such
changes have been observed following a pharmaco-
logical treatment, as described by Agin et al.,
180
as a
consequence of cycloheximide injections.
Squid rarely rest on the bottom of the tank, so their
presence there for an extended period should be
regarded as abnormal, as should extended periods
spent at the surface.
For octopuses, a ‘problem’ in the tank may be
indicated if the animals spend excessive time cling-
ing to the lid of the tank with most of the body out
of the water.
c) Swimming and locomotor activity and coordination.
Each species has a characteristic method of moving
in the tank: by walking, swimming or a combination
of both.
31
Repetitive locomotion in cephalopods,
such as jetting backwards continuously, or in the
case of octopuses performing swimming motions
while attached to a tank wall, can be a sign of
stress. Any abnormalities of coordination should
be noted. For example, a defect in the statocyst
leads to an inability to control orientation during
swimming. This ‘spinner’ behaviour has been
reported in species of cuttlefish, squid and octo-
pus.
181
Although there are likely to be minute-to-
minute changes in the level of locomotor activity
(see e.g. Figure 1 in Boyle
182
), each species has its
own overall daily pattern of activity cued by the
photoperiod.
98,100,130,183–186
Rest/sleep-like–activity
cycles are documented in S. officinalis,
102
O. vul-
garis
101
and Octopus macropus;
103
nocturnal vertical
migration is known to occur in N. pompilius.
29,177
Changes could be an indication that ‘something is
wrong’.
d) Use of arms and tentacles. The behavioural reper-
toire of arm movements in cephalopod species is
reviewed and described in Borrelli et al.
31
A tax-
onomy of arm movements for octopuses is provided
by Mather.
187
An animal spending an extended
amount of time with the arms curled over the
body (a defensive posture), either in the den or the
corner of the tank, should be monitored for other
indications of distress. In Nautilus withdrawal of all
tentacles into the shell with the opening obstructed
by the hood is a defensive behaviour.
188
There are
scattered reports of octopus using an arm to ‘guard’
an injured part of the body (I. Gleadall personal
observation cited in;
8
reports of animals ‘guarding’
the mantle or cranium post-surgery by G. Fiorito,
unpublished data, and also
189
), but this behaviour
has not been systematically investigated and could
also be linked to facilitation of healing by secreted
antimicrobial peptides. Wound-directed behaviour
was not observed in a study of tentacle amputation
in the squid Loligo pealeii,
9
although in two species
of cuttlefish (S. officinalis and S. pharaonis), the use
of a partially (80–90%) amputated arm for prey
manipulation and body posturing was avoided for
up to 3 days post lesion.
190
In cuttlefish, reaching over the dorsal mantle
(‘scratching-like’ behaviour) has been observed a
few days after a transient rise in ammonia concen-
tration leading to skin damage.
145
Tentacles in
cuttlefish and squid should be retracted except
when the animal is engaged in an attack.
The arms are used for skin cleaning in octopus;
excessive cleaning activity and/or frequent presence
of sucker-rings in the tank could be indicative of
abnormality. In all cephalopods, a loss of adhesion
in the suckers should be a cause for concern.
e) Interactions with humans and conspecifics. While
cephalopods, in general, are very responsive to any
novel features introduced in their tank, octopuses
are especially curious about their environment.
Healthy octopuses acclimatised to laboratory hous-
ing will often leave their den when the tank is
inspected and will interact with a hand placed
below the surface of the water (for an historical
account see
191
).
36,192
Reluctance to interact with
humans should be a cause for concern. It should
also be noted that there is some evidence that at
least one species of octopus (E. dofleini) may recog-
nise individual humans;
193
therefore, care should be
taken to ensure that staff who are involved in any
procedure likely to be aversive should not be
involved in routine feeding or inspection as there
is a possibility of inducing a conditioned aversion/
avoidance.
If visually exposed to conspecifics, octopuses may
alter their predatory response due to agonistic inter-
actions, but habituation resulting in a resumption of
normal behaviour has been observed under con-
trolled laboratory conditions.
194,195
Squid and
cuttlefish are social species and changes in social
interactions with conspecifics again may indicate a
welfare/health problem.
f) Squirting, inking, defaecation and regurgitation.
Squirting: all cephalopods use expulsion of water
from the mantle via the siphon in breathing and
locomotion; and this is particularly noticeable in
the jetting escape reaction. Squid and cuttlefish
Fiorito et al. 27

may direct jets of water at a person attempting to
capture them, and this behaviour is particularly not-
able in O. vulgaris where jetting is also a component
of the deimatic display.
31,134
Water jets directed at
an observer is indicative of a moderate aversive
reaction; in some cases this can also be a sign of
‘recognition’.
195
Inking: is a defensive response in cephalopods (apart
from nautiloids which do not have an ink sac), so
inking should always be taken as an indication that
the animal perceives a threat or is stressed.
However, there is individual variability in the
threshold for induction of inking as some O. vulgaris
will ink profusely in response to handling (M.G.
Valentino and P. Andrews, unpublished data) that
does not evoke the same response in other individuals
(G. Fiorito, pers. comm.). In addition, inking does
not necessarily result from the animal receiving a
presumed noxious stimulus, such as an electric
shock (I. Gleadall personal observation cited in;
8
G. Fiorito, pers. comm.). Therefore, absence of
inking should not be interpreted as an absence of
anxiety or distress. A continuous trickle of ink from
the animal should be investigated as it may indicate a
problem with the neural control mechanism or with
ink duct sphincter competence. Animals should not
be allowed to remain in a closed tank in which inking
has occurred, and care should be taken to ensure that
ink does not enter other tanks as it is an alarm
signal.
196
Intramantle inking has been reported as a
post-transport stress behaviour in Octopus bimacu-
loides.
197
Defaecation: there is insufficient knowledge of defae-
cation patterns and their control in cephalopods to
determing whether any changes may be linked to
pain, anxiety or stress, as is the case in many verte-
brates. Although faecal ropes may emerge in octopus
exposed to general anaesthetics, this could be due to
loss of anal sphincter control. The production of
faecal ropes in octopus is a useful indicator of
normal digestive tract functioning in a feeding
animal, but it is not known whether disease can
alter the faecal fluid content or defaecation frequency
(constipation/diarrhoea). However, chemical and
cytological examination of fresh faecal samples can
provide important insights into the health of the
animal and as collecting faecal samples is non-inva-
sive, its utility in cephalopod health monitoring
should be explored.
Regurgitation: there are two isolated observational
reports of regurgitation/vomiting of upper digestive
tract contents one in E. dofleini (I. Gleadall personal
observation cited in
8
) and the other in Sepioteuthis
sepioidea.
198
The location of the beak within the
crown of arms would make this behaviour very
difficult to detect. However, if the ability to regurgi-
tate/vomit upper digestive tract contents is confirmed
then it should be added to the list of possible indica-
tors of illness, as is the case in vertebrates.
199
6.1.2 Appearance
.
a) Skin colour, pattern and texture. Skin colour and
pattern are primarily regulated by motorneurones
from the suboesophageal chromatophore lobes of
the brain, with contributions from reflecting cells,
depending upon the location on the body and the
species.
134,200–202
At the time of writing, there is no evidence to show
that changes in the colour or pattern of the skin in
any cephalopod species are specifically associated
with changes in health or welfare of the animals.
Oestmann and coworkers
145
caution that normally
functioning chromatophores and iridiocytes may
mask underlying skin defects. In Nautilus discolor-
ation of the mantle (with loss of buoyancy) is a
sign of poor health.
203
However, loss of ability to
match substrate or background (see e.g.
204–207
)or
sustained pallor with loss of normal patterning
should be taken as an indication of a problem,
as should excessive or inappropriate flashing in
squid
208
and wandering clouds in coleoid spe-
cies.
209
Note should also be taken of colour
changes in response to a provocative stimulus,
such as the deimantic display often observed in
response to a perceived threat. Skin texture in
octopuses and cuttlefish can be changed by the
formation of papillae, particularly prominent
above the eyes and on the mantle and is indicative
of an aroused or vigilant animal.
31
b) Skin and external shell integrity. Any breach to the
skin of a cephalopod is potentially problematic
because of the possibility of bacterial infection (sec-
tion 7.2.2) causing systemic sepsis, preventing heal-
ing, and local inflammation causing hyperalgesia.
Bleeding from wounds may not be readily apparent
as, although oxygenated haemolymph is pale blue
(extracellular haemocyanin), it will be rapidly
diluted in the tank and deoxygenated haemolymph
is colourless.
Healing of small wounds in octopuses, such as tran-
section of the distal 10% of the length of the arm,
appears to be rapid, with the exposed area in some
animals being almost completely covered by skin in
about 24 hours (T. Shaw and P. Andrews, unpub-
lished data), but larger wounds and particularly
those to the mantle appear to take longer to heal
even without infection.
210
Damage to the skin most
frequently occurs at the distal part of the mantle in
28 Laboratory Animals 49(S2)

animals (particularly squid and cuttlefish) that fre-
quently impact the wall of the tank (‘butt-burn’: J.
Rundle, pers. comm.), with four impacts per hour
being recorded in a study investigating the long-
term health of cultivated cuttlefish in soft-sided
tanks.
211
In cuttlefish and octopus, the ventral sur-
face of the mantle contacting the substrate should
also be examined, and anecdotal evidence that ill or
senescent octopuses avoid rough substrates may
indicate that the skin in this region is particularly
sensitive to damage. Animals showing signs of
healed skin damage should be inspected closely to
ensure that it is healing, as death may ensue rapidly
if the lesion increases in size and penetrates the
underlying muscle.
212
Breaks in the shell of nautiloids may compromise
their buoyancy mechanism and so require treat-
ment.
The cause of any breach in skin or shell occurring
after an animal’s arrival in the laboratory should
be identified and action taken to prevent recurrence
(e.g. carefully examine the tank and items in it for
sharp edges, deep clean and disinfect the tanks, see
section 5.6).
c) Eyes. The eyes should be inspected to ensure that the
cornea and lens are transparent, as opacity is one of
the signs of natural senescence (see below). Pupil
diameter should decrease over a few seconds in
response to a sudden increase in illumination (for
Nautilus see;
213
for cuttlefish and octopus;
214
for
squid
215
), although there is some evidence (S. offici-
nalis and E. cirrhosa) that the response is not con-
sensual.
214
The classic contributions of Beer
216
and
Magnus
217
should be also considered in this frame-
work. The slit-like pupil remains close to horizontal
irrespective of the position of the body, and this is
particularly noticeable in octopus.
150
Both the pupil
diameter and statocyst–ocular responses
(nystagmus) are mediated by the brain and hence
give an insight into central nervous system
functionality.
181,218–220
d) Body posture. Two aspects need to be considered: i.
the relationship between the mantle, head and arms/
tentacles (i.e. the overall appearance of the animal),
and ii. the orientation of the animal in relation to
the floor and sides of the tank.
In Nautilus the shell should be vertical; however, air
bubbles can become trapped in the eyes and under
the hood, leading to adverse health effects.
221,222
Information on treatments related to poor health
conditions is available in Barord et al.
223
Trapped
air can be released by slowly turning the animal,
laterally from side to side.
All cephalopods have well-developed statocyst sys-
tems to maintain body posture and coordinate body
position with eye orientation
224
so an abnormal
body posture may indicate a nervous system prob-
lem or a physical defect that the animal cannot com-
pensate for (e.g. a broken cuttlebone, a fluid-filled
chamber in Nautilus, gas trapped in the distal
mantle of octopus). Damage to arms can also
affect posture as Tressler et al.
190
reported unba-
lanced swimming (body axis tilted to the lesioned
side) lasting up to 3 days in cuttlefish in which 80–
90% of the length of third right arm was removed.
The head in octopuses is particularly mobile and a
raised head, particularly if moving from side to side
or bobbing has been regarded as sign of ‘agitation’
in O. vulgaris by Boyle.
182
However, head bobbing
and similar behavioural patterns are indicative of
increased arousal, as reviewed by Borrelli and
coworkers.
31
6.1.3 Biomarkers
.
a) Body weight. The optimum frequency with which
animals can be handled for routine weighing,
taking into account that anaesthesia may be neces-
sary, is not known. It has been suggested
225
that
frequent handling may impede growth, but this
requires systematic investigation. Cephalopods, par-
ticularly when young, increase body weight daily
(assuming sufficient food) so failure to increase
weight or a loss of weight following an experimental
procedure may be the earliest objective measure-
ment of declining health or welfare, but the poten-
tial additional harms of frequent weighing will need
to be assessed.
Dorsal mantle length (DML) is also frequently used
as an index of body size in cephalopods although
the relationship to body weight (TBW) is not linear.
The K-Fulton condition index, which combines
length and body weight measurement and is used
in fish, has been adapted for cephalopods
(K¼(TBW/DML
3
)100).
226
In O. vulgaris infected
with Aggregata octopiana, the K-Fulton condition
index decreased as the sporocyst counts in the
caecum increased.
226
Consideration should be
given to using this index as part of routine growth
and health and welfare monitoring in cephalopods,
as applied to many species of fish,
227–233
and in
other circumstances to other vertebrates (see,
e.g.
234,235
).
In stock animals, weekly measurement of body
weight may also be a useful index of health and
welfare status, providing this can be done with min-
imal distress to the animal (e.g. in seawater), but the
percentage loss of weight over time that is indicative
of illness is not known. The digestive gland has a
Fiorito et al. 29

lipid reserve;
236
Mangold and Bidder estimated as
9–13% of digestive gland weight in S. officinalis.
148
However, the impact of food deprivation on this is
not known.
Growth data based upon body weight are species
and laboratory specific with the latter depending
upon food type and feeding frequency, water tem-
perature, stocking density, animal age, activity level
(influenced by tank size and photoperiod) and para-
site load. There are limited growth curve data for
representative cephalopod species; for example for:
Nautilus,
237
cuttlefish and squid,
238
and
octopus.
225,239
b) Ventilation (breathing) frequency. Ventilation fre-
quency can be monitored by an observer provided
that the animal is not disturbed, but a video system
may provide an alternative in the absence of other
non-invasive methodology, as standardised by
Borrelli.
26
Although an increase in ventilation fre-
quency may be an indication of physiological stress
(e.g. particularly a fall in inspired pO
284,240,241
), it is
also indicative of arousal to innocuous stimuli and
ventilation frequency also correlates positively with
activity levels, as for S. officinalis and O. vul-
garis.
182,242
Observation of breathing should also
note whether the pattern is even or is interspersed
with periods of apnoea/tachypnoea.
Although frequency is relatively easy to monitor,
26
some assessment should also be made of depth
(mantle stroke volume) as again excessively deep,
shallow or laboured breathing may also indicate a
problem and Smith et al. comment that mainten-
ance of oxygen uptake (in O. vulgaris) relies more
on stroke volume than increasing ventilation fre-
quency.
240
Deep/forceful breathing may manifest as currents in
the water or ripples in the surface if the animal is
close. In the coleoids, the way in which the mantle
distends during inspiration should be noted to
ensure it is bilaterally symmetrical, that the entire
mantle is involved, and inspiration and expiration
are coordinated with the closing and opening of the
siphon respectively. During general anaesthesia,
ventilation frequency, depth and coordination all
become suppressed.
243
Therefore, similar changes
in a non-anaesthetised animal are likely to reflect
depression of brain drive and should be investigated
immediately.
Stress state in Nautilus is usually expressed as a
‘rocking behaviour’ (which reflects hyperventila-
tion), with the animal clearly rocking from front
to back.
c) Cardiovascular parameters. Currently there is no
established non-invasive method for routine meas-
urement of heart rate or blood pressure in an
unanaesthetised cephalopod (but see
244,245
). In
addition, studies of animals with indwelling cath-
eters show that both parameters are very labile
150
suggesting that, even with appropriate method-
ology, they may not be helpful as indices of
health or welfare. In particularly compliant indi-
vidual O. vulgaris, it is possible to observe the
beating of the systemic and branchial hearts in
the mantle without anaesthesia, and to use
Doppler ultrasound to investigate cardiac function
and image the viscera (G. Ponte, pers. comm.).
The resolution achieved is much improved from
previous attempts with cephalopods.
244,246–248
This potentially represents a revolution for future
physiological studies with these animals (D. Fuchs
and G. Ponte, unpublished data; Vevo 2100
Visualsonics, The Netherlands). Such methodology
may be suitable for detailed investigation of ani-
mals showing signs of illness and could be useful
for monitoring some physiological functions
during general anaesthesia.
d) Other biomarkers: analysis of blood. There are no
validated blood biomarkers indicative of the
health or welfare status of a cephalopod, and the
development of such markers is hampered by diffi-
culty in obtaining blood samples using minimally
invasive techniques comparable to those available
for vertebrates (but see also Table 8). Descriptions
of blood sampling in the literature employ some
form of general anaesthesia as, for example, done
in the bobtail squid E. scolopes.
249
In animals
sedated for investigation using a low concentration
of a general anaesthetic (see section 8.5.5), blood
sampling and analysis may be a helpful aid to diag-
nosis and treatment but the relative harms and bene-
fits of undertaking this procedure solely for welfare
assessment need to be considered.
The following parameters should be considered.
Haemocytes. Blood samples enable culture for bac-
teria and examination of smears by electron micros-
copy for viruses. The utility of haemocyte counts
and morphology in general and phagocytes specific-
ally as indicators of infection or stress is limited
because sampling methods (especially repeated sam-
pling involving anaesthesia) themselves seem to
increase haemocyte concentration,
250,251
although
the concentration is increased further by bacterial
infection the effect is transient (i.e. present at 4 but
not 24 hours in E. cirrhosa).
126
The increase in hae-
mocyte counts in response to intramuscular injection
of Escherichia coli lipopolysaccharides in O. vulgaris
begins within 4 hours of injection and is returning to
control levels by 24 hours.
251
Vehicle injection (phos-
phate buffered saline) produced a smaller increase in
30 Laboratory Animals 49(S2)

haemocytes indicating that a rise in haemocytes may
be a useful indicator of generalised stress as well as
of infection.
Chemistry. Levels of the respiratory pigment
haemocyanin can be measured by the haemolymph
copper concentration.
252
Routine measurements of
the common inorganic ions (e.g. Na
þ
,K
þ
,Ca
þþ
,
Mg
þþ
,Cl

and SO
4þþ
) and protein
253–257
would
be helpful in establishing their utility as parameters
for diagnosing disease. For example, in O. vulgaris
a decrease in most inorganic elements and/or in
haemocyte concentrations is observed when infec-
tion by the gastrointestinal parasite A. octopiana
increases.
258
Finally, measuring activity levels of
respiratory enzymes and total protein concentra-
tion may provide additional information on the
health status of an animal.
259,260
Humoral agents. Plasma noradrenaline and
dopamine increase transiently (5 min) in response
to stress induced by air exposure in E. cirrhosa.
255
However, it is difficult to envisage how such transi-
ent changes could be applied in routine health moni-
toring. Circulating levels of a number of hormones
may give insights into health (e.g. steroids) and
reproductive status, but normal ranges need to be
established before these could be useful for health
and welfare monitoring. Other molecules that may
be of relevance for health monitoring include com-
plement system molecules, anti-microbial peptides
(AMPs) and other innate immunity-related proteins
released by haemocytes, as they increase rapidly in
infectious disease.
261–266
e) Other biomarkers: analysis of faeces. One of the
most promising methods to evaluate the physio-
logical conditions of animals in laboratory settings
is through the examination of faeces. Samples may
be utilised to estimate various biomarkers
including steroids (e.g. corticosteroids, estrogens,
testosterone
267
), as well as to evaluate, for
example, the digestibility of alternative diets as in
the case of fish,
267–270
or to identify possible parasite
infections or cytological indicators of intestinal
damage.
271
We recommend the development of faecal analysis
methods to assist in evaluation of health and welfare
of cephalopods in laboratory settings.
6.2 Health and welfare of ageing
cephalopods: a special case?
It is very difficult to determine the age of living ceph-
alopods. Age is a parameter that is known for almost
all other species used in research and should be
included in the methods section of published papers
(see ARRIVE Guidelines
272
), but which is rarely
known in studies of cephalopods, unless they are
laboratory reared.
Within a wild-caught population of a particular
cephalopod species, in a circumscribed location and
time of year, cephalopods of higher body weight are
likely to be older, but the relationship between body
weight and age is not linear, particularly in octo-
puses.
238,273,274
The absence of precise age data compli-
cates experimental design.
While there are variations due to the ecological niche
of individual species, cephalopods generally live for
about a year. With the exception of nautiloids, ceph-
alopods undergo an exponential early growth phase
during which they mature to adult size rapidly.
However, this growth phase can be influenced by
many factors, such as temperature, food availability
and space, which makes body size a poor indicator of
an animal’s age.
238,274–276
The age of sexual maturity is
variable and also appears to depend on the ecological
niche of the species. As reviewed by Rocha et al., some
cephalopods (e.g. Loligo opalescens among squid and
O. vulgaris among octopods) are semelparous (i.e.
breed and then die soon after) while others (e.g.
Nautilus sp., S. officinalis among cuttlefishes, L. vulgaris
among squid and Octopus chierchiae among octopods)
are iteroparous (i.e. breed multiple times, generally with
longer lifespan).
277
For a summary of reproductive
strategies of some cephalopods species refer to
Appendix 3.
In light of these considerations, ageing is relevant in
the context of physical senescence (i.e. ageing changes
in animals over time/after breeding, especially in
females, once their eggs have hatched), but also when
experimental procedures are applied to animals and age
could influence the results.
Possible signs of cephalopods in senescence include
reduced/absent drive to eat, poor skin quality, cloudy
eyes, and changed activity pattern and behaviour.
278–280
It may be difficult to distinguish this state from an
animal that is showing similar signs due to disease.
Good record keeping of time kept in the laboratory
and age whenever possible, alongside general health
records of individuals may help to differentiate the
two situations.
It is unknown whether cephalopods experience any
form of pain or suffering during senescence, but the
precautionary principle should be applied when deter-
mining humane end-points (see section 8.3) for studies
involving senescent cephalopods.
The senescent state makes animals more susceptible
to a number of problems which, if they occurred in
non-senescent animals, would be regarded as indicators
of illness.
145,278,280
These include: skin breaches
Fiorito et al. 31

including ulceration; primary and secondary cutaneous
(e.g. Aeromonas sp., Vibrio sp. and Staphylococcus sp)
and systemic (e.g. Flexobacter,Vibrio) bacterial or
fungal (e.g. Labyrinthula sp., Cladosporium sp.) infec-
tions; increased parasite load.
281,282
The senescent animals are not only more susceptible
to infections, particularly of skin,
283
but they also
appear to have a reduced ability to recover once
infected.
In general, animals showing signs of senescence
should be humanely killed, unless there is sound scien-
tific or animal welfare justification for keeping them
alive.
A discussion of ethical aspects of both caring for and
using senescent cephalopods in research is available in
Smith et al.
3
Careful routine monitoring of the physical condition
of captive cephalopods at all life stages is essential for
their proper care.
6.3 Post-mortem evaluation
Post-mortem evaluation of cephalopods is often a neg-
lected aspect of health and welfare monitoring.
It enables thorough inspection, revealing abnormalities
not readily visible when the animal was living, the
cause of death can be confirmed or ascertained, histo-
logical samples collected, and a database of findings
can be gathered to support future post mortem
evaluations.
The overall aim of such detailed evaluation is to
facilitate better health and welfare assessments, and
implementation of humane end-points, in future stu-
dies. Tissues such as the beak, statoliths and vestigial
shells can also be collected, which may provide infor-
mation on the age of the animal (for example for:
O. vulgaris;
273,284
Sepioteuthis lessoniana;
285,286
other
cephalopod species
287–300
).
Cephalopod tissues are rich in protease enzymes,
which cause rapid tissue autolysis post mortem.
301,302
Autopsies should be performed immediately after
humane killing an animal, for example, on welfare
grounds when humane end-points have been reached,
or at the end of a novel procedure and/or when the
cause of welfare effects is uncertain; or as soon as an
animal is found dead (see section 8.11), but only once
death is confirmed (see section 8.12).
Cephalopods do not exhibit post mortem rigidity so
rigor mortis cannot be used to confirm death, and other
methods need to be employed (see section 8.12).
Autopsy findings should be reported in the first
instance to the person responsible for overseeing the
welfare and care of the animals, and any actions
needed to safeguard animal welfare in future should
be agreed, recorded and implemented.
Information on likely cause of death may be
required for consideration by the local Animal
Welfare Body or the National Competent Authority,
especially if there is unexpected mortality following a
procedure but note that mortality should never be used
as an end-point for a procedure.
Steps to be considered for inclusion in post mortem
evaluations include:
Haemolymph sampling: when animals are humanely
killed for welfare reasons, it will be possible to collect
haemolymph immediately surgical anaesthesia is
achieved, but before death ensues (see section 8.5.5 for
techniques). A bacterial septicaemia is suspected when
the haemocytes have aggregated into visible clumps.
Systemic bacterial infections should be confirmed by
bacterial culture of the haemolymph.
Skin examination: external lesions should be blotted to
remove excess mucus, then, aseptically, samples obtained
using swabs and submitted for bacterial culture. Smears
obtained from the swab or skin scrapings should be air
dried and stained for bacteria or fungi.
Anatomical examination: descriptions of the gross
internal anatomy of the main cephalopod species can be
found in the following classic references: N. pompilius;
303
S. officinalis;
304
L. vulgaris;
305
O. vulgaris;
306
E.
cirrhosa.
307
In brief, following a detailed external inspection
including skin breaches, abnormal colouration,
damage to appendages and deformities the mantle
cavity is opened by an incision, following the anatom-
ical approach that gives best accessibility in each species.
The viscera are examined visually and particular
note taken of the state of the hearts, gills and the digest-
ive gland (hepatopancreas), which is the largest and the
main metabolic organ. Organs should be inspected for
abnormal colour (particularly hepatopancreas), shape,
size, texture (e.g. oedema, hard lump caused by a cyst
or tumour), and presence of parasites (particularly
intestine) or foreign bodies.
The presence, or not, of food (digested/
undigested) in the crop, stomach and caecum/intestine
should be noted as well as faecal ropes in the rectum to
assess gastrointestinal tract functionality. Digestive
tract samples should be analysed for the presence of
parasites.
The degree of filling of the ink sac should be noted as
an empty one may indicate that the animal has inked
profusely in the tank prior to death, which might not
otherwise be apparent if the animal was found dead in a
tank with circulating seawater.
Haemorrhage is impossible to detect as the blood is
colourless when deoxygenated, and oedema may be
hard to detect without histology.
Tissue samples can be fixed by immersion in neutral-
buffered 10% formalin, and standard histo-
32 Laboratory Animals 49(S2)

pathological techniques applied although fixation in
buffered glutaraldehyde will be required for ultrastruc-
tural studies and for identification of viruses.
Creation of a repository of data and/or reports on
cephalopod pathology would provide an important
resource in the effort to ensure good health and welfare
in captive cephalopods used in laboratories. This
is a current project of the non-profit Association
for Cephalopod Research (see www.cephalopodre
search.org/projects), which is also included as
goal of the COST Action FA1301 (CephsInAction;
http://www.cost.eu/COST_Actions/fa/Actions/
FA1301; www.cephsinaction.org).
7. Disease: causes, prevention and
treatment
The major known risks to health and welfare in ceph-
alopods are environmental parameters, and especially
water quality issues, physical injury and infection (espe-
cially due to parasites), all of which may be interrelated.
In addition, the psychological well-being of the animals
should be considered (for a preliminary discussion
see:
308,309
), as presented in section 6.
7.1 Introduction to the issues related to
diseases of cephalopods
7.1.1 Environmental influences on disease. Host-
pathogen interactions can be strongly influenced by
the environment in which an animal lives. In addition,
stressful conditions deriving from inadequate physico-
chemical parameters (e.g. increased or decreased tem-
perature, presence of undesirable chemicals, low
oxygen saturation) may result in impaired defense
responses against pathogens (review in
310
), thus
increasing the probability of a disease outbreak.
Maintenance of water quality is essential for minimis-
ing infectious diseases and tank design (including enrich-
ment) is important for avoiding self-induced physical
trauma (e.g. ‘butt-burn’, see 6.1.2b above) and reducing
general ‘stress’.
Close attention to these factors should make animals
more resistant to infection as well as improving psycho-
logical well-being.
7.1.2 Effects of physical trauma. Injuries may be
inflicted by some methods of capture (e.g. tentacle loss
by squid jig;
311
see also
312
) and handling without appro-
priate care (e.g. skin damage by net reported in
313
), or
during transport as, for example, for O. vulgaris that
may fight if transported together and eventually bite or
cannibalise each other (see description of fighting in
31
).
Skin damage makes the animal susceptible to sec-
ondary infections (particularly bacterial) which can be
fatal if untreated.
314
7.1.3 Disease caused by feeding live food. Although
feeding with live food may be preferred to an artificial diet
(see section 4.2), it is important to avoid the use of species
that are recognised for their role as hosts of important
pathogenetic parasites. For example, coccidian
Aggregata sp.
258,315,316
(for review see
73
)orviruses
317–319
may infect cephalopods through food items such ascrust-
aceans. In the case of the use of crabs as a prey item,
special attention should be given to distinguish between
those carrying parasites and to remove them from ceph-
alopod facilities.
It is also noteworthy that penetrative injuryto the brain
has been reported to occur in O. vulgaris
320
due to the setae
of ingested polychaete scaleworms (Herminone hystrix)so
their presence in laboratory tanks should alsobe avoided.
See also section 7.2 below for discussion on infec-
tious diseases of cephalopods.
7.1.4 Action points when disease is
suspected. Animals showing signs of illness should
be placed in quarantine (see section 3.6) to reduce the
potential spread of the causal agent, and any animals
found dead removed and autopsied (see section 6.3).
Water from tanks of quarantined animals should not
contaminate water supplies to other animals or
the environment, and personnel handling potentially
infectious animals should wear protective clothing
(see section 9). Seriously ill animals not responding to
treatment (where treatment is possible) should be
humanely killed (see section 8.11) and autopsied imme-
diately (see section 6.3).
Investigation of disease outbreaks should not only
include identification of the immediate cause (e.g. infec-
tious agent), but also underlying origin such as adverse
water quality, contamination of food or effects caused
by other animals. Failure to correct such factors will
often result in further outbreak of disease.
Careful records must be kept of all occurrences of
illness or mortality irrespective of identification of
cause, so that patterns can be identified over time
(e.g. there might be higher mortality/infection rates
from certain suppliers).
7.2 Infectious agents in cephalopods
Immunity in cephalopods differs from vertebrates due
to the absence of an adaptive immune response.
321,322
However, these animals do have an innate (non-
specific) immune response, mediated by both humoral
(e.g. haemagglutinin) and cellular (haemocyte)
mechanisms.
73,252,323–325
Fiorito et al. 33

As in other molluscs, circulating haemocytes are
responsible for infiltration, aggregation, encapsulation,
cytotoxic reactions and phagocytosis of foreign par-
ticles. Cowden and Curtis estimated that the phagocytic
capacity of octopus haemocytes was low;
326
while high
phagocytosis of carbon particles has been described in
E. cirrhosa.
327
Phagocytic capacities of the haemocytes
of the common octopus, O. vulgaris, challenged in vitro
using zymosan as a test particle,
325
and those of the
haemocytes of E. dofleini (see citations in
328
) have
been reported. Recently, an extensive analysis of octo-
pus haemocytes at morphological, flow cytometry and
functional level (including phagocytic capability as well
as reactive oxygen species (ROS) and nitric oxide pro-
duction) after challenging with different stimuli has has
been carried out by C. Gestal and coworkers.
73,329
In addition, several biologically active molecules likely
to be involved in responses to infection and injury are
known to be present in the haemolymph of cephalo-
pods, such as lectins, proteinases, including antipro-
tease and lysozyme activities.
126,266,330–332
The immunobiological system in cephalopods is
quite effective, as reflected by the scarce reporting of
illness in captivity for this class over many years, but
this low incidence could also reflect under-diagnosis,
particularly of systemic disease that may not have an
external manifestation, or under-reporting.
7.2.1 Viruses. Viruses are the most abundant compo-
nent of aquatic microbial communities (for review refer
to
333–335
). However, there are few records of virus-indu-
cing pathologies in cephalopods as reviewed in Hanlon
and Forsythe.
336
The first known evidence of viral
infections in cephalopods was provided in specimens
of O. vulgaris and in the cuttlefish S. officinalis.
337,338
In the octopus, oedematous, modular tumors
embedded in arm musculature and tissue degeneration
were observed in animals showing anorexia, apathy and
often autophagy.
337,339
The lesions were linked to the
presence of viral particles suggested to belong to the
group of iridovirus, according to their size, morphology
and location.
340
In S. officinalis, virus-like particles
were identified in the gastric epithelium and described
as similar to reoviruses of vertebrates, but details on the
symptoms induced are not provided.
338
Virus-like par-
ticles have been also reported in the epithelial cells of
the tubules of the digestive gland of Loligo pealei, and
in the renal appendages of several octopod species.
336
More recently, Gregory and coworkers
341
reported
another possible infection of iridovirus in cephalopods
(i.e. Nautilus sp.). Intracytoplasmic inclusion bodies
were observed in tissues from an animal found dead
in aquarium without premonitory signs of disease.
Furthermore, Todarodes pacificus (utilised as a food
item in aquaculture) have been reported to be positive
for Betanodavirus,
342
which is the aetiologic agent of a
serious viral disease known as VER (viral encephalop-
athy and retinopathy) that has been detected in a wide
range of vertebrate and invertebrate hosts worldwide
and caused severe mass mortalities in both farmed
and wild marine organisms.
343
Betanodavirus was
also identified in skin lesions, in the eye and in the
branchial heart of O. vulgaris.
344
Squid have been sug-
gested also as possible vectors of zoonotic viral agents
such as Norovirus.
345–347
Infections from viruses may sometimes be
asymptomatic (see examples from fishes:
348,349
) but
pathogenicity may be higher if temperature increase,
as in the case of global environmental changes.
350
In
fishes, symptoms of viral infection may include cloud-
ing of the eye, anorexia, changes in body colour and
uncoordinated swimming.
7.2.2 Bacteria. In cephalopods, pathogenic bacterial
infections are caused by several microbes; for an over-
view see Table 6. These include various species of
Gram-negative Vibrio (review in
336,351
). However,
Vibrio bacteria can also be symbiotic, as for the case
of the Hawaiian bobtail squid (E. scolopes) where
Vibrio fischeri is a mutualist in the light organ,
352,353
as well as Psedomonas sp. and other bacteria that are
symbionts in Nautilus sp.
354
Secondary bacterial infections in skin lesions have
been reported in squid,
355
cuttlefish
356
and octopus,
357
and skin lesions are considered to be the most common
conditions in which infections occur.
351
Bacterial infec-
tions may spread to conspecifics sharing the tank.
77
In addition, bacteria may cause skin ulcers on mantle,
head and arms, hyperplasia of the epidermis and
increased mucus production (e.g. in Lolliguncula
brevis;
355
in O. joubini and O. briareus;
357
for review
see
336,358
).
While infections occurring on the skin are most
commonly reported, they are not the only tissues sus-
ceptible to bacterial infection, since Rickettsiales-like
organisms have been found in the gills of laboratory
reared O. vulgaris, observed as basophilic intracytoplas-
matic microcolonies within epithelial cells, on which
they cause hypertrophia and occasionally necrosis. No
significant harm has been observed in the host, but
under conditions of stress or intensive husbandry, it
has been suggested that these bacteria may have a det-
rimental effect on the host’s respiratory gaseous
exchange although this has not been shown
experimentally.
359
Gram-negative bacteria Vibrio lentus have been also
identified in the branchial heart of wild O. vulgaris and
reported to induce mortality in 50% of octopuses in the
first six hours, with lesions showing a typical round
pattern on the arms or head.
360
34 Laboratory Animals 49(S2)

Table 6. Most common bacteria reported in cephalopods. Information included hereunder is deduced from various works and reviews
336,355,357,359–361,363,530
.
Sepia
officinalis
Loligo
forbesi
Loligo
pealei
Lolliguncula
brevis
Sepioteuthis
lessoniana
Octopus
vulgaris
Octopus
briareeus
Octopus
bimaculoides
Octopus
joubini
Octopus
maya
Enteroctopus
dofleini
Acinetobacter anitratus 3
Aeromonas cavia 3
Aeromonas sp. 3
Micrococcus sp. 3
Myxobacteria spp. 3
Pseudomonas sp. 333 3 3
Pseudomonas stutzeri 333
Rickettsia sp. 3
Vibrio parahaemolyticus 3
Vibrio alginolyticus 3
Vibrio anguillarum 3
Vibrio carchariae 333
Vibrio damsela 33
Vibrio harveyi 3
Vibrio lentus 3
Vibrio pelagius 3
Vibrio sp. 33 3
Vibrio splendidus 33
Fiorito et al. 35

Finally, cloudy-to-opaque corneal tissue as well as
opaque lenses in Loligo forbesi and S. lessoniana have
been reported due to infection with Gram-positive bac-
teria (Microccocus sp.) found in the vitreous-induced
swelling of the infected eye and causing opacity of the
cornea.
361
7.2.3 Fungi. Reports of fungal infections in cephalopods
are scarce and mostly relate to eggs and embryos. Hanlon
and Forsythe
336
refer to infection by Labyrinthula sp. in
adult O. vulgaris; in these animals grey patches of inacti-
vated chromatophores appeared followed by progres-
sively larger and whiter patches in which the entire
epidermis and dermis was missing. Thraustochytrid and
labyrinthulid fungi have also been isolated from skin
lesions in E. cirrhosa, but it is not clear whether these
organisms are causal agents or secondary infections.
362
Harms et al.
282
reported a case of mycotic infection in
adult captive cuttlefish showing skin lesions in the
dorsal mantle. Cytology revealed hemocyte granulomas
surrounding fungal hyphae, and culture yelded
Cladosporium sp. Infection from the same organism
was also reported by Scimeca and Oestmann (1985,
cited in
351
) in octopus, while Fusarium sp. has been
found infecting the chambered nautilus, N. pompilius.
351
7.2.4 Parasites. Most wild cephalopods host parasites
include protozoans, dicyemids and metazoans.
Generally, these are found in skin, gills, digestive
tract, digestive gland and kidneys.
73,363–370
Among the protozoans, one of the main parasites
infecting both wild and cultured cephalopods is the
gastrointestinal coccidian of the genus Aggregata,
which produces severe disease in cuttlefishes and octo-
puses, by causing a malabsorption syndrome, diminish-
ing nutrient absorption and reducing the immune
response capability.
226,258
In addition, the parasite
may produce behavioural alterations in the infected
host inducing excitation, impaired ability to camouflage
and aggressive behaviour.
316
Mortality has been attrib-
uted to the infection, and it has been reported that a
few days before dying an infected octopus became inac-
tive in the shelters and unresponsive to stimuli.
316
Dicyemids are endosymbionts that inhabit the renal
sacs of cephalopods including cuttlefish, loliginid squid
and octopuses.
370–372
No host damage has been
reported due to dicyemids, but a possible contribution
to ammonium ion elimination from the host urine has
been proposed
370
. However, dicyemids could be a prob-
lem if the parasite load is elevated enough to cause
physical obstruction of the renal sac.
Cephalopods are intermediate or parathenic hosts
for a variety of metazoan parasites, namely trematodes,
digenea, cestodes and nematodes transmitted via the
food chain.
370,373
Amongst these, one of the most abundant and fre-
quent parasites are anisakid nematodes, which have
been reported to cause important pathological effects
in several cephalopod host species.
374–376
Larvae of
Anisakis simplex are pathogenic to humans when raw,
under-cooked or lightly marinated fish or squid are
ingested.
377
Crustaceans, such as copepods and iso-
pods, also parasitise the gills and mantle cavity of ceph-
alopods, affecting the body condition of the host.
378
Apart from the potentially pathogenic organisms (e.g.
Vibrio,Aeromonas,Pseudomonas and Flavobacterium
sp.; ciliates and dicyemids), none of these diverse organ-
isms is known to cause severe health problems in captive
cephalopods. However, as some of the mechanisms
exploited by parasites to produce changes in host’s
behaviour would seem to be highly conserved through-
out the evolution of both vertebrates and inverte-
brates,
379–382
it would be unwise to exclude such
interactions in cephalopods without specific research.
7.3 Antibiotic treatment of infectious
diseases
Antibiotics have been utilised in some instances to treat
cephalopods in laboratory experiments as reviewed in
Table 7. Several routes of administration have been
used (i.e. oral, parenteral or tank/bath immersion); in
addition Berk and coworkers
383
have suggested a tech-
nique for gavage in squid that could be adapted to
octopus and cuttlefish. Intramuscular injections of anti-
biotics have been given at the base of the arms taking
special care to avoid the axial nerve cord.
358
Sherrill
et al.
356
have suggested the use of oral, parenteral, or
tank/bath immersion prophylactic antibiotics as
reasonable for captive cuttlefish subjected to physio-
logical stress, since this treatment may delay disease
progression and improve longevity. This method
should be avoided unless there are exceptional, scien-
tifically justified circumstances, as it is clearly preferable
to identify and remove the source of the stress.
Despite published evidence, caution should be
applied when using oral or parental routes of adminis-
tration since these are stressful for animals and may be
difficult to perform safely.
In any case, prophylactic use of antibiotics is not
recommended because of the risk of promoting bacter-
ial and fungal resistance, masking infection and
allowing secondary infection. It should not be used to
‘prop up’ poor tank hygiene.
Cephalopods used for scientific purposes should
be maintained free from infections and contact with
infection sources avoided. To this end, the use of
high-performing filtration systems (i.e. combining
mechanical, biological and physical filtrations) is
highly recommended in combination with careful
36 Laboratory Animals 49(S2)

screening of animals entering the facility and efficient
quarantine procedures. The importance of tank design
in minimising the potential for skin damage and subse-
quent increased probability of infection should not be
overlooked.
8. Scientific procedures, severity and
harm-benefit assessment, anaesthesia
and humane killing
8.1 Definition of a ‘procedure’
Directive 2010/63/EU defines a regulated ‘procedure’
as, ‘Any use, invasive or non-invasive, of an ani-
mal [e.g. living cephalopod] for experimental or
other scientific purposes, with known or unknown
outcome, or education purposes, which may cause
the animal a level of pain, suffering or lasting harm
equivalent to, or higher than, that caused by the
introduction of a needle in accordance with good
veterinary practice’.
It should be noted that this definition is not confined
to procedures that induce pain, but also includes pro-
cedures that cause other forms of suffering, such as
anxiety, fear, stress and distress. Table 8 illustrates
this point by listing some studies which include proced-
ures that are likely to be subject to regulation under the
Directive.
8.2 Identifying and reducing the adverse
effects of procedures
All adverse effects that could be caused to animals by
particular scientific procedures must be identified and
predicted at the project planning stage (prospective
assessment), then adequately monitored throughout
Table 7. Substances given to some cephalopods to treat infection, but not recommended for routine use in laboratory
facilities; see text for details. The table summarises for each species the treatment (i.e. dosage, route and duration) and
substances tested. All drugs included in this table belong to the class of pharmaceuticals utilised as antibacterial agents,
unless otherwise stated. The studies cited refer to treatment of infections, with the exception of Gore et al.
530
who
investigated a pharmacokinetics. For a short discussion see also Scimeca
351
and Forsythe et al.
358
Therapeutic inter-
ventions should be discussed with a veterinarian.
Drug Dosage Route
a
Duration Species References
Sepia
officinalis
Sepioteuthis
lessoniana
Lolliguncula
brevis
Octopus
joubini
Chroramphenicol 40 mg/kg PO 7d ˇˇ
145
75–100 mg/kg PO/IM twice* 6 d ˇ
358b
Enrofloxacin 5 mg/kg IM/IV 8–12 h ˇ
530
10 mg/kg PO 8–12 h ˇ
530
2.5 mg/L Imm 5 h/d* 7–10 d ˇ
530
Gentamicin 20 mg/kg IM 7d ˇˇ
145
Tetracycline 10 mg/kg PO ˇ
531
Furazolidone 50 mg/L Imm 10 min* 2 d ˇGore et al. 2004,
cited in
351
Nitrofurazone 2 mg/L Imm 1h* 2d ˇ
355
2 mg/L Imm 72 h ˇˇ
145
25 mg/L Imm 1h* 2d ˇGore et al. 2004,
cited in
351
Metronidazole
c
100 mg/L Imm 16 h ˇGore et al. 2004,
cited in
351
a
Administration route: PO, per os (i.e. provided through food items); Imm, immersion in a solution; IM, intramuscular injection;
IV, intravenous injection.
b
But see also Table 1–3 in Hanlon and Forsythe.
336
c
Antiprotozoal agent.
Fiorito et al. 37

the procedure. Steps must be taken to: i. refine each
procedure, so as to minimise and preferably eliminate
its adverse effects, and ii. alleviate any animal suffering
that occurs during the conduct of procedures or whilst
animals are recovering.
This is a legal, as well as ethical, requirement under
Directive 2010/63/EU, which requires implementation
of replacement, reduction and refinement (3Rs) strate-
gies (section 2) wherever possible, with the aim of
‘eliminating or reducing to a minimum any possible
pain, suffering, distress or lasting harm’ [PSDLH]
caused to the animals. Note that 3Rs strategies
should be implemented whenever feasible, at all times
from birth to death of the animals including: sourcing,
transport, housing and care, handling, and fate of ani-
mals, as well as the procedures themselves.
Table 8. Selected examples of research in cephalopods which involves an intervention that it is considered would come
within the definition of a procedure (see also section 8 of this work) within the Directive. Studies published recently have
been selected where possible to show that the Directive will impact on current research areas. It should be noted that
non-surgical interventions that may induce PSDLH fall within the definition of a procedure. The fact that a particular
technique has been used in a previously published study does not guarantee that the same technique would now be
permitted by the national competent authority under the Directive.
Research topic or technique and species studied References
Amputation of a portion of an arm under general anaesthesia followed by recovery
Octopus vulgaris,Sepia officinalis,S.pharaonis,Doryteuthis pealeii
10,190,431
Deprivation of ‘sleep’ for 48 h by continuous visual stimulation
Sepia officinalis
102
Administration of E. coli lipopolysaccharide by intramuscular injection into the arm under
general anaesthesia followed by recovery and subsequent repeated sampling of haemo-
lymph
Octopus vulgaris
251
Investigation of the efficacy of different general anaesthetic substances techniques and
mechanisms of anaesthesia
Sepia officinalis
440
Production of hatchlings with deleterious phenotypes/genotypes by exposure of the eggs
to: a harmful environment, or mutagen, or genetic manipulation
Loligo vulgaris
532
Implantation of electrodes for either recording or stimulation into the brain under anaes-
thesia followed by investigation of the effects in the conscious/sedated animal
Octopus vulgaris, Doryteuthis pealeii
208,533
Removal of samples of haemoloymph from the dorsal aorta under anaesthesia with recov-
ery
Euprymna scolopes
249
Administration of drugs to modify nervous system functionality
Doryteuthis pealeii, Sepia officinalis
396,402
Implantation of temperature and depth-logging archival tags under general anaesthesia
with recovery and monitoring for up to 5 months
Sepia officinalis
130,132
Non-invasive measurement of brain and arm morphology under anaesthesia with recovery
Octopus vulgaris
246,247
Immobilisation of animal and exposure to light stimuli to investigate the pupillary reflex
Lolliguncula brevis
215
Food deprivation for 7 or more days
Sepia officinalis
534,535
Aversive training paradigms to test acquisition, consolidation and memory recall
Sepia officinalis, Octopus vulgaris
26,420,504,506,507
38 Laboratory Animals 49(S2)

8.3 Monitoring animals undergoing
procedures and setting humane
end-points
Assessment of the severity of adverse effects must be
carried out before, during and after procedures (see also
details in section 2.2 above, which describes require-
ments for setting prospective ‘severity limits’ and retro-
spective reporting of the severity of procedures, along
with questions for consideration).
Schemes for monitoring adverse effects during the
procedure should cover the criteria outlined in section
6 and Table 5 for routine daily assessment of animal
welfare; these should be supplemented with any other,
specific, adverse effects that might be caused by the par-
ticular procedure(s). Criteria for assessment, and fre-
quency and timing of observations, should be agreed
before studies commence; then regularly reviewed as stu-
dies progress, and, wherever necessary, added to or
amended.
Ahumane end-point must also be defined for each
procedure, to describe (in terms of indicators of the
nature and degree of suffering) the earliest point at
which a specific intervention must be made to end an
animal’s suffering, e.g. by: i. removing the animal from
the study, ii. providing analgesia, iii. humanely killing
the animal and/or terminating the study. The use of
‘score sheets’ for monitoring can be particularly helpful
in determining when humane end-points have been
reached, and when severity limits are being
approached.
384
To be effective, this monitoring requires
a team approach, with good planning, and appropriate
training for all involved. Methods for the observation and
assessment of adverse effects are relatively well developed
for many vertebrate species including fish.
384–387
There is a need for further development of objective
criteria for assessing severity that can be used by the
entire EU cephalopod community to ensure consist-
ency. This is a current project of the non-profit
Association for Cephalopod Research (see www.cepha
lopodresearch.org/projects).
Working on a consensus view for severity assessment
of procedures is also a goal of the FA1301 COST Action
(CephsInAction; http://www.cost.eu/COST_Actions/fa/
Actions/FA1301; www.cephsinaction.org). An equiva-
lent initiative has been set up for describing characteristics
of laboratory mice (http://www.mousewelfareterms.org/
doku.php),
8
and and for severity classification classifica-
tion of scientific procedures involving fish.
384
8.4 Harm-benefit assessment
Of course, one way of eliminating animal suffering is
not to carry out the procedure at all. In this context, it
must be remembered that ‘procedures may only be
carried out within the framework of a [authorised] pro-
ject’ (Directive, Article 12.2), which is subject to a
harm-benefit analysis, ‘to assess whether the harm to
the animals in terms of suffering, pain and distress is
justified by the expected outcome taking into account
ethical considerations, and may ultimately benefit
human beings, animals or the environment’.
Hence, for legal as well as moral reasons, investiga-
tors should carry out a harm-benefit analysis, as
described above, prior to conducting any procedure.
In the following discussion, we cite published studies
that provide evidence about possible harms caused by
common procedures. These are included as examples
that will help to predict and identify harms in future
projects, but we are not suggesting that these studies
would necessarily be considered justified according to
the harm-benefit analysis conducted under the new EU
Directive.
8.5 Some common procedures in
cephalopod research
The following sections summarise current knowledge of
the regulatory status, adverse effects and possibilities
for refinement of some common procedures. It is evi-
dent that this knowledge is patchy and there is need for
further work, especially to help refine procedures.
Nevertheless, where possible, provisional recommenda-
tions for good practice are made.
8.5.1 Behavioural studies. Cephalopods have been
used extensively for a variety of behavioural studies,
as reviewed in several works.
2,25,36,175,192,195,388
Classical behavioural studies have used a variety of
aversive stimuli (e.g. electric shock;
72
acid solutions;
389
bitter;
390
mechanical
391
) as part of training protocols.
Because of their potential to cause distress and possible
suffering, such studies would certainly fall within the
definition of a regulated procedure under the Directive
and should be avoided wherever possible. For example,
electric shock or application of acid solutions should be
unacceptable, and these and other harmful stimuli
should be replaced with reward-based conditioning
and, at the least, stimuli that do not cause pain.
Exposure of an animal to a stimulus known to evoke
an escape response (i.e. inking, jetting locomotion,
dymantic display) could also be argued to cause dis-
tress, especially if the exposure is repeated, and hence
is likely to be a regulated procedure. Deprivation of
food for prolonged periods, deliberate exposure to ele-
vated noise or adverse change in water temperature, pH
or chemistry, and direct exposure to a predator would
also be likely to fall within the definition of a proced-
ure, depending on the severity/degree of change.
Fiorito et al. 39

Table 9. Routes used for haemolymph sampling and drug administration in exemplar species of laboratory cephalopods species. Species are listed on the basis
of the most commonly utilised for such procedures. Abbreviations: NS, not stated; NA, not applicable; R, sample removed; A, drug administered; EtOH, ethanol;
SW, seawater.
Species Body weight (g)
Anaesthetic
when injection
given Site Needle size
Volume removed [R]
Administered (A)
Substance injected
Fluid removed Reference
Octopus vulgaris 98–1268
(mean 533)
55 mM MgCl
2
þ1% EtOH
Branchial hearts 30G R 70–100 ml Haemolymph
251
Octopus vulgaris 200–500 Cold water
No anaesthetic
Branchial heart Microlance 3 A 500 ml Filtered seawater
395
Octopus vulgaris 98–1268
(mean 533)
No anaesthetic Arm (2) NS A 1 ml/kg divide between
two arms
Phosphate buffered saline
or Lipopolysaccharide
(15 mg/kg)
251
Octopus vulgaris 200–400 No anaesthetic Branchial heart Microlance 3 A 1 ml/kg Filtered seawater or
scopolamine (2 mg/kg)
536
Octopus vulgaris 290–1040 NA Implanted cannula in
dorsal aorta or afferent
branchial vessel
NA R 40% of blood volume in
total (20 ml in a 1Kg
animal)
Haemolymph
537
Octopus vulgaris 200–1500 No anaesthetic Intramuscular (site NS) 25G 5/8 in. NS L-NAME (75 mg/kg), D-
NAME, artificial seawater
503,538,539
Octopus vulgaris
a
NS No anaesthetic Intramuscular at base
of arm
NS A 400–600 ml
300 ml
Reserpine (4 mg/kg)
Pargylline hydrochloride
(100 mg/kg)
540
Octopus vulgaris 200–500 No anaesthetic Arm in the region of the
brachial nerve to pro-
duce nerve block
NS NS 2% xylocaine
243
Octopus vulgaris
b
200–500 NA Implanted cannula in
dorsal aorta
NA A 100 ml substance
þ100 mlSW
Acetylcholine, carbachol,
dopamine,encephalin, nor-
adrenaline, GABA, 5-HT,
kainic acid, L-glutamate,
methysergide, nicotine,
nor-adrenaline, pentagas-
trin, taurine, tubocurarine
(doses: from 10–100 mg)
401,456
Eledone cirrhosa >250 2.5% EtOH Branchial vessel 21G 1.5 in R 300 ml/100 g Haemolymph
250
Eledone cirrhosa 500–800 2.5% EtOH Branchial vessel 26G 0.5in R 1 ml/animal haemolymph
255
(continued)
40 Laboratory Animals 49(S2)

Table 9. Continued
Species Body weight (g)
Anaesthetic
when injection
given Site Needle size
Volume removed [R]
Administered (A)
Substance injected
Fluid removed Reference
Eledone cirrhosa 493–1050 2.5% EtOH Web tissue at arm base 21G 1.5in A (NS) 1% Alcian Blue
for marking
250
Eupymna scolopes NS 2% EtOH Cephalic vessel
between eyes
26.5G R 10–20 ml (for multiple
sampling) 50–100 ml (for
single sample)
haemolymph
249
Sepia officinalis 590–900 No anaesthetic Into the side of the
neck at a depth of
10 mm
NS A 1 ml/kg Cycloheximide (10 mg/kg)
Octopressin (3–60 mg/kg)
Cephalotocin (3–60 mg/kg)
150 mM NaCl
180,396
Sepia officinalis 220 3% EtOH Cephalic vein 25G 9 mm R 500 ml haemolymph
282
Sepia officinalis 220
?
3% EtOH
?
Cephalic vein NS A NS Enrofloxacin (10 mg/kg)
Enrofloxacin (5 mg/kg)
282,530
Sepia officinalis NS No anaesthetic Intramuscular at base
of arm
NS A NS Chloramphenicol (40 mg/
kg, daily)
Gentamicin
(20 mg/kg, daily)
145
Sepia officinalis NS
(DML 4–8 cm)
1% EtOH Fin nerve branch A 2 ml 5% Texas red dextran
541
Sepia officinalis 200–1200 2%EtOH þ17.5
0/
00
MgCl
2
Brain vertical lobe Microcannula
(OD: 125 mm)
A2ml over 3 min Kainic acid (25–100 mM)
L-Glutamate (100–800 mM)
(injections also contained
methylene blue and DiI
for site marking)
398
Doryteuthis pealeii 65 No anaesthetic Crop/stomach Tygon tube (OD:
760 mm))
A 250 ml T-817 3.6 mM (a neuropro-
tective agent)
383,402
Doryteuthis pealeii
c
200–400 NA Anterior vena catheter
previously impanted
under anaesthesia
PE50 cannula
extruded
R NS Haemolymph for measure-
ment of pH, PCO
2
, [HCO3

]
417
Doryteuthis pealeii 68.5
(mean of N ¼3)
No anaethetic Intramuscular, head,
arms or mantle
NS A 1 ml/kg Gallamine triethiodide
(2.37 mmol)
208
a
Intramuscular injection of reserpine at the same dose also studied in Eledone cirrhosa and Sepia officinalis
542
b
Similar but more limited studies also performed in Eledone cirrhosa,Sepia officinalis and Alloteuthis sp.
401
c
Similar studies performed also using Illex illecebrosus
417
Fiorito et al. 41

Protocols using positive reinforcement (for example
see:
104,392–394
) should be used wherever possible.
2
8.5.2 Administration of substances. Administration
of substances can cause harms to animals as a result of
the route of administration and/or the substance itself.
Table 9 lists commonly-used routes of administration,
but does not recommend a particular technique since,
despite the number of examples included, there have
been no systematic studies investigating the optimal size
of needles or injection sites and volumes in cephalopods.
In addition, although adverse effects of injections have
not been reported, in view of the limited literature this
needs further investigation from a welfare
perspective.
251,395
Cephalopods do not have readily accessible large
superficial blood vessels; therefore, in general
experimental agents and therapeutic drugs are given
to unanaesthetised animals by bath application
(assuming absorption via skin and gills), or injection
via subcutaneous and intramuscular routes.
The efficacy of subcutaneous injections is not known,
but drug absorption may be slow because of a low
capillary density. However, this is not based on phar-
macokinetic studies and only a limited number of sub-
stances have been studied.
123,395
Locations for intramuscular injections include the
arms (particularly proximal parts in octopus) and
‘neck’ (in cuttlefish) reported to have a high vascular
density.
123,180,208,251,396
Care should be taken not to
damage arm nerve cords and ganglia by injection in
the arms (especially in octopus), nor the cuttlebone or
gladius in cuttlefish and squid respectively. The use of
ultrasound may be helpful in directing injections to
avoid vulnerable structures.
246,247
Substances have been injected into the branchial
hearts of O. vulgaris, but this requires eversion of the
mantle. The animals recover rapidly from this proced-
ure and no adverse events have been reported; however,
although behaviour rapidly (<1 hour) returned to
normal,
395
a systematic study of welfare of the animal
has not been fully carried out.
8.5.3 Other methods. It should be noted that the wel-
fare effects of the following methods has not been
assessed. Intravenous (including vena cava) and intra-
dorsal aortic routes have been used for substance
administration by direct injection.e.g.
282
Direct admin-
istration into the brain has been successfully attempted
in a number of studies. e.g.
397,398
. Finally, surgical
implantation of vascular catheters has been used as a
method of drug administration and for pressure
recording.
399–401
A method for gavage administration of drugs to ceph-
alopods has been described by Berk et al.
383
and used to
investigate the efficacy of a putative neuroprotective
agent in blocking the effects of human tau protein on
transmission in the squid giant synapse.
402
This method
requires restraint of the animal and may require removal
from the water and sedation. As the brain in cephalo-
pods encircles the oesophagus care should be taken to
use the narrowest gauge tube as possible, and it should
also have a round end to minimise the chances of punc-
turing the gut particularly at the oesophageal/crop junc-
tion. Substances administered by this route will reach the
crop and stomach directly.
Drugs and other substances such as nanoparticles
have also been administered per os (i.e. by inclusion
in the food,;
403
G. Ponte, unpublished data). Further
experiments may help in better refine this approach.
8.5.4 Administration of drugs as investigational
agents. When undertaking studies using pharmaco-
logical agents, the wider effects of the drug must also
be considered in assessing the overall impact of a treat-
ment on the welfare of the animal, and any unexpected
side-effects should be recorded and minimised/avoided
where possible. For example, Agin et al.
180
used cyclo-
heximide, a protein synthesis inhibitor, to investigate
the machinery involved in long-term memory in the
cuttlefish. However, it induced positive buoyancy that
interfered with animals’ ability to catch prey.
Relatively little is known about the pharmacology of
central and peripheral neurotransmitter systems.
1,404,405
In addition, very limited is the knowledge on the
pharmacological characteristics of drug receptors in
cephalopods. Therefore, caution should always be exer-
cised when drugs, whose pharmacological properties
have been defined in mammalian systems, are used as
investigational agents in cephalopods.
If such studies are undertaken in group housed spe-
cies, it will be necessary to identify individual animals
(see section 4.1.2) so that individual variations in drug
response can be identified. Additionally in group-
housed species, special attention should be paid to
agents that may increase aggression (e.g. by alteration
of brain neurotransmitters) or that may impair the abil-
ity of an individual to escape or defend itself and action
taken to reduce or eliminate these adverse effects.
8.5.5 Haemolymph (blood) collection. It should be
noted that cephalopod blood does not clot, but haemo-
cytes aggregate and vessels constrict to prevent blood
loss.
150,406
However, as haemolymph is pale blue (oxy-
genated) or colourless (deoxygenated), haemorrhage
may be very difficult to detect.
A summary of routes used in several studies for
haemolymph sampling is presented in Table 9. There
are no systematic data on the welfare impacts of blood
sampling methods in cephalopods, but in several
42 Laboratory Animals 49(S2)

studies behaviour was reported to return to normal fol-
lowing the sampling.
249,251,395
In large cephalopods (i.e. E. dofleini), cuttlefish and
E. scolopes, small blood samples have been obtained
under anaesthesia via a needle inserted into the cephalic
vein dorsal to the funnel.
249,282,407,408
This method
has also been successfully applied for haemolymph
sampling in O. vulgaris (G. Ponte and G. Fiorito,
pers comm.). In addition, small single samples of
haemolymph can be taken directly from the branchial
hearts in O. vulgaris (250 ml) and E. cirrhosa (<300 ml/
100 g body weight; 1 ml/animal in animals of 500–800 g,
as shown in
250
) using a hypodermic needle, although
this requires general anaesthesia to permit manipula-
tion of the mantle to expose the hearts.
250,251,255,403
In
the bobtail squid (E. scolopes) single haemolymph sam-
ples of 50–100 ml can be withdrawn from the dorsal
aorta by direct needle puncture under anaesthesia.
249
Where frequent sampling of blood is necessary, it is
likely that this will require implantation of
catheters.
240,407,408
Again, the welfare impact of this
procedure has not been fully assessed.
The maximum volume of blood which can be collected
at a single sampling in particular cephalopods has not
been assessed from a welfare perspective. In mammals,
it is recommended that no more than 10% of the blood
volume is removed at any one time.
409
For fish the
Canadian Council on Animal Care (CCAC)
zz
recom-
mends a maximum withdrawal of 1 ml blood/kg weight.
Blood volume in octopus is estimated at 5–6% of
body weight,
410,411
hence using the mammalian values
as a guide, a maximum of 5–6 ml blood/kg weight
would be advised, but, as noted above, only 1 ml/kg
according to CCAC guidelines for fish.
However, in publications where multiple sampling
has been used, authors either collected a reduced
volume on each occasion (e.g. 10–20 ml for multiple
sampling vs 50–100 ml for single samples in bobtail
squid) as done by Collins and Nyholm
249
or have set
a time for recovery between samples (e.g. 4 hours in
E. cirrhosa).
126
8.6 Surgery
Cephalopods have been subjected to a broadly similar
range of surgical procedures as have been performed in
vertebrates, but in general surgical techniques are not
described in detail in publications; this is no longer
considered acceptable and we strongly recommend fol-
lowing ARRIVE Guidelines on reporting.
272
Surgical approaches and techniques, along with
understanding of their welfare effects, are not as
advanced in cephalopods when compared with verte-
brates, particularly in relation to: i. intra-operative
monitoring and maintenance of physiological param-
eters (e.g. blood pressure, PO
2
, PCO
2
, pH, temperature;
see also section 8.8.4); ii. identification and control of
haemorrhage; iii. controlled general anaesthesia and
analgesia (see section 8.8); iv. optimal techniques for
the repair of muscle and skin tissue and wound closure
(see section 8.6.2) and healing in general; v. post-opera-
tive monitoring and special care that may be needed if,
for example, feeding is transiently impaired; vi. infec-
tion risk (intra- and post-operatively) and requirement
for prophylactic antibiotic cover (see section 7.3).
Although it will be some time before there is
welfare guidance on all the above topics, anyone
contemplating undertaking a surgical procedure in
cephalopods will need to consider all of the factors,
and submit relevant information, as part of the project
evaluation and authorisation process, and during the
conduct of any authorised surgical procedures. This
will include:
(i) ethical ‘justification’ for the procedures, in terms
of harm vs. benefit of the outcomes;
(ii) identification of potential adverse effects and the
steps taken to refine the procedures, so as to min-
imise the adverse effects;
(iii) processes for intra- and post-operative monitor-
ing of the animals;
(iv) timely application of methods to minimise post-
operative suffering, e.g. analgesia and other spe-
cific care; and
(v) clearly defined humane end-points (section 8.3) to
set an upper limit to the suffering that an animal
experiences.
Some general principles for performing surgery on
aquatic animals can be found in the fish literature, and
these should be adapted where possible to
cephalopods.
412–414
As for all other procedures, anyone attempting any
surgery in cephalopods must be ‘adequately educated
and trained’ in the principles and practical techniques
of surgery, and must be ‘supervised in the performance
of their tasks, until they have demonstrated the requis-
ite competence’ (Directive Article 23§2), for example, to
the designated veterinarian or other competent person
(see section 10 for further discussion).
A full description and evaluation of surgical
techniques in cephalopods is outside the scope of this
document but the following four specific aspects are
highlighted, along with a detailed discussion of anaes-
thesia in section 8.8 below.
zz
see: http://www.ccac.ca/Documents/Education/DFO/4_Blood_
Sampling_of_Finfish.pdf
Fiorito et al. 43

8.6.1 The operating room and environment. Surgical
procedures should be performed in a dedicated room
located close to the animals’ home tanks to minimise
animal transport, and induction of anaesthesia should
take place in the surgical procedure room. Good surgi-
cal lighting is essential, but care should be taken that
this does not cause localised heating of the animal.
Although a sterile operating environment is highly
desirable to prevent cross-contamination from human
to cephalopod (and potentially vice versa, see section 9),
and to ensure a consistent scientific baseline, mainten-
ance of sterility is not practical unless: sterile seawater
and anaesthetic solutions are used throughout the oper-
ation, and the surgeon and any assistants are fully
trained in sterile technique and are wearing appropriate
clothing. General information on asceptic surgical tech-
niques can be found in a LASA 2010 report.
415
As a minimum, it is recommended that:
i. all apparatus or surgical instruments contacting the
animal during surgery should be sterilised or at a
minimum cleaned thoroughly with anti-bacterial
and viricidal agents (e.g. diluted Halamid
§§
or
Virkon
***
, F. Mark, pers. comm.);
ii. all material contacting the wound site such as surgi-
cal swabs and sutures should be sterile; and
iii. all personnel contacting the animal in the surgical
environment should have cleaned their hands in
accordance with aseptic technique and/or use sterile
surgical gloves (blue nitrile gloves are recommended
for handling animals, to avoid pale colours but
these are not sterile and so should not be used for
surgery);
iv. the surgical site should be cleaned; but at present
there is no information on the effect of commonly
used skin cleaning preparations on cephalopod skin
integrity and healing;
v. the surgical site should be isolated as far as possible
from any source of contamination (e.g. seawater/
anaesthetic solution).
8.6.2 Maintaining physiological function. Water used
to irrigate cephalopod gills during surgery should be
circulated, treated and monitored to maintain appro-
priate anaesthetic levels, oxygen, pH, temperature and
salinity, and to remove particulates. Po
¨rtner et al. note
that the sensitivity to hypoxia is greater in squid than
either cuttlefish or octopus.
417
Consideration should be given to using a cooled
operating table (temperature monitored) in
combination with mantle perfusion with a chilled
anaesthetic solution, as cooling can act as an adjuvant
to anaesthesia,
243,418
but should not be used as an
anaesthetic alone. Cooling will also reduce metabolic
rate, which may be advantageous when cardiorespira-
tory function is impaired as a result of anaesthesia.
The skin must be kept moist and this can be done
with a sterile surgical drape moistened with sterile sea-
water. Care should be taken to minimise the potential
for seawater to enter the wound site. Although anaes-
thesia to a depth sufficient to markedly suppress or stop
ventilation may not be desirable from a physiological
perspective, surgery involving incision of the mantle
muscle and suturing may be very difficult in the absence
of such marked suppression.
Whilst the efficacy of mantle perfusion with gassed
anaesthetic/seawater solution for maintenance of blood
gases may be questioned when there is severe suppres-
sion of cardiac function by the anaesthetic/anaesthesia,
such perfusion may still be advisable to reduce anaes-
thetic wash out from the blood/tissues, to prevent des-
iccation of visceral structures and prevent/reduce
hypoxic damage to critical tissues such as branchial
hearts and gills. This may not be necessary for short
duration (<10 min) minor surgical procedures, pro-
vided that full surgical anaesthesia can be assured.
8.6.3 Wound closure and healing. There are no studies
assessing optimal materials to use for repair/closure of
skin, muscle, connective tissue (e.g. hepatopancreas
capsule), blood vessels or cranial cartilage in cephalo-
pods, and publications do not often detail the materials
used. The inclusion of such information in future pub-
lications is essential.
Closure techniques should follow normal practice for
surgery in vertebrates, using round-bodied needles to
reduce tissue trauma. Braided non-absorbable silk
123,243
,
polyglyconate sutures
282
and cyanoacrylate adhe-
sives
132,401,419–422
have been utilised. Several authors
report that cephalopods will try to remove sutures with
their arms (F. Mark, unpublished data),
240
although thisis
not a universal finding and may reflect differences in sur-
gical technique. Sutures sealed with cyanoacrylate have
been used, but in some instances they may cause skin irri-
tation (possibly due to being inflexible) and may not main-
tain integrity for longer periods of time in seawater.
An evaluation of five commonly used suture materials
including braided silk, monofilament nylon and poligle-
caprone has been undertaken in the mollusc Aplysia
californica.
423
All materials induced similar adverse
skin and subcutaneous tissue reactions, but the authors
recommended the use of braided silk because it induced
a less intense granuloma reaction. This result was unex-
pected as in studies of aquatic vertebrates monofilament
sutures are less reactive than braided silk.
423
§§
N-chloro tosylamide, sodium salt; see: http://www.halamid.com/
Aquaculture-desinfection.htm, last visited August 2014.
***
Peroxygenic acid; see Hernndez et al.
416
and http://www2.dupont.-
com/Virkon_S/en_GB/, last visited August 2014.
44 Laboratory Animals 49(S2)

We would encourage anyone undertaking surgery in
cephalopods to undertake histology on the wound site at
the end of study, and include findings in any publications,
with the aim of generating a database of good surgical
practice. In addition to suture material, the most appro-
priate suture patterns (e.g. discontinuous, continuous)
and needle size for skin closure in cephalopods has yet
to be established, and this is knowledge especially import-
ant as the skin is particularly delicate in cephalopods.
Studies involving chronic implantation of devices
such as archival (‘data logging’) tags
185,417,424–426
need
to consider possible chronic reaction of the tissues to
the device and the impact that this may have on the
normal behaviour of the animal. Mounts implanted in
cuttlefish to support archival depth and temperature
tags were well tolerated over a period of up to 5
months although post-mortem tissue thickening (pos-
sibly fibrosis) was observed around the implant and this
increased in thickness with the duration of implant-
ation.
130,132
Chronic tissue reactions are also a potential
issue for implanted identification tags (see section 5.7).
Descriptions of the natural course of wound healing
following skin damage to the dorsal mantle in E. cir-
rhosa can be found in Polglase et al.
189
Authors also
found that bacterial infection of a mantle wound
impairs healing.
210
Healing and regeneration of the fin
dermis in S. officinalis is described by Yacob et al.
427
and regeneration of the mantle connective in several
species of octopus by Sereni and Young.
428
The arms in cephalopods have a remarkable capacity
to heal and regenerate when a segment is removed by
transection with the histological changes described in
detail in O. vulgaris,E. cirrhosa,S. officinalis and S.
pharaonis
190,429–431
In O. vulgaris following transection
of the distal 10% of the length of the arm as part of
study to investigate regeneration, the exposed area was
almost completely covered by skin in about 24 hours in
some animals (T. Shaw and P. Andrews, unpublished
data).
Cephalopods frequently injure arms, tentacles and
fins in the wild;
311,312,432–435
therefore a well-developed
healing and regeneration mechanism is perhaps
expected. This suggests that, provided sufficient time is
allowed, surgical wounds, appropriately repaired, should
heal successfully provided they do not become infected.
In addition to general anaesthesia surgery also
requires the use of analgesics and this is discussed in
section 8.9 below.
8.7 Tissue biopsy
Tissue biopsies for DNA isolation and PCR analysis
for genotyping cephalopods have been obtained by
taking small samples from the tip of an arm or tentacle
with a sharp, sterile blade.
436,437
The harms vs benefits of performing this procedure
under general anaesthesia have not been assessed.
We propose that irrespective of the use of general anaes-
thesia the arm tip should be treated with a local anaes-
thetic (in the absence of systemic analgesics). Only the
absolute minimum amount of tissue should be taken
(taking into account animal size) especially in cuttlefish
as removal of large amounts of arm tissue transiently
interferes with food manipulation and results in abnor-
mal body posture.
190
In squid and cuttlefish, the fins may
provide an alternative site for biopsy as they also appear
to heal rapidly and regrow.
427
Although arms, fins and
other tissues will regenerate, attention should be pro-
vided to avoid unnecessary sampling.
8.8 General anaesthesia
The Directive requires that ‘procedures are carried out
under general or local anaesthesia’ unless anaesthesia is
judged to be ‘more traumatic to the animal than the
procedure itself’ and/or ‘is incompatible with the
purpose of the procedure’ (Article 14§2). ‘Analgesia
or another appropriate method’ must also be ‘used to
ensure that pain, suffering and distress are kept to a
minimum’ (Article 14§1), for example, peri- and post-
operatively (see section 8.9 for further discussion).
Furthermore, drugs that stop or restrict the ability of
an animal to show pain (e.g. neuromuscular blocking
agents) must not be used without an adequate level of
anaesthesia or analgesia. When such agents are used, ‘a
scientific justification must be provided, including
details on the anaesthetic/analgesic regime’ (Article
14§3). After completion of the procedure requiring
anaesthesia ‘appropriate action must be taken to min-
imise the suffering of the animal’, for example, by use of
analgesia and special nursing care (Article 14§5).
During and after general anaesthesia and surgery,
animals should be carefully and regularly monitored
using a welfare monitoring scheme, and a record kept
of observations and interventions to reduce or alleviate
adverse effects. Of particular relevance is wound-direc-
ted behaviour but other potential welfare indicators are
listed in Table 5. It is currently believed that operated
animals should be housed individually at least until full
recovery from general anaesthesia is assured, but this
will clearly need particular consideration in gregarious
species.
Many scientific procedures in cephalopods will
require the use of general anaesthesia but despite more
than 60 years of literature detailing studies involving
anaesthesia, covering at least 17 agents, information is
lacking on which agents in which species provide the
most effective, humane general anaesthesia (i.e. which
definitely blocks nociception and pain perception,
Fiorito et al. 45

generates no aversion, enables rapid induction and
allows animals to recover quickly without adverse
effects).
Recent studies have reviewed the use of agents
claimed to have anaesthetic properties in cephalopods
and criteria for evaluation of anaesthe-
sia.
8,61,131,418,438–440
In a recent contribution, isofluor-
ane has been utilised to induce ‘deep anesthesia’ in O.
vulgaris.
441
However, more detailed studies are required
to assess the application of this agent to induce anaes-
thesia in cephalopods.
In some cases, a range of behavioural responses is
reported to occur during exposure of the animals to
anaesthetic agents (i.e. inking, jetting, escape reactions,
increased ventilation);
208,243,438
however, it should be
noted that most studies report immersing the animal dir-
ectly in the anaesthetic at the final anaesthetic concentra-
tion, and this may not be the best technique to minimise
trauma (see below). It should be also noted that criteria
for general anaesthesia are not consistent between studies
and that behaviours observed during induction and to
assess depth may differ between species.
439
Moreover, studies of general anaesthesia in cephalo-
pods have investigated single agents and the familiar
concept of ‘balanced anaesthesia’ involving more than
one agent, used in mammalian studies (for reference
see
409,442
) has not been explored in cephalopods
(but see exceptions in Packard
200,443
).
8.8.1 General anaesthetic agents. Detailed descrip-
tions of the commonly used anaesthetic agents (magne-
sium chloride, ethyl alcohol and clove oil) and their
advantages and disadvantages in the common labora-
tory species can be found in recent reviews,
8,439,440
and
key features are described in Table 10.
Although a large number of agents have been
investigated for anaesthetic efficacy in cephalopods,
some are now considered unacceptable on either
welfare or safety grounds (e.g. urethane),
444
and so
are not considered here. Similarly, we are not consider-
ing those utilised in a single experiment, and pending
more evidence they are not considered herein.
Magnesium chloride (MgCl
2
)is the most extensively
studied and used agent, probably because it appears to
be the least aversive. However, it disturbs haemolymph
Mg
2þ
levels, and so may not always be appropriate, for
example, when blood samples are required to investi-
gate normal magnesium ion levels.
Furthermore, there has been a recurrent concern
that MgCl
2
may be acting at least in part as a neuro-
muscular blocking agent;
441,445
but see comments in
Graindorge et al.
398
However to date there is no
direct experimental evidence to support this concern
in cephalopods, as reviewed in Andrews et al.
8
The
supposition may have arisen from the original use of
magnesium chloride to relax small invertebrates prior
to fixation.
446
Messenger et al.
447
concluded that MgCl
2
exerts an
effect on the central nervous system, and subsequent
studies (G. Ponte, M.G. Valentino and P.L.R.
Andrews, unpub. obs.) showed that electrical stimula-
tion of efferent nerves in ‘anaesthetised’ O. vulgaris
(as per criteria below) with MgCl
2
(3.5%) evoked chro-
matophore contraction (arm and mantle), arm exten-
sion and mantle contraction showing a lack of effect at
peripheral sites of motor control.
However, more recently, Crook et al. have shown
that local subcutaneous and intramuscular injection
of isotonic MgCl
2
suppressed the afferent nerve activity
in nociceptors activated by crushing a fin.
11
This dem-
onstrates that should such high local concentrations
occur in animals immersed in MgCl
2
, it may have anal-
gesic/local anaesthetic effects. Further studies of its
mechanism of action are urgently required.
Ethanol is widely used as an anaesthetic agent in
cephalopods, but it is not clear whether it blocks pain
perception and/or is aversive. Some variability is
reported in the response of animals exposed to it (see
Table 10A), but this could reflect impurities in the dif-
ferent sources of ethanol used
439
and other factors such
as temperature.
For example, ethanol is considered effective in
O. vulgaris, but has been reported to produce inking
and escape reactions,
243
suggesting that it is aversive.
However, these reactions are not noted if the tempera-
ture is below 12C.
448
Moreover, ethanol has been
reported to be ineffective in cold water octopus species,
as cited in Lewbart and Mosley following a personal
communication from I.G. Gleadall.
418
However, this
observation seems contradicted by other personal
experience on Antarctic octopods (F. Mark, pers.
comm.).
Clove oil has been the subject of limited study so it is
difficult to make a judgment about its use especially as
there appear to be marked species differences in the
response (see Table 10D). Further studies with clove
oil, and its active constituent eugenol, are required to
fully assess its utility as an anaesthetic for cephalopods.
Both MgCl
2
and ethanol have been reported to induce
general anaesthesia as defined in section 8.8.2 below to a
level sufficient to perform relatively short duration
(30 min; see Table 10A,B) surgical or invasive proced-
ures (see also Tables 8 and 9). However, it must again be
emphasised that the analgesic, aversive and amnesic effects
of these agents have not been studied in any detail and the
molecular mechanism of their general anaesthetic action in
cephalopods has not been elucidated.
In addition to further research on the agents
themselves, it is also apparent from the limited data
in Table 10 that there is emerging evidence of
46 Laboratory Animals 49(S2)

Table 10A-D. Examples of studies using ethyl alcohol (EtOH), magnesium chloride (MgCl
2
) alone or in combination, or clove oil for induction and maintenance of
general anaesthesia in exemplar cuttlefishes, squid and octopuses. Only publications with primary data are included; for recent reviews of other agents and species
see: Andrews et al.
8
, Gleadall
438
, Sykes et al.
61
Studies utilising the same agents solely for euthanasia are not included.
For each case we report: species, temperature at which the study have been carried out (T), body size (expressed in grams, unless otherwise stated), concentration of
the agent utilised (expressed in %, unless otherwise stated), time to anaesthesia (in seconds), criteria for time to anaesthesia, duration of anaesthesia (in seconds,
unless othewise stated), purpose of the study, recovery time (in seconds or minutes, as required), references and comments, if available. Number of subjects utilised
and their gender are reported, if available from the original work, with body size. Abbreviations: F, females; M, males; NS, not stated; NA, not applicable; DW, distilled
water; SW, seawater. See original works for full description. Note that other criteria for time to anaesthesia are also used to identify onset of general anaesthesia and
are discussed in the text.
Table 10A. Ethanol as an agent.
Species T (C) Body size
Concentration
(%)
Time to
anaesthesia (s)
Criteria for time
to anaesthesia
Duration of
anaesthesia (s)
Purpose of
anaesthesia Recovery time
References and
comments
S. officinalis 19.3 1.4
35.1 8.3
a
42.2 9.4
a
42.7 6.6
a
1.0
2.0
3.0
434.0 192.3
88.3 41.2
73.3 29.1
Body colour,
swimming
behaviour, funnel
suction intensity
b
180
Investigation of
anaesthetics
(no procedures
applied)
64.7 26.2 s
91.2 36.7 s
101.7 49.3 s
440
Aim of the study was
to sedate animals for
handling not for
surgery
S. officinalis NS
NS
M(N ¼7)
F(N ¼6)
2.0 NS
Cessation of arm
movement and no
righting response
NS Branchial heart
injection NS
543,544
S. officinalis 21 DML: 4–8 cm
(N ¼6) 1 NS NS NS Fin dye injection NS
541
S. officinalis 220
F
3% for
1 min then 1.5% NS NS 44 min Mantle granuloma
excision NS
282
S. officinalis 20–22 200–400
(N ¼3) 1.5 NS NS NS Mantle cannulation NS
545
S. officinalis 10–14 DML: 2–5 cm 2 NS NS NS Arm tip removal NS
430
S. officinalis
22–23
DML: 5.2–6.4 cm
(see
c
)0.8–1.0 %
(in gradual
increments)
NS
No response to:
light, pinch by
forceps; pallor
NS Arm tip removal 60–300s
190
S. pharaonis DML: 5.1–5.8 cm
(see
d
)
E. scolopes NS adult 2 600
Cessation of
swimming þunrespon-
sive to touch
(note: ventilation
þchromatophore
activity continues)
NS
Haemolymph
sampling from
cephalic blood
vessel
<30 min
249
(continued)
Fiorito et al. 47

Table 10A. Continued
Species T (C) Body size
Concentration
(%)
Time to
anaesthesia (s)
Criteria for time
to anaesthesia
Duration of
anaesthesia (s)
Purpose of
anaesthesia Recovery time
References and
comments
D. pealeii 14 68.6
e
1
3
696
240
Unresponsive to
handling, immobile,
loss of righting
response
43.1 min
Investigation of
anaesthetics (no
procedures applied)
NS
208
Animals attach to
container, jetting
behaviour, unusual
colour/patterns; 0%
mortality
S. sepioidea NS 42.2–290.9
f
1–3 <120 s
Absence of body
hardness, flexible
tentacles, pallor
90
(handling)
Anaesthetic
efficacy study
4–12 min
(proportional to
exposure time and
concentration)
198
‘no pernicious
side effects’
S. lessoniana NS 3.2–16.9 cm
g
1.0
1.5
2.0
30 s
Immobility þtranspar-
ent with dark band on
mantle and head
120 s
Implantation of
Visible Implant
Fluorescent
Elastomer tags
20–30 s
547
Higher mortality
amongst young
animals with 2%
O. vulgaris NS 200–350
(N ¼15) 1.0–2.0 NS NS NS
Electrode
implantation
in brain
NS
101
O. vulgaris 20–24 673–1369
(N ¼8) 2.5 NS NS NS
Anterior and
posterior basal
lobe removal
NS
156
O. vulgaris 20–22 500–1500
(N ¼6) 2 NS NS NS Mantle cannulation NS
545
O. vulgaris 290–1040 2.5 NS NS NS Branchial vessel
cannulation NS
537
O. vulgaris 15 1000–1500
(N ¼28)
2.5
(in cold
seawater)
NS
Total relaxation and
mantle manipulable
to access branchial
vasculature
NS Branchial vessel
sampling NS
325
O. vulgaris 22–25 150–500
h
2.0 234 33 Cessation of
ventilation Up to 10 min
Branchial heart
injection, brain
lesion, dorsal aorta
catheter implantation,
pallial nerve section
27 13 s
456
(continued)
48 Laboratory Animals 49(S2)

Table 10A. Continued
Species T (C) Body size
Concentration
(%)
Time to
anaesthesia (s)
Criteria for time
to anaesthesia
Duration of
anaesthesia (s)
Purpose of
anaesthesia Recovery time
References and
comments
O. vulgaris 22.30.5 1268291
i
1.0
1.5
2.0
from 240 to 50 Skin pallor NS PIT tag implantation 180–360 s
131
At 1.5% (considered
optimal) showed
correlation between
body weight (700 g-
1130 g) and time (60–
100 sec) to anaesthesia
O. vulgaris 18 500–800
F(N ¼6) 2300 Body pattern
change NS Arm tip amputation 120–300 s
431
O. vulgaris
25–26 730–817
(N ¼2) 1 420–480 Unresponsive to
external stimuli,
loss of righting
reflex, cessation
of ventilation
j
1min
Mixture of
surgical interventions
(not described)
k
180–420 s
439
shallow anaesthesia at
1%; also provides
information on other
octopus species
19–21 150–490
(N ¼7) 2 90–600 14 to 20 min
(and more) 300
21 335
(N ¼1) 3360 >14 min NS
E. cirrhosa 10–11 350 (mean) (N ¼12) 2 NS NS NS Dorsal mantle skin
lesion NS
189
E. cirrhosa 14–15 500–800 2.5 NS NS NS Branchial vessel hae-
molymph sampling NS
255
a
Juveniles (N ¼6 for each concentration).
b
Detailed description of stages of anaesthesia provided in the original study.
c
10–12 weeks post hatch; N ¼9.
d
13–14 weeks post hatch; N ¼4.
e
DML: 18.5 cm (mean of N ¼5).
f
DML: 7.6– 17.1 cm.
g
Young and sub-adult; N ¼46.
h
MþF,N¼10.
i
N¼3 for each concentration.
j
Additional detailed description in the original study.
k
A study of anaesthesia only.
Fiorito et al. 49

Table 10B. Magnesium chloride as an agent.
Species T (C) Body size
Concentration
(% or g/L)
Time
to anaesthesia
(s)
Criteria used for
time to anaesthesia
Duration of
anaesthesia
(s)
Purpose of
anaesthesia
Recovery
time
References and
comments
S. officinalis 15 365–890
a
7.5
b
300–720
Pallor, arm flaccidity,
cessation of ventila-
tion, loss of righting
response, unrespon-
sive to noxious
stimulus
0–25 min Surgery
(no details) 120–1200 s
447
for use to implant fin
EMG electrodes see
548
MgCl
2
reported to be ‘more
reliable’ than EtOH
S. officinalis NS DML: 122–240 mm
(adult animals) 1.9 600
Floating at surface,
skin pallor, cessation
of breathing and
medial fin motion,
unresponsive to gentle
mantle pressure
<5min
Implantation of data
storage tag-includes
skin incision and dril-
ling cuttlebone
20 min
(full recovery)
132
S. officinalis NS
DML: 170–205 mm
(adult animals)
DML: 173–457
132–180 mm
(sub adults)
1.9
3.3
9–19 min
12.1 3.25 min
N¼9
4–8 min
5.9 1.2 min
N¼10
Floating at surface,
skin pallor, cessation
of breathing and
medial fin motion,
unresponsive to gentle
mantle pressure
<5min
Implantation of data
storage tag-includes
skin incision and dril-
ling cuttlebone
NS recovery
time longer for
sub -adults
130
In sub-adults using 1.9%
MgCl
2
time to anaesthesia was
29–58 min (mean
36.7 14.17 min, N ¼4). Note:
the sub-adults were caught in
October but the adults were
caught in May, therefore
season could be a confounding
factor
S. officinalis 19.31.4
48.2 5.1
(N ¼6; juveniles) 20 g/L 468.7 88.9 Body colour,
swimming behaviour,
funnel suction inten-
sity. Detailed descrip-
tion of stages in paper
180
(handling) Investigation of
anaesthetics
(no procedures
applied)
381.2 62.1 s
440
Study aimed to sedate
animals for handling not
for surgery. MgCl
2
considered
the ‘best’ agent of the six
studied
Juveniles
62.9 12.9 (N ¼6) 27g/L 368.7 77.8 353.2 101.7 s
L. forbesi 13 DML: 210 mm
M7.5
b
90
Pallor, arm flaccidity,
cessation of ventila-
tion, loss of righting
response, unrespon-
sive to noxious
stimulus.
300 Anaesthetic efficacy 98 s
447
D. pealeii 14 54.6
c
30.5 g/L
(0.15 mol) 186
Unresponsive to hand-
ling, immobile, loss of
righting response
162.8 min
(max 302 min)
Anaesthetic efficacy
and implantation of
electrodes in statocyst
nerves
NS
208
3.8% mortality
Repeated induction investi-
gated; sedation time increased
with induction number
(continued)
50 Laboratory Animals 49(S2)

Table 10B. Continued
Species T (C) Body size
Concentration
(% or g/L)
Time
to anaesthesia
(s)
Criteria used for
time to anaesthesia
Duration of
anaesthesia
(s)
Purpose of
anaesthesia
Recovery
time
References and
comments
S. sepioidea NS 42.2–290.9
d
1.5–2 300–420
Absence of body hard-
ness, flexible tenta-
cles, pallor
90 handling Anaesthetic efficacy
study
4–18 min
(proportional to
exposure time
and
concentration)
198
Inking and mantle colouration
changes on initial exposure.
Also investigated MgSO
4
at 3–
4% (similar to MgCl
2
)
I. illecebrosus 8–15 300–500 7.5
e
A few minutes Muscle relaxation NS Cannula implantation
in vena cava 300–420 s
417
O. vulgaris 22 120
F7.5%
b
780
Pallor, arm flaccidity,
cessation of breathing,
loss of righting
response, unrespon-
sive to noxious
stimulus.
NS Anaesthetic efficacy 480 s
447
Study also investigated 20%
MgSO
4
reported to be ‘slightly
less effective’
O. vulgaris NS
58–1532
M(N ¼71)
F(N ¼78)
3.5% 900
Lack of spontaneous
movement, complete
relaxation and cessa-
tion of breathing
NS Brain ultrasonography NS
246f
O. vulgaris 17 NS 7.5%
b
NS NS NS Removal of arm
segment NS
549
O. vulgaris NS 500–1000 7.5%
b
NS NS NS Brain lesion NS
539
O. vulgaris NS 158–428
(N ¼17)
3.5%
(in SW) 1200
Lack of spontaneous
movement, complete
relaxation and cessa-
tion of breathing
20 min Removal of distal 10%
of one arm 15–30 min T. Shaw et al.,
pers. comm.
E. cirrhosa
14
15
301 (F)
845 (F)
7.5%
b
1200
900
Pallor, arm flaccidity,
cessation of ventila-
tion, loss of righting
response, unrespon-
sive to noxious stimuli
0min
3min
Anaesthetic efficacy
460 s
420 s
447
a
DML: 145–180 mm, M(N ¼7).
b
Isotonic solution in DW with an equal volume of SW (i.e. 3.75%).
c
DML: 14.8 cm, average of N ¼26.
d
DML: 7.6–17.1 cm.
e
In DW mixed 1:1 with SW; final concentration 3.75%.
f
See also Margheri et al. for similar study of arm ultrasonography.
247
Fiorito et al. 51

Table 10C. Magnesium chloride and ethyl alcohol mixture as agents. In the column ‘Concentration’ values for EtOH and MgCl
2
are indicated with their respective
concentrations.
Species T (C) Body size
Concentration
(EtOH þMgCl
2
)
Time to
anaesthesia (s)
Criteria used
for time to
anaesthesia
Duration of
anaesthesia
(s)
Purpose of
anaesthesia
Recovery
time
References
and comment
S. officinalis NS 200–1200 2% þ17.5 ø80–90 NS NS
Drug microinjection
in brain; electrolytic
lesion
NS
398
Addition of
MgCl
2
is to ‘prevent any
muscular contraction
during surgery’
O. vulgaris 24 0.5
166–1268
M(N ¼20)
89–1256
F(N ¼15)
1% þ55 mM 900 NS NS
Haemolymph sampling;
injection of LPS/PBS
into arm
NS
251
O. vulgaris NS 200–500 1% þ55 mM 25–45 min NS NS Tetanisation
(Vertical lobe)
A few
minutes
See supplementary
data in Shomrat
et al.
420
52 Laboratory Animals 49(S2)

Table 10D. Clove oil as an agent. Clove oil is diluted in SW unless otherwise stated. For the four studies with clove oil it must be noted that only two cite the source.
Species T (C) Body size Concentration
Time to
anaesthesia (s)
Criteria used
for time to
anaesthesia
Duration of
anaesthesia
(s)
Purpose of
anaesthesia
Recovery
time
References
and comment
S. officinalis
a
19.4 1.4 46.5 9.0
31.1 8.4
0.05 ml/L
b
0.15 ml/L
b
Anaesthesia
not achieved
Body colour,
swimming behaviour,
funnel suction
intensity
c
NA
Investigation of
anaesthetics-no
procedures
NA
440
Study aimed to sedate
animals for handling
not for surgery.
Clove oil obtained from
Omya Peralta, Germany
D. pealeii 14 83.5
(DML: 190 mm) 1 ml/L Anaesthesia
not achieved
Unresponsive to
handling,
immobile, loss of
righting response
NA
Anaesthetic efficacy
and implantation of
electrodes in statocyst
nerves
NA
208
Traumatic reaction; died
within 4 min.
Clove oil source NS
O. vulgaris 22.3 0.5 NS
20 mg/L
40 mg/L
100 mg/L
Anaesthesia
not achieved Skin pallor NA PIT tag implantation NA
131
Surgical anaesthesia not
achieved at any concentration
tested; exposure times not
given. Clove oil source NS
O. minor
d
25 NS
50 mg/L
100 mg/L
150 mg/L
200 mg/L
250 mg/L
300 mg/L
559 52.6
317 32.8
277 17.5
265 23.4
240 24.0
230 15.9
Pallor, arm
inactivity, loss
of suction,
cessation of
breathing
NA
Study of
anaesthetic
efficacy
586 53.6 s
620 60.8 s
678 65.2 s
724 60.3 s
797 70.8 s
964 72.5 s
449
Mortality at >550 mg/L
Also studied efficacy at
15 and 20C and showed
that, as temperature
decreased, induction and
recovery times increased.
200 mg/L considered optimal.
Clove oil from Sigma USA
a
Juvenile animals were utilised in this study.
b
N¼6 for each concentration.
c
Detailed description of stages of anaesthesia provided in the original study.
d
Diluted in 95% EtOH and then 1:10 in SW; N ¼10 for each concentration.
Fiorito et al. 53

differences in ‘efficacy’ of these agents according to
age
102
and body weight
131
of animals; and temperature
of the solution.
449
It should also be noted that the majority of system-
atic studies of anaesthesia in cephalopods involve the
commonly used laboratory species, and particular care
should be taken when attempting to anaesthetise other
species, as responses to the same agent may differ
markedly. For example, benzocaine (ethyl p-amino
benzoate) produces a violent reaction followed by
death in the squid D. pealeii,
208
but violent reactions
are not reported in the octopus E. dofleini where it has
been used for euthanasia.
450
8.8.2 Criteria for general anaesthesia. A detailed dis-
cussion of criteria for general anaesthesia in cephalo-
pods, based on a review of recent studies on
cuttlefishes, squid and octopuses is now available.
8,440
They will only be outlined here, and further work is
needed to define species-specific criteria for general
anaesthesia.
Quantification of the physical (externally visible)
parameters listed below might enable assessment of
putative ‘stages’ or ‘planes’ of anaesthesia, and studies
in cuttlefish
440
and octopuses
131,243,449
illustrate this
approach. In particular, ventilation depth and fre-
quency and chromatophore activity are the parameters
most amenable to continuous real time quantification
to monitor the onset of ‘anaesthesia’. Currently there
are no studies of electroencephalogram (EEG) activity
in cephalopods under general anaesthesia, and all the
parameters illustrated below rely on the assumption
that the agents used to induce anaesthesia do not act
only as neuromuscular blocking agents.
(i) Decreased or absent response to a noxious stimu-
lus. This is the most important test that the
animal is sufficiently anaesthetised for surgery
or other procedures to commence. Without
blocking pain perception general anaesthesia
cannot be considered to have been achieved.
Studies have used a mechanical stimulus (e.g. a
pinch) applied to the arm, mantle or supraorbital
skin as a test of insensibility to a noxious stimu-
lus.
132,243,447
The selection of this type of chal-
lenge appears appropriate, as it is now known
that cephalopods possess mechano-sensitive per-
ipheral nociceptors,
10,11
and in future this know-
ledge will enable the identification of better-
defined stimuli to test for insensibility.
Assuming that there is evidence to suggest that
the animal is insensible, the following criteria
should also be evaluated.
(ii) Depression of ventilation. The initial reaction to
exposure to the anaesthetic may be an increase
in depth and frequency of mantle contractions
with attempts to eject the solution via the
siphon. Subsequently the frequency decreases
progressively with time after exposure, and the
coordination between the mantle and siphon
become uncoordinated. With prolonged expos-
ure, mantle and siphon contractions cease.
(iii) Decreased chromatophore tone. Although animals
become pale overall with increasing time of
exposure to the anaesthetic, flashing colour
changes have been reported on initial exposure
to anaesthetic agents (e.g. in D. pealeii
208
or in
S. officinalis
440
). The overall paling of the
animal is indicative of a decrease in the central
nervous system drive to the chromatophore
motorneurones.
201
(iv) Decreased locomotor activity, arm/tentacle tone
and sucker adhesiveness. The initial reaction to
anaesthetic exposure may be an increase in activ-
ity (i.e. agitation, for review see Gleadall
439
), but
activity gradually decreases, including swimming
activity and fin movement, as for example in
cuttlefish and squid.
132,451
Octopuses will tend
to settle on the bottom of the tank as the arms
and suckers begin to lose tone and adhesion, but
anaesthetised cuttlefish may float near the surface
of the tank.
132
The arms should be flaccid and
readily manipulated.
449
(v) Loss of normal posture and righting response.As
animals (e.g. squid and cuttlefish) become deeply
‘anaesthetised’ they lose the ability to maintain a
normal position in the water column or adopt an
abnormal position with the arms, head and
mantle at angles not normally seen in conscious
animals. For example, in squid and cuttlefish the
arms and head may appear unsupported by the
mantle collar muscles; octopuses adopt a flat-
tened appearance on the floor of the tank rather
than the usual posture with the head raised. In
addition, animals placed on their dorsal surface
make no attempt to right themselves. The right-
ing response returns after ventilation and chro-
matophore activity return,
243
and this is
probably a good indicator of overall recovery
from anaesthesia, as both effects require complex
coordination of neuromuscular activity.
(vi) Absence of a response to light. The absence of a
reaction to a strong light has been used as one
sign of general anaesthesia (e.g. in Messenger
et al.
447
), but this is poorly characterised.
8.8.3 Induction. A common practice for induction is to
immerse the animal in the anaesthetic solution (made
up in seawater) at its final concentration. However, to
54 Laboratory Animals 49(S2)

minimise trauma it is preferable to expose the animal to
a rising concentration of the agent (as for example in
Yacob et al.
427
), which will also allow any adverse reac-
tion to be quickly identified.
The animal should remain completely immersed in
the anaesthetic solution for rapid effect. In addition,
the use of a specialised closed anaesthetic chamber
should be considered. The chamber could also be used
as a transport box from the home tank to the operating
room, and it may be possible to habituate at least some
species (e.g. octopus and cuttlefish) to the box and train
them to enter. This will reduce stress to the animals, and
a closed chamber will prevent octopus escaping. As a
general rule, animals should always be transported in
seawater and movement should be minimised.
Anaesthetic solutions should always be freshly
made, using filtered seawater, which is gassed (prefer-
ably with oxygen rather than air) and equilibrated to
home tank temperature before immersing the animal.
It is not good practice to anaesthetise an animal in a
solution that has been used to anaesthetise another
animal, as the water may contain chemical alarm sig-
nals. Animals must not be left immersed in an anaes-
thetic solution in which they have inked.
Although limited in scope, the pre-anaesthetic sed-
ation technique applied by Packard
200
has not been
followed by any other systematic study of methods to
minimise the stress of general anaesthesia in cephalo-
pods, as has been done for fishes.
452
Moreover, there is no general agreement about
whether cephalopods should be deprived of food
prior to anaesthesia. Some studies remove food for 24
hours
440
while others do not.
208
We are aware of only one report of food regurgita-
tion by S. sepiodea during ‘anaesthesia’ in magnesium
sulphate,
198
although defaecation is relatively common.
In view of the above, the anaesthetic protocol for
cephalopods requires careful planning, including con-
sidering whether or not to withdraw food and, if so, for
how long.
8.8.4 Maintenance and monitoring. Once the animal is
fully anaesthetised (see above for criteria), it will usu-
ally be necessary to remove it from the anaesthetic
chamber to perform a procedure. Anaesthesia must
be maintained for the entire duration of the procedure,
and physiological functions supported.
282,453,454
Several authors describe apparatus (adapted from
fish anaesthetic apparatus) for maintenance of anaes-
thesia during surgery (e.g. for cuttlefish:
132,418,454
for
squid:
417,451
), and these could also be better tested,
and also adapted for octopus.
Marked suppression or cessation of ventilation
(indicated by mantle/siphon contraction) is a common
feature of general anaesthesia in cephalopods, so it is
essential that the mantle is perfused with oxygenated sea-
water/anaesthetic. Such perfusion will only be effective if
the branchial and systemic hearts continue to function,
but little is known of cardiac function under anaesthesia.
However, it has been observed that heart rate is very low
in O. vulgaris anaesthetised with MgCl
2
(M.G. Valentino
and P.L.R. Andrews, unpublished observations) and in
animals immersed in cold water.
243
Monitoring of physiological function under anaes-
thesia and during surgery is clearly an area requiring
research so that the extent of hypoxia/hypercapnia is
known and its impact on post-operative recovery and
procedures can be assessed. However, O. vulgaris
appears to be able to recover rapidly from protracted
periods of apnea
150
and Po
¨rtner et al.
417
comment that
cuttlefish and octopus are less sensitive to hypoxia
than squid (as might be expected from their different
lifestyles). Doppler ultrasound (e.g. as in D. Fuchs and
G. Ponte, unpublished observations; Vevo 2100
Visualsonics, The Netherlands) offers the best tech-
nique for monitoring cardiovascular function, but
non-invasive methods for real time monitoring of
blood gases (such as oximetry) and metabolic status
(e.g. NMR spectroscopy as in Melzner et al.
455
) need
further development.
8.8.5 Recovery. The mantle should be flushed of resi-
dual anaesthetic solution and the animal then placed in
clean aerated/oxygenated seawater. Ventilation can
often start without intervention (depending upon the
duration of anaesthesia), but gentle massage of the
mantle is frequently used in cuttlefish, squid and octo-
pods until spontaneous ventilation, as indicated by
mantle and siphon movements, restarts. Other func-
tions (sucker adhesion, chromatophore tone, righting)
recover after ventilation recommences, usually in the
reverse order to which they were lost.
Many studies monitor the time at which various
functions return, but the time taken for full recovery
of normal function from particular anaesthetic proto-
cols is not known and, as most studies of anaesthesia do
not involve surgery, the impact of surgery upon recov-
ery is not known, but see Shomrat et al.
420
As currently
assessed, recovery appears rapid (less than 15 mins, e.g.
in
243,420,440
) and dependent on the procedure(s) per-
formed, animals will usually take food quickly after
‘anaesthesia’ when returned to their home tank.
395,420
However, further monitoring criteria are needed to
ensure that animals have fully recovered from the
anaesthesia and any surgical procedure. If recovery
from anaesthesia is as rapid as appears to be, and the
anaesthetic agents used do not have residual analgesic
properties, it is vital that suitable analgesics are admin-
istered – which at this time of writing may best be done
Fiorito et al. 55

by infiltrating surgical sites with local anaesthetic (see
next section below).
8.9 Analgesia and local anaesthesia
Directive 2010/63/EU, Article 14§4, requires ‘an animal
which may suffer pain once general anaesthesia has worn
off, shall be treated with pre-emptive and post-operative
analgesics or other appropriate pain-relieving methods,
provided that it is compatible with the procedure’.
At the time of writing, there is no information on
the efficacy of any analgesics in cephalopods, although
both ketoprofen and butorphanol have been
recommended.
445
As nociceptors have been recently studied in
cephalopods,
10,11
it should be possible to investigate
the efficacy of systemically administered substances
for potential analgesic activity. In addition to identifi-
cation of mechano-nociceptors, Crook et al. also
showed that injury to a fin in squid induced spontan-
eous activity and sensitisation at sites distant from the
lesion including the contralateral body;
11
similar sensi-
tisation of both the wound site and at distant sites has
been reported in octopus.
10
This implies that poten-
tially surgery at any surface site (but possibly anywhere
including the viscera) could evoke a more general sen-
sitisation of nociceptors. If this is the case, it is essential
that suitable analgesic agents are quickly identified.
In the absence of the availability of systemic
analgesics, it is recommended that local anaesthetics
are used to produce localised analgesia either by infiltra-
tion into a wound site or local nerve block. Xylocaine
(2%) and mepivacaine (3%) have both been shown to be
effective in producing a block of transmission in the arm
nerve cord lasting at least 1 hour (G. Ponte, M.G.
Valentino and P.L.R. Andrews, unpub. obs.).
149,456
It
should be noted that local anaesthetics acting on the
fast tetrodotoxin (TTX) sensitive voltage gated sodium
channels may not be effective in species such as the blue-
ringed octopus which possesses endogenous TTX.
457
Selective nerve block with infiltration of a local
anaesthetic should also provide an interim means of
preventing more generalised nociceptor sensitisation
(see above), as it has been demonstrated that afferent
nerve block by injected isotonic MgCl
2
prevented both
local and distant sensitisation.
11
8.10 Fate of animals at the end of a
procedure
At the end of a procedure as defined in section 8.1
above, a decision about the fate of the animal is
required. The Directive identifies three possibilities:
Humane killing: Article 17 requires that ‘at the end of a
procedure’ an animal must ‘be killed when it is likely to
remain in moderate or severe pain, suffering, distress or
lasting harm’. Other animals should also be humanely
killed at the end of procedures, unless ‘a decision to keep
an animal alive’ has been taken ‘by a veterinarian or
another competent person’, when the possibilities
described below should be considered. Methods of huma-
nely killing cephalopods are discussed in section 8.11
below.
Release or re-homing: An animal ‘may be... returned
to a suitable habitat or husbandry system appropriate to
the species’, provided that: ‘its state of health allows it;
there is no danger to public health, animal health or the
natural environment; and appropriate measures have
been taken to safeguard [its] well-being’ (Article 19).
This could include release to the animals’ natural envir-
onment, transfer to public aquaria, educational or other
competent holding facilities. However, in general, ceph-
alopods should not be returned to the wild, except in
studies where, following a procedures, animals are
released immediately at the exact location where they
were captured, during a relevant season for migratory
species, having been certified fit by a veterinarian or
other competent person (Articles 17§2 and 19). Any
requirements of other national and international legisla-
tion regarding the release of animals to the wild must also
be met.
Re-use: Animals may also be considered for re-use,
provided certain conditions are met.
Article 16 defines re-use as ‘use of an animal already
used in one or more procedures, when a different
animal on which no procedure has previously been car-
ried out could also be used. By definition, re-use applies
to situations in which the objectives of the first and
second procedures are unrelated.
yyy
Reuse is only permitted when:
.‘the actual severity of the previous procedure(s) was
‘mild’ or ‘moderate’;
.the animal’s ‘health and wellbeing has been fully
restored;
.the further procedure is classified as ‘mild’, ‘moder-
ate’ or ‘non-recovery’ (see section 8.1); and
.the proposed re-use ‘is in accordance with veterinary
advice, taking into account the lifetime experience of
the animal’.
zzz
yyy
This distinguishes reuse from situations where the scientific object-
ive can only be achieved by using the same animal in more than one
procedure (this is known as continuing use, although the term is not
used in the Directive).
zzz
For further discussion and examples, see pp. 8–10 in EU National
Competent Authorities endorsed document (2011), available at:
http://ec.europa.eu/environment/chemicals/lab_animals/
interpretation_en.htm
56 Laboratory Animals 49(S2)

8.11 Methods of humane killing
The ultimate fate of cephalopods in the majority of
studies will be humane killing. All personnel involved
in humane killing should be trained and be familiar
with the principles of good practice, such as those set
out by Demers et al.
458
Article 6 requires that, whenever animals are killed:
.it should done with minimum pain, suffering and
distress by a competent person; and
.one of the methods of killing listed in Annex IV
(section 3) of the Directive should be used, followed
by confirmation of death, using one of the methods
listed in Annex IV section 2.
However, whilst methods listed for fish might be
applied to cephalopods, Annex IV offers no specific
guidance on methods for humanely killing cephalo-
pods. Although the CCAC
459
take the view that the
priority is a rapid loss of consciousness, these guidelines
concur with the view of Hawkins et al. from a discus-
sion of CO
2
killing in vertebrates that ‘it is more
important to avoid or minimise pain and distress than
it is to ensure rapid loss of consciousness’ (p. 2).
460
We have described some possible methods below,
but the efficacy, and level and nature of any suffering
caused, have not been comprehensively evaluated for
all of these techniques, and further research is needed.
Animals should not be killed in the rooms used to
house other animals nor within sight of conspecifics.
It must also be ensured that blood or chemical alarm
signals cannot be detected by other animals (e.g. by
entering the water system). Whichever method is used
in a given species, the potential impacts of factors such
as body weight, age, sex, season and water temperature
on the efficacy of the method must be considered.
8.11.1 Chemical methods. Chemical methods for
killing cephalopods are based on an overdose of anaes-
thetic agents, by using either a higher concentration and/
or longer exposure time than that needed for anaesthesia.
As also outlined above, these methods all have the poten-
tial to cause adverse effects prior to unconsciousness, such
as skin or eye irritation, or sensation of asphyxia. To
reduce and avoid any such suffering, animals should be
exposed to a gradually rising concentration of the anaes-
thetic, and not directly immersed in a solution at the full
concentration needed to cause death. In tropical and tem-
perate species, cooling may be used as an adjuvant to the
anaesthetic for humane reasons, and could also reduce
post-mortem tissue damage if tissue is required for
in vitro studies.
A previous review of this topic proposed the following
protocol as suitable for S. officinalis,D. pealei, O. vulgaris
and E. cirrhosa: ‘At least 15 minutes immersion in MgCl
2
,
with a rising concentration [optimal rates to be deter-
mined], ending with a final concentration of at least
3.5% in the chamber used for humane killing; possibly
enhanced by using chilled solutions or with the clove oil’s
active ingredient eugenol, followed by immediate mech-
anical destruction of the brain’ (Andrews et al., p. 61).
8
It was further proposed that if the brain was needed,
the immersion period should be extended to more than 30
minutes to ensure unconsciousness as required in
Directive 2010/63/EU (Annex IV, 1(a)) prior to removal
of the brain. If the brain is not removed for study, con-
firmation of permanent cessation of circulation (see 8.12
below) is also considered as a possible method for com-
pleting killing according to Directive 2010/63/EU (Annex
IV, 2(a)).
8.11.2 Mechanical methods. When carried out by
highly skilled operators, death by mechanical destruction
of the brain takes only a few seconds, but the nature and
degree of any suffering is unknown. For this reason we
take the view that cephalopods should not be killed by
this, or any other, mechanical method without prior sed-
ation/anaesthesia. However, it may be possible to utilise a
‘mechanical method’ if it can be justified and is authorised
by the National Competent Authority as a specific regu-
lated procedure within a project application.
Electrical methods such as ‘Crustastun’ used for
humanely killing crustacea, such as lobsters and
crabs,
461
might also be considered and evaluated for
their suitability in terms of animal welfare. It will be
particularly challenging to develop humane methods
for use when the brain is required intact, but where
the use of anaesthesia may be a confounding factor.
8.12 Confirmation of death
Use of a method for confirming death following
humane killing is mandatory, and options are listed in
Annex IV§2 of the Directive. Two of the methods listed,
i.e. ‘dislocation of the neck’ and ‘confirmation of onset
of rigor mortis’ are impossible for cephalopods – the
latter because it does not occur in cephalopods.
This leaves three possible methods: i. confirmation
of permanent cessation of the circulation; ii. destruction
of the brain; or iii. exsanguination.
Confirmation of permanent cessation of the circulation
and exsanguination. Octopuses, cuttlefish and squid have
two branchial hearts that move blood through the capil-
laries of the gills.
462
A single systemic heart (the only one
in nautiloids) then pumps the oxygenated blood through
the rest of the body. The heart(s) may continue beating
for some time after permanent cessation of breathing, so
transection of the dorsal aorta/vena cava may be used.
Transection of the dorsal aorta/vena cava will be effective
in inducing exsanguination if the systemic heart is able to
Fiorito et al. 57

pump effectively (i.e. the anaesthetic used does not
supress cardiac function); also note that the systemic
heart is distension sensitive.
463
Finally, the possibility of
transection of the branchial aorta afferent to the heart, at
the level of the auricle, should be further explored con-
sidering the easy access to them through the mantle cavity
nearby the gills (G. Ponte and G. Fiorito, pers. comm.).
The effectiveness of exsanguination as a method of
killing is not known.
Freezing (below 18C for several hours) after kill-
ing may be a further means of confirming cessation of
circulation and hence death that does not necessarily
entail destruction of the body.
Destruction of the brain may be difficult to ensure in
some species because of the location and relatively
small size (e.g. Nautilus sp.) although this can be over-
come by training and a detailed knowledge of the
cranial anatomy of the relevant species.
The methods used for humane killing and confirm-
ation of death should always be included in publications.
Article 18 of the Directive requires member states ‘to
facilitate the sharing of organs and tissues of animals
killed’ where appropriate. Researchers should be encour-
aged to use tissue from animals killed in other projects
for in vitro research (e.g. tissue bath pharmacology),
rather than killing an animal specifically/only to obtain
tissues; and consideration should be given to setting up
banks of frozen and fixed tissue to optimise animal use.
9. Risk assessment for operators
This section will focus on the potential risks from the
direct handling of cephalopods in a laboratory setting,
but will not cover the more generic risks associated with
working in either a laboratory (e.g. tissue fixatives,
reagents) or a marine aquarium environment (e.g.
tank cleaning agents, slipping, electricity in a wet envir-
onment). However, all personnel involved in research
should be appropriately educated, trained and compe-
tent to perform any task relevant to the research.
Personnel should be actively involved in risk assessment
and management, and incident reporting encouraged.
9.1 Personnel to consider
The following personnel should be considered and their
risks assessed:
a. Fishermen, divers or others responsible for the cap-
ture of cephalopods in the wild. Although such
people may not be employees of the institution/facil-
ity, as far as possible, the institution/facility should
be assured that safe practices are being employed.
The Directive specifies that, in cases where justifica-
tion is provided, to obtain animals from the wild
‘competent persons’ (Article 9) should be involved.
As part of competency assessment issues related to
health and safety practices of cephalopod capture
should be explored.
b. Animal carers and technicians. This includes any-
one involved in cleaning and feeding animals;
cleaning animal rooms and equipment (e.g. tanks,
filters).
c. Animal technologists or research laboratory techni-
cians. These are personnel who may be involved in
manipulating animals during experiments, sampling
biological fluids, euthanasia and necropsies.
d. Principal and other Investigators: All personnel
involved in performing research experiments includ-
ing in vivo regulated procedures and in vitro hand-
ling of live tissue.
e. Designated veterinarian (or other suitably qualified
expert).
f. Personnel responsible for the disposal of animal
remains.
9.2 Risk identification, prevention and
protection
9.2.1 Physical risk. Bites of cephalopods are produced
by the hard beak-like jaws associated with powerful
musculature of the buccal mass located at the center
of the arm crown. Such bites do not always penetrate
the skin of human beings (e.g. see p. 68 in Wells).
150
The effects of penetrating bites are exacerbated by
enzymes, venoms, other bioactive substances and
microorganisms in the saliva. The possibility of injury
should not be overlooked even when animals are trans-
ported in a plastic bag.
464
The arms of all cephalopods are relatively strong, and
this is especially the case in larger octopuses where the
grip is enhanced by the numerous suckers. Areas of ery-
thema may be induced if attempts are made to detach an
animal by pulling in air, rather than allowing it to leave
the arm naturally under water. Special precautions
should be used if handling members of the teuthoid
family as their arms/tentacles have hook-like appendages.
Staff should be trained in how to remove animals that
have become attached to their arms using the minimum
of force and without inducing the animal to bite.
The use of Personal Protective Equipment (PPE),
such as gloves or gauntlets resistant to penetration,
may be suggested, but care should be taken to ensure
that the gloves do not harm the delicate skin of ceph-
alopods and that safe handling is not impaired by wear-
ing gloves. In addition, the typical behaviour of the
animals should be well recognised and mostly for
signs of imminent aggression, escape attempt and
other putative abnormal behaviours.
58 Laboratory Animals 49(S2)

9.2.2 Chemical risk
Direct contact with mucus, faeces and biological
fluids. Biological materials can represent a risk of allergy,
intolerance and/or toxicity particularly with repeated
exposure. Exposure to mucus, faeces and ink may occur
during routine cleaning and handling and other fluids
during autopsy. Faeces and blood (haemolymph) are
also potential routes of infection. We are not aware of
reports of reactions to these biological fluids, but in
case of doubt the use of waterproof gloves is recommended;
in addition, in case of inadvertent contact with any bio-
logical substances, hands should be properly washed
immediately.
Handlers and those undertaking autopsies should
also be aware of any experimental procedures
previously undertaken involving the administration of
potentially hazardous substances (e.g. infectious agents,
radioactive material, drugs, nanoparticles) to the ani-
mals so that assessment of potential risks can be
undertaken.
Venom, enzymes and other pharmacologically
active substances. The secretion from the posterior sal-
ivary glands of coleoid cephalopods (see Table 4.1 in
Wells
150
) is injected into prey via the salivary papilla to
immobilise and digest it with a mixture of venoms (i.e.
cephalotoxin
465
), enzymes (e.g. chitinase, carboxypepti-
dase, hyaluronidase, phospholipase A2) and other bio-
logically active substances (e.g. 5-hydroxytryptamine,
dopamine, substance P) as reviewed by Ruder et al.
466
A localised or systemic response could be induced by
one or more of these substances particularly in sensitive
individuals, but documented examples of systemic reac-
tions to bites are rare except in the case of the blue-
ringed octopus.
In fact, the venom of blue-ringed octopuses
(Hapalochlaena spp.) can be fatal
467
unless there is
prompt medical attention.
467,468
The toxin involved is
the potent sodium channel blocker TTX
469
that is
found in the posterior salivary glands, skin, branchial
hearts, gills and Needham’s sac,
470
so care should also
be taken with handling these animals post mortem.
Recently, TTX has been found in the eggs with the
levels increasing after laying;
471
therefore, the risk with
this species does not only come from adults. Other data
show that the venom is produced by symbiotic bacteria
(Aeromonas,Bacillus,Pseudomonas and Vibrio) found
in the salivary glands.
472
Clinically, the bite of the blue-
ringed octopus is most often painless but freely bleed-
ing. Erythema and edema at the bite site usually occurs,
but the most important effects are those that are sys-
temic. Most severe envenomations are characterised by
generalised weakness, slurred speech, circumoral par-
aesthesias, respiratory difEculty and dysphagia. Such
symptoms may last for 12–24 hours. There is no anti-
venom, therefore treatment includes pressure immobil-
isation and immediate transport to a medical facility
while monitoring respiratory and neurologic status.
473
No reactions specifically attributable to the venoms
from other species of cephalopod have been described,
but there are several reports of localized reactions to
bites from cephalopods although these seem rare. Wells
reports having been bitten himself 10 or 20 times
without event, but also describes a reaction to an O.
vulgaris bite on the forearm in a student who had
previously never been exposed to cephalopods.
150
The response included swelling of the forearm (com-
parable to a bee sting) and overnight pain, both of
which resolved the next day. A local skin reaction
probably related to proteolytic activity has been
described after red octopus (O. rubescens) bite.
474
Haemolytic activity against mammalian red cells has
been reported in vitro with low concentrations of
saliva from E. cirrhosa,
475
but we are not aware of
any evidence for haemolysis in vivo, although caution
should be exerted.
Reactions to a bite will depend upon the sensitivity
of the individual and this may be a particular issue with
atopic individuals or those who have become sensitised
by repeated exposure. The reaction to a bite may not be
immediate as indicated by a case report of giant cell-
rich granulomatous dermatitis/panniculitis one month
after a bite from an octopus (species not given) on the
wrist.
476
Bites are also a source of infection (see below)
and again the reaction may be delayed.
Precautions should be the same as for the protection
against bites (see above). All occurrences of bites by
cephalopods should be recorded in the laboratory
safety book, medical/paramedical advice sought parti-
cularly in cases where the skin is broken and the clinical
outcome monitored. Anderson et al. reported anec-
dotally that immediate hot water treatment was effect-
ive in neutralising the localised effects of the bite of O.
rubescens.
474
Seawater and ink. Cephalopods in general and
octopuses in particular can forcibly squirt seawater
and/or ink directly at handlers. In addition to melanin,
ink contains an array of bioactive substances including
enzymes (e.g. tyrosinase, tyrosine hydroxylase) and
other chemicals (e.g. dopamine, 5,6-dihydroxyin-
dole).
477,478
There is a theoretical risk that the seawater
may be contaminated by pollutants or infectious agents
in open systems but seawater or ink in the eye could
cause irritation and also distract the handler from their
task increasing the risk of a bite or animal escape.
Wearing eye or face protection should be considered
when handling cephalopods.
Fiorito et al. 59

9.2.3 Biohazards
The animals. Animals, especially those taken from
the wild, can transmit infectious agents (zoonoses) to
humans and as cephalopods host a number of bacteria
(Gram þand ), viruses and parasites; this is a potential
risk for anyone in contact with cephalopods. The major
risk of transmission is via accidental ingestion or a bite.
This section will not cover the potential pathogen-
icity of different agents, however, mention should be
given to, for example, Anisakis and Aggregata that
are known to be the cause of the zoonotic disease, as
reviewed by Yang et al.
479
Most information regarding infection with Anisakis
comes from human consumption of uncooked cephalo-
pods, but poor hand hygiene and laboratory practice –
especially when undertaking autopsies of fresh animals –
means that hand-to-mouth transmission is a possibility.
An additional potential source of parasites (and possibly
bacteria, viruses and toxins) is the fish, crustacea and
molluscs used as food for cephalopods so wearing
gloves and hand hygiene should also be considered.
The main documented risk is bacterial infection
from bites although the bacteria may originate from
contaminated water as well as the animal itself.
480–482
Particular attention should be paid when handling
cephalopods with skin lesions as these wounds are often
infected (see section 4.4).
Seawater. Seawater itself is a potential reservoir and
transmission vehicle for infectious organisms, chemical
pollutants and toxins (e.g. from algae) particularly in
open systems. In seawater, many pathogens can be respon-
sible for infections, e.g. Staphylococcus aureus,
Streptococcus pyogenes,Mycobacterium marinum,Vibrio
vulniOcus,Erysipelothrix rhusiopathiae,Aeromonas hydro-
phila,Pseudomonas aeruginosa,Prototheca wickerhamii.
473
These represent a potential hazard for both animals
and humans who have contact with the seawater but
the risk should be minimal if the water quality is moni-
tored and maintained within strict limits. Additional
safety precautions may need to be put in place if
there is a breakdown in water quality management
especially for those who may need to decontaminate
and clean the tanks.
Allergens. Repeated exposure to animals, chemicals
(e.g. some disinfectants) and some disposables can
result in the appearance of several clinical forms of
allergic reaction (e.g. contact dermatitis or urticaria).
To minimise the allergy risk operators should have a
medical assessment before starting work and at regular
intervals. Cephalopod mucus can be an irritant and has
been occasionally described as an allergen (A. Affuso,
pers. comm.). Since there are cases of an acquired
allergy to cephalopod eggs, it is recommended that
they are handled wearing gloves. Arginine kinase from
Octopus fangsiao has been shown to react with IgE in
the serum of octopus-allergenic subjects,
483
emphasising
the importance of identifying individuals who may be
especially sensitive to cephalopods before they begin
work so that the risk can be assessed and managed.
9.3 A summary of practical advice
9.3.1 Assess and manage the potential risks. Each
person involved with cephalopods should be assessed,
and monitored for potential risks, notes taken of the
species involved and the work to be undertaken before
any work assigned. For example, the risks in moving a
potentially lethal blue–ringed octopus or a 30 kg E.
dofleini between tanks are different from moving a
200 g cuttlefish. Protocols of good laboratory practice
(GLP) – Standard Operating Procedures (SOPs),
should be developed in conjunction with the Health
and Safety officer or other person, incorporated into
training programmes (see section 9) and common
protocols (e.g. handling, cleaning, humane killing) dis-
played or kept in a file in the facility and in the room
dedicated to procedures. Risk assessment will also need
to comply with local ‘out of hours’ policies. All working
areas should be equipped with required PPE (overalls,
hypoallergenic gloves, safety glasses, eye wash) and
have a telephone with an emergency number displayed.
9.3.2 Operator health. Most staff undergo some form
of health assessment at the beginning of employment.
Anyone working with cephalopods should be asked if
they are allergic to these animals or if they are atopic so
that risks can be managed. All staff with regular expos-
ure to cephalopods should be monitored regularly for
signs of sensitisation.
9.3.3 Recording incidents. All incidents (including
‘near misses’) should be recorded partly to facilitate
development of improved protocols. For example,
skin-penetrating bites must be recorded and reported
immediately to the relevant person so that appropriate
action can be taken and any delayed reactions (e.g.
infections) documented.
The ‘incident book’ should be reviewed regularly
and protocols and policies modified as required.
Lessons for the wider cephalopod community should
be posted on the CephRes web site (www.cephalopo
dresearch.org) and if possible published.
9.3.4 Dealing with incidents. Staff should be familiar
from training and laboratory SOPs/GLP with action to
be taken in particular circumstances. The most likely
incident requiring immediate action is a bite in which
the skin is broken and the wound infiltrated with
60 Laboratory Animals 49(S2)

secretions from the posterior salivary glands and/or
seawater. A protocol for this and other eventualities
should be drawn up in consultation with the trained
on-site first responder and a medical practitioner.
10. Education and training: carers,
researchers and veterinarians
The Directive requires that all staff involved in
regulated research are adequately educated and trained
for tasks they are required to perform (Article 23)
including: i. carrying out procedures on animals; ii.
designing procedures and projects; iii. taking care of
animals; or iv. humane killing of animals.
Education and training therefore includes all staff
involved in daily care (including veterinarians; since
courses on marine invertebrate medicine are rare),
researchers performing procedure and principal
investigators designing studies. Anyone having direct
contact with the animals will need to be able to dem-
onstrate that they are practically competent in perform-
ing tasks or regulated procedures and have this
competence assessed periodically. There may also be
an argument for providing limited theoretical training
for members of an institutional project/ethical review
committee about novel species.
Trained individuals should understand and be able
to demonstrate the importance of the animal welfare
regulations and guidelines for housing, care and use,
assessment of animal welfare including signs of illness,
PSDLH and their palliation or treatment.
Developing training programmes to meet these require-
ments is a particular challenge for cephalopods as although
knowledge of general care and welfare is relatively well
developed (as reviewed in Fiorito et al.
2
), knowledge of
PSDLH and their application to assessment of animal wel-
fare aspects of a project are less well established.
3,8
In add-
ition, there are multiple species of cephalopod with a
varietyofcare(seealsoAppendix2)andwelfarerequire-
ments. Many aspects of training will need to be delivered by
animal technologists, researchers and veterinarians who
have gained experience prior to the regulation of research
involving cephalopods, but it would be desirable to involve
some trainers with expertise in working with aquatic ver-
tebrates (including expertise in ethical review).
Courses will need to align with specific national
requirements, and will need to be recognised by the
National Competent Authority (http://ec.europa.eu/
environment/chemicals/lab_animals/ms_en.htm) of
Member States as fulfilling the requirements of the
Directive 2010/63/EU for all persons involved in the
use, care and breeding of cephalopods for scientific pro-
cedures. Recognition at EU level will facilitate move-
ment of personnel between member states. The content
and delivery of the modules should be validated by a
university or other competent awarding body, and
linked to Continuing Professional Development
(CPD) programmes of professional bodies.
Training modules should be designed taking into
account of the EU Commission working document of
a development of a common education and training
framework to fulfil the requirements under the Directive.
Ideally courses should be offered at EU level but as
many aspects of training are ‘hands-on’ courses will need
to be based in facilities with aquaria and access to several
species. It is proposed that a training course should be
structured into three modules; basic training, a species-
specific module, procedures, PSDLH assessment and
management module. These modules cover the key wel-
fare assessment competencies and welfare training topics
outlined in Tables 12 and 13 of Hawkins et al.
484
Delivery and assessment of modules are not dis-
cussed here.
10.1 Indicative content of modules
10.1.1 Basic module: an introduction to cephalopods
in research. This should cover: i. national and EU
legislation on protection of animals used for scientific
purposes; ii. a brief introduction to cephalopod biol-
ogy; iii. why cephalopods were included in the
Directive; iv. the philosophy of the institution regarding
animal care and use; v. the requirement to comply with
all national regulations and institutional guidelines; vi.
the key differences between undertaking research in a
legally regulated and an unregulated environment; vii.
record keeping; viii. the requirement to respond imme-
diately to any PSDLH issues; ix. reporting animal care
and use concerns at institutional and national levels; x.
health, safety, risk assessment and security; xi. roles of
the institution and project (‘ethical’) review committee,
veterinarian (or other competent expert), animal
care, and research staff in the animal care and use pro-
gramme; xii. public engagement.
10.1.2 Species-specific module. This focuses on the
species utilised for research in a particular institution
and should include the following species-specific topics:
i. biology and behaviour; ii. supply, capture and
transportation (including any additional permits and
regulations); iii. environment (tanks, water, enrich-
ment) and control; iv. signs of health, welfare and dis-
ease; v. assessing when an animal should be killed
humanely; vi. specialised techniques for identification
of individuals; vii. anaesthesia and humane killing;
viii. tagging and marking; ix. genotyping; x. anal-
gesia/anaesthesia/euthanasia/confirmation of death.
10.1.3 Procedures, PSDLH assessment and manage-
ment module. This module has three main elements:
Fiorito et al. 61

a) Project/licence and ethical committee applications.
This will cover: i. experimental design from a 3Rs
perspective (see section 2.2.2; see also Smith et al.
3
);
ii. principles of harm-benefit analysis (e.g. Bateson
cube); iii. principles of severity assessment (prospect-
ive, actual and retrospective); iv. setting humane end-
points; v. writing a lay summary of the project/ethical
application; vi. public engagement.
b) Recognising PSDLH and their management. This
component deals with these aspects in depth as
this module is intended for those designing projects
or performing procedures some of which may never
have been performed in a cephalopod previously.
The following topics will be covered: i. evidence
for the capacity of cephalopods to experience
PSDLH (see review and discussion in Andrews
et al.
8
); ii. recognising PSDLH and techniques to
minimise and treat them in the context of a regu-
lated procedure; iii. special considerations regarding
senescent cephalopods.
c) Procedures. This covers: i. what is a procedure
within the meaning of the Directive?; ii. an introduc-
tion to basic surgical techniques and post-operative
monitoring and care; iii. non-surgical procedures, tech-
niques and assessing their impact on the animal.
d) Reporting studies under the Directive. Annual
statistical return (Article 54) and an introduction
to the ARRIVE Guidelines (www.nc3rs.uk/
ARRIVE;
272
) and the Gold Standard Publication
Checklist.
485,486
Education and training of all personnel involved in
the research programme at whatever level is essential to
ensure the optimal care and welfare of animals and for
the standards to improve with time by identification
and dissemination of examples of good practice.
These Guidelines and recent publications
1,2,487
establish
the core material required for the delivery of the mod-
ules outlined here as a basis for education and training
of personnel involved in research now regulated by the
Directive. The next step will be to develop the above
outlines into a document that can be used for accredit-
ation of a course compliant with FELASA recommen-
dations for laboratory animal science education and
training, as outlined by Nevalainen et al. and recently
updated.
488,489
11. Concluding comment
This paper represents the first attempt by members of
the international cephalopod community to develop
guidelines for Care and Welfare of cephalopods utilised
in scientific research. Although the guidelines primarily
address the requirements of Directive 2010/63/EU, we
anticipate they will also be utilised by the wider ceph-
alopod research community outside the EU. It is recog-
nised that in contrast to equivalent guidelines for
vertebrates the evidence base for some aspects of
these guidelines is not strong.
It is hoped that this paper will prompt research dir-
ected specifically at the Care and Welfare of cephalo-
pods in the laboratory to provide a solid evidence base
for future revisions of these guidelines.
Disclaimer
These Guidelines represent a consensus view, but inclu-
sion in the authorship or contributor list does not
necessarily imply agreement with all statements.
Funding
CephRes, AISAL and FELASA support this work. This pub-
lication is also supported by COST (European Cooperation in
Science and Technology).
Acknowledgements
This work originates from a joint effort to develop the first
Guidelines for the Care and Welfare of Cephalopods in
Research according to Directive 2010/63/EU made by the
Association for Cephalopod Research - CephRes, The Boyd
Group (UK), and the Federation for Laboratory Animal
Science Associations – FELASA. This has been initiated
and subsequently developed following April 2012 CephRes
Meeting in Vico Equense (Italy). We would like to thank
Drs John Messenger, Andrew Packard and Sigurd von
Boletsky for their contributions to the initial meeting where
ideas for Guidelines for the Care and Welfare of Cephalopods
were discussed. We are also grateful for comments and con-
tributions by Piero Amodio (CephRes, Italy), and by Ariane
Dro
¨scher (University of Bologna, Italy) for historical data.
PA and GF conceived and edited this work as well as a
making a major contribution to the authorship. GF and GP
organised the meeting; GP and AC provided assistance to the
leading authors on behalf of CephRes.
This manuscript provides the basis for the activities of the
FA1301 COST Action ‘A network for improvement of cepha-
lopod welfare and husbandry in research, aquaculture and
fisheries (CephsInAction; http://www.cost.eu/COST_Actions
/fa/Actions/FA1301; www.cephsinaction.org)’ aimed to facil-
itate and support the cephalopod community in meeting the
challenges resulting from the inclusion of cephalopod mol-
luscs in Directive 2010/63/EU.
We are grateful to Professor Torsten Wiesel for his enthu-
siasm and genuine interest for cephalopod biology research.
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535. Grigoriou P and Richardson CA. Effect of body mass,
temperature and food deprivation on oxygen consump-
tion rate of common cuttlefish Sepia officinalis.Mar
Biol 2009; 156: 2473–2481.
536. Fiorito G, Agnisola C, d’Addio M, Valanzano A and
Calamandrei G. Scopolamine impairs memory recall in
Octopus vulgaris.Neurosci Lett 1998; 253: 87–90.
537. Wells MJ and Wells J. Fluid uptake and the mainte-
nance of blood volume in octopus. 1993; 175:
211–218.
538. Robertson JD, Bonaventura J and Kohm AP. Nitric
oxide is required for tactile learning in Octopus vulgaris.
Proc R Soc Lond B 1994; 256: 269–273.
Fiorito et al. 79

539. Robertson JD, Bonaventura J, Kohm A and Hiscat M.
Nitric oxide is necessary for visual learning in Octopus
vulgaris.Proc R Soc Lond B 1996; 263: 1739–1743.
540. Tansey EM. A histochemical study of the cephalopod
brain. PhD Thesis, University of Sheffield, UK, 1978.
541. Gaston MR and Tublitz NJ. Peripheral innervation pat-
terns and central distribution of fin chromatophore
motoneurons in the cuttlefish Sepia officinalis.J Exp
Biol 2004; 207: 3089–3098.
542. Juorio AV. Catecholamines and 5-hydroxytryptamine in
nervous tissue of cephalopods. J Physiol 1971; 216:
213–226.
543. Beuerlein K, Lohr S, Westermann B, Ruth P and Schipp
R. Components of the cellular defense and detoxi-
fication system of the common cuttlefish Sepia officina-
lis (Mollusca, Cephalopoda). Tissue Cell 2002; 34:
390–396.
544. Beuerlein K, Ruth P, Westermann B, Lohr S and Schipp
R. Hemocyanin and the branchial heart complex of
Sepia officinalis: are the hemocytes involved in hemo-
cyanin metabolism of coleoid cephalopods? Cell Tissue
Res 2002; 310: 373–381.
545. Wells MJ and Wells J. Ventilatory currents in the mantle
of cephalopods. J Exp Biol 1982; 99: 315–330.
546. Ikeda Y, Sugimoto C, Yonamine H and Oshima Y.
Method of ethanol anaesthesia and individual marking
for oval squid (Sepioteuthis lessoniana Ferussac, 1831 in
Lesson 1830-1831). Aquac Res 2009; 41: 157–160.
547. Kier WM, Smith KK and Miyan JA. Electromyography
of the fin musculature of the cuttlefish Sepia officinalis.
J Exp Biol 1989; 143: 17–31.
548. Gutfreund Y, Matzner H, Flash T and Hochner B.
Patterns of motor activity in the isolated nerve cord of
the octopus arm. Biol Bull 2006; 211: 212–222.
80 Laboratory Animals 49(S2)

Appendix 1.
Summary of key information required in a
project application under the Directive (see
for further detailed information Smith
et al., 2013)
1
Information required in applications for
project authorisation
Directive 2010/63/EU Annex VI and Article 38(2)e
.
1. Relevance and justification of the following:
(a) use of animals including their origin, estimated
numbers, species and life stages;
(b)procedures.
2. Application of methods to replace, reduce and
refine the use of animals in procedures.
3. The planned use of anaesthesia, analgesia and other
pain relieving methods.
4. Reduction, avoidance and alleviation of any form
of animal suffering, from birth to death where
appropriate.
5. Use of humane end-points.
6. Experimental or observational strategy and statistical
design to minimise animal numbers, pain, suffering,
distress and environmental impact where appropriate.
7. Re-use of animals and its accumulative effect on the
animals.
8. The proposed severity classification of procedures.
9. Avoidance of unjustified duplication of procedures
where appropriate.
10. Housing, husbandry and care conditions for the
animals.
11. Methods of killing.
12. Competence of persons involved in the project
Purposes of procedures permitted
Directive 2010/63/EU Article 5
Procedures may be carried out for the following pur-
poses only:
(a) basic research;
(b) translational or applied research with any of the
following aims:
(i) the avoidance, prevention, diagnosis or
treatment of disease, ill-health or other abnorm-
ality or their effects in human beings, animals or
plants.
(ii) the assessment, detection, regulation or
modification of physiological conditions in
human beings, animals or plants.
(iii) the welfare of animals and the improvement of
the production conditions for animals reared
for agricultural purposes
(c) for any of the aims in (b), in the development,
manufacture or testing of the quality, effectiveness
and safety of drugs, food- and feed-stuffs and
other substances or products;
(d) protection of the natural environment in the inter-
ests of the health or welfare of human beings or
animals;
(e) research aimed at preservation of the species;
(f) higher education, or training for the
acquisition, maintenance or improvement of voca-
tional skills;
(g) forensic inquiries.
Questions that should be addressed in
relation to the Three Rs in writing a project
application, and in the project review and
approval process
Replacement
.
(i) What on-going efforts will you make to identify
‘scientifically satisfactory’ alternative methods
that could replace the use of some or all animals?
(Article 4§ 1)
(ii) Could you avoid the use of animals by asking
different type of question or making better use
of existing data or literature to address the scien-
tific objectives?
(iii) Could in vitro studies or in silico (computer)-mod-
elling be used to replace some or all of the animals?
Reduction
.
(iv) How will you ensure that the number of animals
used in the project, and in individual studies
within the project, is ‘reduced to a minimum with-
out compromising the scientific objectives’?
(Article 4§2)
(v) Could any further reductions be made, e.g. by
taking expert statistical advice to help optimise
experimental and statistical design?
Refinement
.
(vi) How have you refined the ‘breeding, accommoda-
tion and care of the animals’ and the ‘methods
used in procedures’, so as to ‘reduce to the
1
For full reference to the cited document see Reference List (#3) at
the end of the paper.
Fiorito et al. 81

minimum any possible pain, suffering, distress or
lasting harm to the animals’ throughout their
lives? (Article 4§3)
(vii) Have you considered and implemented all the
possibilities for refinement described elsewhere
in these guidelines? (see Smith et al. 2013 for
examples of refinement in the context of specific
procedures).
(viii) How will you ensure that all relevant personnel
working on the project are adequately educated
and trained, and are supervised until they have
demonstrated their competence in the proce-
dures? (see section 10)
82 Laboratory Animals 49(S2)

Appendix 2. Recommended species-specific minimal requirements for care and management of cephalopods under Directive 2010/63/EU. The appendix is organized
into sub-sections providing information about housing, environmental parameters, transport, and feeding.
Appendix 2A (Housing) Recommended species-specific minimal requirements for housing for the establishment, the care and accomodation of cephalopods.
a
Data
for different body size/life stages are included when available.
Nautilus sp. Cuttlefishes Squids Octopuses
Life stages Juveniles, adults Juveniles Adults
b
Juveniles
c
Adults Juveniles, adults
Structural materials Fibreglass, PVC,
acrylic, glass or any
non-toxic material
Polycarbonate, glass or
any non-toxic materials
Polycarbonate, glass or
any non-toxic materials
Fibreglass, PVC,
acrylic, glass or any
non-toxic material
Fibreglass, PVC,
Acrylic, glass or any
non-toxic material
PVC or glass (for glass
tanks see recommen-
dations in main text)
Pipe materials Copper and heavy
metal free, PVC
d
Copper and heavy
metal free
Copper and heavy
metal free
Copper and heavy
metal free
Copper and heavy
metal free
Copper and heavy
metal free, PVC
c
Recommended tank
design
Cylindrical (height/
width >1)
Maximal horizontal
surface area, rounded
ends
Maximal horizontal
surface area, rounded
ends
Circular/elliptical Circular/elliptical Any shape
Recommended inter-
nal tank colour
Dark Opaque Opaque Opaque; Contrasted or
dark sides so that the
squid are able to detect
Opaque; Contrasted or
dark sides so that the
squid are able to detect
Opaque-grey; Mirrored
surfaces should be
avoided
e
Tank internal surface Smooth Smooth Smooth Smooth Smooth Smooth
Minimum suggested
size
0.005 square metre per
animal
0.48 square metre per
animal
0.30 square metre
per animal is
recommended
Lid cover No (to control light
levels a dark plastic
drape or a solid lid
with a few holes drilled
in the drape/lid to allow
dappled light into the
tank and communicate
the daily light cycle)
No (but one shelter per
animal in the tank)
No (but one shelter per
animal in the tank)
No No Yes. Transparent
covers, firmly secured
to the tank
Drain cover Yes No No Suspended drains
with netting
Yes
Mesh size No mesh 120–160 mm 120–160 mm Small Small No mesh
Light source Standard fluorescent
lighting is acceptable
Halogen lamps are
recommended.
Standard fluorescent
lighting should be
avoided.
Halogen lamps are
recommended.
Standard fluorescent
lighting should be
avoided.
Can be overhead light-
ing via normal strip
lights or by 400w
halide (10–100 lx)
Can be overhead light-
ing via normal strip
lights or by 400w
halide (10–100 lx)
Halogen lamps are
recommended.
Standard fluorescent
lighting should be
avoided.
Recommended mini-
mum water volume
30 L/animal 2 L/animal 80 L/animal 220 L 1500 L 100 L/animal
(depending from the
body size of the animal)
(continued)
Fiorito et al. 83

Appendix 2A (Housing) Continued
Nautilus sp. Cuttlefishes Squids Octopuses
Life stages Juveniles, adults Juveniles Adults
b
Juveniles
c
Adults Juveniles, adults
Recommended mini-
mum water high
1.5 m 0.05 m 0.4 m 0.6 m for a 1.8 m tank 0.6 m for a 1.8 m tank 0.40 m
Group rearing Yes Possible but it is
preferable individual
housing
Possible but it is
preferable individual
housing
Yes Yes Depending on species
(not recommended for
Octopus vulgaris)
Recommended maxi-
mum stocking density
Few individuals 200/square metre 2/square metre Dependent on size and volume of tank; e.g in a 2-
m circular tank, 10–15 individuals of Loligo species
(150 to 250 mm ML). No less than 1 squid per
58 L
Individual rearing,
preferred
Enrichment Yes (Few smooth verti-
cal PVC pipes attached
to the side of the tank
to allow the animal for
its natural movement).
Adding texture (artifi-
cial coral reef) to at
least one wall of the
tank may make it
more attractive to the
animal.
A shadow area should be provided in each tank. A
fine layer of gravel/pebbles where cuttlefish can
settle is recommended in each tank. Sand can be
provided in the tanks
Substrate can be placed on bottom but be aware
that it needs to be maintained to reduce bacterial
problems. Fake seaweed and overhead shelter to
provide shadow may also be used to give areas of
shelter
Each tank should be
provided with a den for
the animal and a layer
of sand
a
These requirements do not take into account those adopted and suggested for rearing cephalopods for purposes of aquaculture (for review see Iglesias et al.
1
).
b
Sexually mature females and males should be in distinct tanks to avoid fighting.
c
Schooling behaviour in Sepioteuthis lessoniana occurs around 20 days after hatching and that synchronisation of individuals into shoaling increased over time.
2
d
Indicated as suggested material.
e
Although recommendation to avoid mirroring surfaces in the tank is available only for O. vulgaris, we suggest the same precaution will be utilized for all cephalopod species.
84 Laboratory Animals 49(S2)

Appendix 2. Recommended species-specific minimal requirements in relation to environmental parameters
a
to be applied for the establishment, the care and
accomodation of cephalopods.
Appendix 2B (Environmental parameters)
Nautilus sp. Cuttlefishes Squids Octopuses
Temperature range 14–26C (preferably 14–17C)
In accordance with the spe-
cies of which are being held
12–25C
In accordance with the spe-
cies of which are being held
In accordance with the spe-
cies of which are being held
16–26C
In accordance with the spe-
cies of which are being held
Salinity range 34–36 ppt 29–33 ppt 30–36 ppt 32–35 ppt
pH range 8.0–8.5 (preferably 8.2) (within
the range of the natural fish-
ing site)
7.8–8.1 (within the range of
the natural fishing site)
7.8–8.3 (within the range of
the natural fishing site)
7.9–8.3 (within the range of
the natural fishing site)
Max [NH
4
]<0.10 mg/L <0.5 mg/L <0.01 mg/l <0.10 mg/L
Max [NO
2
]<0.10 mg/L <0.2 mg/L <0.1 mg/l <0.10 mg/L
Max [NO
3
]<20 mg/L <80 mg/L <40 mg/l <20 mg/L
Min [O
2
]8 ppm (saturated)
b
7.0 ppm Saturated 8 ppm (saturated)
Recommend
light wave length
450–650 nm No specific requirements Normal overhead light so
yellow/white
Natural light at 3–5 m depth
(for deep living species, blue
light or shadow is
recommended)
Recommended light
intensity at water
surface
Dim
c
<350 lux 100–200 lux 200–400 lux
Photoperiod According to the natural geo-
graphical location of the
animal
According to the natural geo-
graphical location of the
animal. A dimming period is
recommended
According to the natural geo-
graphical location of the
animal. A dimming period is
recommended
According to the natural geo-
graphical location of the
animal. Add crepuscular
effect as enrichment
d
Noise level and
vibration
Avoid vibration Avoid vibration Avoid vibration Avoid vibration
a
These requirements do not take into account those adopted and suggested for rearing cephalopods for purposes of aquaculture (for review see Iglesias et al.
1A
).
b
Care should be taken that the source of oxygenation be kept away from the animals (in a separate tank or sump). Nautiluses are attracted to and can retain air bubbles in their eyes, and
under their hood leading to adverse health effects. Nautilus is particularly sensitive to air and any exposure should be avoided.
c
Tanks should allow for both dark refuges and dim light.
d
It is reported that crepuscular light prevent from premature hatching of paralarvae from octopus females caring eggs in tanks.
Fiorito et al. 85

Appendix 2 Recommended species-specific minimal requirements for short-duration transport for the establishment, the care and accomodation of cephalopods.
Data for different body size/life stages are included when available.
Appendix 2C (Short-duration transport)
Nautilus sp.
Cuttlefishes Squids Octopuses
Life stages
Eggs,
Juveniles Adult Eggs Adults Eggs Paralarvae
Juveniles,
Adults
Source Wild Wild and
captive bred
Wild and
captive bred
Wild and
captive bred
Wild Wild; Captive
bred
Captive bred Wild
Transport
container
A bucket that
allows the animal
to move freely and
attach to the side
of the bucket
Large buckets Large buckets Plastic bags Plastic bags
a
250–500 ml tissue
culture flask
Plastic bags
Plastic bags Large buckets
Water
quantity/quality
b
Water covering the
bucket surface and
at least 2 shell depths
beneath
1 L oxygenated
seawater
(Hatchlings,
max 10)
Maximum 10
animals in 50 L
oxygenated
seawater
Eggs should be
transported in their
natural seawater
(if from wild)
15 L oxygenated
seawater/
animal (but
depends on size
of squid and
distance at
which it is travelling)
50% water or
embryo media
and 50% air. 1–2 per
ml of sterile water.
Methylene blue
(0.5 mg/L or 0.5 ppm)
can also be added to
the solution to reduce
fungal growth
Seawater (1/3)
and O
2
(2/3) at
a density up to
3000 individuals
1–5 animals/
15 L oxygenated
seawater.
Water
temperature
14–17C 12–18C 12–18C 17–19C 17–19C Around 15C Around 15C 15–24C
Food
deprivation
No No No No No No No No
Acclimatisation
after transport
Yes, brief
c
Yes, brief Yes, brief Yes, brief Yes, brief Yes, brief Yes, brief Yes, brief
Quarantine Not required Not required Not required Not required Not required Not required Not required Not required
a
During transport within lab squid may be transported in plastic containers to awaiting larger containers such as an ice chest or bucket which can be submerged in the recipient tank. In
some cases it may be necessary to anaesthetize the animal with MgCl
2
.
b
No special requirements for quality, except the normal clean seawater utilized in establishments.
c
To equilibrate pH and temperature values and let the animal adapt to the new environment.
86 Laboratory Animals 49(S2)

Appendix 2 Recomended species-specific minimal requirements for long-duration transport for the establishment, care and accomodation of cephalopods. Data for
different body size/life stages are included when available.
Appendix 2D (Long-duration transport)
Nautilus sp.
a
Cuttlefishes Squids Octopuses
Life stages Eggs, Juveniles Adult Eggs Adults Eggs Paralarvae Juveniles, adults
Transport
container
Double-bagged
common aquarium
bags placed into styro-
foam boxes. The exter-
nal container should
be able to carry and
hold the entire water
volume in it even if all
inside containers/bags
are raptured.
Dark bags Dark containers (50
to 100 liters) with
opaque covers.
Recommend either horizontal cylindrical tank
or rectangular horizontal tank
with closed top
Double-bagged common aquarium bags placed into
styrofoam boxes. The external container should
be able to carry and hold the entire water volume in
it even if all inside containers/bags are ruptured.
Water
quantity/quality
1/3 pre-oxygenated
seawater and 2/3
air (1 animals
per bag)
1/3 Oxygen
2/3 seawater
Max density:
3000/L
1/3 Oxygen 2/3 sea-
water
Max 10 animal/
containers
Depending on size
of squid and
distance at which
it is traveling
15 L oxygenated
seawater/animal
(depending on
size of squid and
distance at which
it is traveling)
To be
developed
To be
developed
Octopus (BW 50–600 g)
should be double-
bagged in a good
quality plastic
fish bag at a density
of about 1 octopus
per 2 L
seawater þ2/3 of
oxygen AmQuel (a
commercially
available ammonia
sequestrator) can be
added to bind any
ammonia that is
produced during
transport.
Water
temperature
b
Should never
exceed 23C
12–18C 12–18C Should match that of
where the squid
naturally occurs
Should match that
of where the squid
naturally occurs
13–19C 13–19C 15–22C
Food
deprivation
Yes (2–3 days) Yes (24 h) Yes (24 h) Only if possible
(animals not
caught from the wild)
Not Applicable Not Applicable No
For O. dofleini it
is suggested 10–12 days.
(continued)
Fiorito et al. 87

Appendix 2D (Long-duration transport) Continued
Nautilus sp.
a
Cuttlefishes Squids Octopuses
Life stages Eggs, Juveniles Adult Eggs Adults Eggs Paralarvae Juveniles, adults
Sedation Not
recommended
Not
recommended
Not
recommended
Not
recommended
Not
recommended
Not
recommended
Not
recommended
Not
recommended
Acclimatisation
after transport
Yes (approximately 1 week) Yes (3–4 h) Yes (3–4 h)
c
Yes Yes Criteria to be
developed
Criteria to be
developed
Yes
Quarantine Yes (only for ill
or injured animals)
If applicable a
diagnosis of the
levels of parasite
infection and of
haemocyte count
may be carried out
by the animal
care Staff to
determine if
quarantine is
required.
If applicable a
diagnosis of the levels
of parasite
infection
and of haemocyte
count may be carried
out by the animal care
staff to determine if
quarantine is required.
Separate quarantine
tanks should be
available if squid
is damaged and
needs veterinary
attention
Separate quarantine
tanks should be
available if squid
is damaged and
needs veterinary
attention
Criteria to be
developed
Criteria to be
developed
Yes (only for ill
or injured animals)
a
It is important to check local requirements for the transport of animals in all countries along the route while transporting live cephalopods. Nautiluses are particularly sensitive to
exposure to air and this should be avoided if possible by transporting them in vessels containing seawater.
b
Should match that of where the species naturally occurs.
c
Change of environment, particularly slight changes in temperature, can induce egg laying.
88 Laboratory Animals 49(S2)

Appendix 2 Recommended species-specific minimal requirements for feeding
a
to be provided to animals as applied to the establishment, care and accomodation of
cephalopods. Data for different body size/life stages are included when available.
Appendix 2E (Feeding)
Nautilus sp. Cuttlefishes Squids Octopuses
Life stages Hatchlings
Post
hatchlings Juveniles, Adults Juveniles Adults
Food items Dead food only.
Nautilus requires
food with a high
level of Calcium
Carbonate, such
as shrimp with
carapace, lobster
moult shells or
fish heads.
No food for
1–7 days
Shrimp-like
prey
Different prey items.
Frozen food is
acceptable (live
preys preferred)
b
Live larvae such
as artemia, mysis
or fish
Live or dead
fish
Live prey. Crabs, mostly
utilized Carcinus
mediterraneus.
c
Feeding
regimes
Daily-2 times/week Daily, ad
libitum
Daily, ad
libitum
Daily, ad
libitum
Three to five
times a day
Three to five
times a day
At least every other day.
Providing food once a day
should reccomended.
a
Information on nutritional requirements of cephalopods species reared for purposes of aquaculture is reviewed in Iglesias et al.
1A
.
b
They can accept frozen food from 2 months of age, after familiarisation.
c
A list of potential live prey traditionally utilized for octopuses is also given by Borrelli.
3A
Remove any solids after the last feeding.
References
1A. Iglesias J, Fuentes L and Villanueva R. Cephalopod Culture. Dordrecht: Springer Netherlands, 2014.
2A. Sugimoto C and Ikeda Y. Ontogeny of schooling behavior in the oval squid Sepioteuthis lessoniana. Fish Sci. 2012; 78: 287–294.
3A. Borrelli L. Testing the contribution of relative brain size and learning capabilities on the evolution of Octopus vulgaris and other cephalopods [PhD Thesis] Stazione Zoologica Anton
Dohrn, Napoli, Italy; Open University, London, UK, 2007.
Fiorito et al. 89

Appendix 3 - Reproductive strategies of some cephalopods species as deduced from Rocha et al. (2001)
a
. The list of
species included below (listed in taxonomic order) are those derived from Smith et al. (2013)
b
that counted more than 150
species utilised for the scientific purposes in EU over a 5-year period. The life span of cephalopods typically ranges from 6
months to 2 years; smaller tropical species tend to have shorter lives while larger, cold-water species live longer. The
sole exception known to this rule is Nautilus with a life span known to be longer than 20 years.
Reproductive Strategy
Polycyclic
spawning
c
Intermittent
terminal
spawning
d
Simultaneous
terminal
spawning
e
Multiple
spawning
f
Continuous
spawning
g
Nautilus sp.ˇ
Sepia officinalis ˇ
S. elegans ˇ
Idiosepius pygmaeus ˇ
Sepiola rondeleti ˇ
S. atlantica ˇ
S. robusta ˇ
Sepietta oweniana ˇ
Rossia macrosoma ˇ
Loligo vulgaris ˇ
L. (Alloteuthis) subulata ˇ
L. forbesi ˇ
L. opalescens ˇ
L. pealei ˇ
Lolliguncula brevis ˇ
Photololigo sp. ˇ
Sepioteuthis sepioidea ˇ
S. lessoniana ˇ
Illex illecebrosus ˇ
I. argentinus ˇ
I. coindetii ˇ
Todarodes sagittatus ˇ
T. angolensis ˇ
T. pacificus ˇ
Todaropsis eblanae ˇ
Dosidicus gigas ˇ
Octopus vulgaris ˇ
O. cyanea ˇ
O. macropus ˇ
Enteroctopus dofleini ˇ
Eledone moschata ˇ
E. cirrhosa ˇ
a
For full reference to the cited document see Reference List (#277) at the end of the paper.
b
For full reference to the cited document see Reference List (#3) at the end of the paper.
c
Iteroparity (egg capsules are released with a significant time interval between successive eggs spawned one by one or successive egg
batches), with asynchronous ovulation and growth between egg batches.
d
Iteroparity, with synchronous ovulation and no growth between egg batches.
e
Semelparity (egg capsules are released simultaneously), with synchronous ovulation and no growth between egg batches.
f
Iteroparity, with group-synchronous ovulation (at least two populations of oocytes can be distinguished at some time) and growth
between egg batches.
g
Iteroparity, with asynchronous ovulation and growth between egg batches.
90 Laboratory Animals 49(S2)