ICES WKAREA REPORT 2009
ICES A DVISORY C OMMITTEE
ICES CM 2009\ACOM: 48
R EF . PGCCDBS, WGEEL, S CI C OM
Workshop on Age Reading of European
and American Eel (WKAREA)
20-24 April 2009
Bordeaux, France
International Council for the Exploration of the Sea
Conseil International pour l’Exploration de la Mer
H. C. Andersens Boulevard 44–46
DK-1553 Copenhagen V
Denmark
Telephone (+45) 33 38 67 00
Telefax (+45) 33 93 42 15
www.ices.dk
[email protected]
Recommended format for purposes of citation:
ICES. 2009. Workshop on Age Reading of European and American Eel (WKAREA),
20-24 April 2009, Bordeaux, France. ICES CM 2009\ACOM: 48. 66 pp.
For permission to reproduce material from this publication, please apply to the
General Secretary.
The document is a report of an Expert Group under the auspices of the International
Council for the Exploration of the Sea and does not necessarily represent the views of
the Council.
© 2009 International Council for the Exploration of the Sea
| i
Conte nts
Preparation of this document ............................................................................................... 1
Executive summary ................................................................................................................ 2
1
Opening of the meeting................................................................................................ 4
2
Terms of Reference ........................................................................................................ 4
3
Glossary ........................................................................................................................... 5
3.1
Eel Terms ............................................................................................................... 5
3.2
Otolith Terms ........................................................................................................ 6
4
Introduction .................................................................................................................... 8
5
Otolith Preparation Techniques ................................................................................. 9
5.1
Introduction ........................................................................................................... 9
5.2
Otolith preparation techniques – pre 1988 ........................................................ 9
5.3
Otolith preparation techniques – post 1988 .................................................... 10
5.4
Validation of Age Determination ..................................................................... 14
5.4.1
5.4.2
5.4.3
5.4.4
6
Pre-1988 Validation ............................................................................... 14
Comparison between methods ............................................................ 16
Recent Validation (post-1988) .............................................................. 21
Supporting Case Studies ....................................................................... 24
Guidelines for Interpretation .................................................................................... 30
6.1
Related Field Data .............................................................................................. 30
6.1.1 Options for reading with/without a priori knowledge of
vital information .................................................................................... 30
6.2
Continental age – where to start? "Zero bands" ............................................. 30
6.3
Annual (winter) rings and false checks ........................................................... 34
6.3.1 False annuli ............................................................................................. 35
6.4
7
8
Otolith reading calendar .................................................................................... 41
Accuracy and Precision ............................................................................................... 42
7.1
Reference collection. ........................................................................................... 42
7.2
Inter-calibration................................................................................................... 42
References ..................................................................................................................... 44
Annex 1: Agenda................................................................................................................... 50
Annex 2: List of participants............................................................................................... 52
Annex 3: Individual Institute standard operating procedures for the
preparation of eel otoliths, by grinding and polishing ........................................ 55
Annex 4: Manual for the Ageing of Atlantic Eel (separate document) ....................... 63
ICES WKAREA Report 2009
| 1
Preparation of this document
This publication is the report of the ICES Workshop on Age Reading of European and
American Eel, held in CEMAGREF, Bordeaux, France from 20-24 April 2009. The
Workshop was chaired by Francoise Daverat and co-chaired by Russell Poole, Hakan
Wickström and John Casselman (not present at the workshop).
This report is designed as a stand alone report covering the Terms of Reference, and
should be read in conjunction with the EIFAC 1988 publication on eel age
determination (Vøllestad, Lecomte-Finiger and Steinmetz, 1988). An illustrated
manual of best practice and otolith images has been drafted as a separate document
and this could be published as a separate but related document.
This document is a report of an Expert Workshop under the auspices of the
International Council for the Exploration of the Sea.
Images contained within the report should not be extracted and/or used for
publication without the prior permission of ICES and the creator of the original
image.
ICES WKAREA Report 2009
| 2
Executive summary
The Workshop commenced with a review of general eel biology and otolith structure,
confirming the main terms and definitions in a glossary. The Workshop also
reviewed the report of the 1987 EIFAC workshop on eel age determination and used
this as a point from which to move forward.
The two main otolith preparation protocols for the Atlantic species of eel, Anguilla
anguilla and A. rostrata, currently in use are, with slight variations between institutes,
the burning and cracking (or better now the cutting and burning), and the grinding
and polishing (and in most cases staining) protocols. Clearing whole otoliths "in toto"
has limited use for small eels of young age. The validation methods were reviewed
along with information for otoliths prepared by each technique and also images were
compared between the two main techniques so as to identify common structures (e.g.
zero band). The Workshop recommended a preparation with a transverse section of
the otolith for slow growth, or old eels, with burning and cracking being the most
efficient in that case. Detailed protocols were discussed for each otolith preparation
technique and these have been included in the manual.
The estimation of growth is based on the count of winter annuli, excluding the
oceanic and glass eel phase. A number of studies were reviewed that used otolith
marking, otolith time series, and eels of know age in order to confirm the presence of
a zero band for the start of the continental growth phase and to identify and discount
the inclusion of false annuli growth checks, often due to stress associated with
quarantine/marking and tagging of stocked glass eel, periods in aquaculture, and in
the wild due to natural stresses and growth variability. The identification of the zero
band, may be confirmed by the use of the measurement of the nucleus size, or the
average measurement of the radius from the centre of the nucleus to the zero band
(170µm) which is quite consistent for both eel species, irrespective of the otolith
preparation technique used.
The date of reference for age is set as the 1st of January, meaning that a cautious
approach is recommended for eels sampled in winter and spring before the period for
which the winter annuli is not obvious on the otolith margin. The Workshop
recommended that the age estimation is obtained using both the otolith annuli count
and additional data such as location and date of capture, eel life stage (i.e. yellow or
silver), length, sex, and previous history if known (e.g. stocked from wild, stocked
from aquaculture) as this supports a more accurate interpretation of the growth
pattern and helps to discriminate winter annuli from false checks. "Blind reader" tests
may be appropriate in some circumstances but for routine age determination,
possession of the full information reduces unnecessary misinterpretation and
variability.
A preliminary inter-calibration exercise was carried out during the workshop using
21 Swedish otoliths of eels of known age where the 25 readers were of varying
experience and had varying levels of knowledge on the supporting eel data and
information. This indicated considerable variation and the results were only used to
support the workshop discussions. An experienced reader inter-calibration procedure
should be undertaken in the near future based on the age criteria described in the
manual and the reading of at least a hundred otolith pictures for each species,
including both eels of known and unknown age. This could be done using image
exchange and culminate in a workshop for discussion of the outcomes, follow-up
training and update of the manual.
ICES WKAREA Report 2009
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P ARTICIPANTS AT THE ICES W ORKSHOP ON A GE R EADING OF E EL IN
CEMAGREF, B ORDEAUX .
Ode to Sally
There once was an eel in Bordeaux
Whose growth rate was steady but slow
Poor Sally asked “how is this so?”
“Are there experts who are bound to know?”
So a meeting was called full of brains
To decipher train tracks without trains
With her ears on a desk, and her brains in a mess
Sally asked “can you do more than just guess?”
“We’ll burn and we’ll crack, we’ll grind and we’ll polish
These myths to your age we’ll firmly abolish
Fret no more of these lines in your ears
They don’t all correspond to your years”
“All the stress from your life leaves a mark
But a true year shows up nice and dark”
So the brains argued for and against
Through discussions they eventually found sense.
So rest easy young Sally my dear
Just lie down and lend me your ear.
J. Ava
Sally – the WKAREA mascot
ICES WKAREA Report 2009
1
| 4
Opening of the meeting
The Workshop was opened by the Chair, Francoise Daverat, and a welcome was
given by Mr Hugues Ayphassorho, Regional Director of CEMAGREF, Bordeaux. The
Agenda plan for the week was discussed and agreed (see Annex 1). The workshop
was attended by 21 participants from 12 countries and an additional 9 participants
joined the group on the 22nd April for the training sessions (Annex 2). Countries
represented at the Workshop were Belgium, Canada, Denmark, France, Germany,
Ireland, Italy, Netherlands, Portugal, Sweden and the UK and Northern Ireland.
2
Terms of Reference
2008/2/ACOM46: The Workshop on Age Reading of European and American Eel
[WKAREA] (Chair: Françoise Daverat (France); co-chairs Hakan Wickström
(Sweden), Russell Poole (Ireland) and John Casselman (Canada)) will be established
and will meet 20–24 April 2009 in Bordeaux, France to:
a ) review literature and current practices on eel age reading;
b ) review the methods used by participating institutes to prepare and store
eel otoliths, and the processes and protocols currently used to read them
(including quality assurance methods);
c ) review approaches for validating eel age reading;
d ) conduct an otolith exchange study between participating institutes before
the workshop to compare age reading results;
e ) establish a common methodology for age reading and determine the
precision and bias of corresponding age reading data;
f ) establish a common methodology for otolith preparation and age reading;
g ) determine the precision and bias of eel age reading data derived from
different readers and methods;
h ) develop a manual to describe the agreed methodology; and
i ) recommend, if needed, a framework to improve and to validate age
reading.
WKAREA will report by 4 May 2009 for the attention of the PGCCDBS, SciCom, and
WGEEL.
Descriptions of national sampling, otolith preparation technique and age
determination protocols were updated in Chapter 5 (ToR a and b). The methods used
by the participating institutes were reviewed in Chapter 5 as well as the validations
of eel age reading in Chapter 6 (ToR b and c). An exchange of otolith pictures from
both species, based on the age estimation of 25 readers was conducted and the age
reading results were discussed during the meeting (ToR d), leading to the conclusion
that additional data (e.g. location, date of capture, history of stocking) are necessary
to be known before reading. In Chapter 7 an inter-reader calibration was carried out
based on a limited number of samples, and we recommend to conduct a reading of a
significant number of otolith pictures (>100) of known and unknown age to
accurately demonstrate inter-reading calibration, using the otolith manual (ToR f &
g). A manual was agreed based upon plenary discussion of individual participants’
current methodologies (ToR h). The exchange that is recommended to take place after
the workshop should have improved age reading based on the manual (ToR i).
ICES WKAREA Report 2009
3
Glossary
3.1
Eel Terms
| 5
Eels are quite unlike other fish and
consequently come with a specialised
jargon (WGEEL 2008). This section
provides a quick introduction and is not
intended to be exhaustive.
The life cycle of the European eel.
The
names of the major life stages are indicated.
Spawning and eggs of Anguilla anguilla and
A. rostrata have never been observed in the
wild (supplied by Dekker).
Atlantic
Eel
The collective term Atlantic eel will be used in this report to cover both A. anguilla and
A. rostrata where there are no differences. Differences between the species will be
indicated by their specific names.
Glass eel
Young, unpigmented eel, recruiting from the sea into continental waters
Elver
Young eel, in its 1st year following recruitment from the ocean. The elver stage is
sometimes considered to exclude the glass eel stage, but not by everyone. Thus, it is a
confusing term.
Bootlace,
fingerling
Intermediate sized eels, approx. 10-25 cm in length. These terms are most often used
in relation to stocking. The exact size of the eels may vary considerably. Thus, it is a
confusing term.
Yellow eel
Life stage resident in continental waters. Often defined as a sedentary phase, but
migration within and between rivers, and to and from coastal waters occurs. This
phase encompasses the elver and bootlace stages.
(Brown
eel)
Silver eel
Migratory phase following the yellow eel phase. Eel characterised by darkened back,
silvery belly with a clearly contrasting black lateral line and enlarged eyes.
Downstream migrating phase, moving towards the sea,. This phase mainly occurs in
the second half of calendar years, though some are observed throughout the winter
and following spring.
Eel River
Basin
“Member States shall identify and define the individual river basins lying within their
national territory that constitute natural habitats for the European eel (eel river basins)
which may include maritime waters. If appropriate justification is provided, a
Member State may designate the whole of its national territory or an existing regional
administrative unit as one eel river basin. In defining eel river basins, Member States
shall have the maximum possible regard for the administrative arrangements referred
to in Article 3 of Directive 2000/60/EC [i.e. River Basin Districts of the Water
Framework Directive].” EC No1100/2007
River Basin
District
The area of land and sea, made up of one or more neighbouring river basins together
with their associated surface and groundwaters, transitional and coastal waters, which
is identified under Article 3(1) of the Water Framework Directive as the main unit for
management of river basins. Term used in relation to the EU Water Framework
Directive.
Stocking
Stocking is the practice of adding fish [eels] to a water body from another source,
to supplement existing populations or to create a population where none exists.
ICES WKAREA Report 2009
3.2
| 6
Otolith Terms
(updated from Vøllestad, Lecomte-Finiger & Steinmetz, 1988) and see also Figure 3.1
Annual zone:
Structural feature of the otolith corresponding to the growth
during a complete year of life
Annulus:
The theoretical boundary between two successive annual
zones
Burning & cracking:
The traditional otolith preparation of burning and cracking
has been improved by cutting the otolith before burning.
Both methods are covered in this manual by the term
"Burning and cracking".
Frontal Plane:
The flat cut, or cracked, face of a transverse section of an
otolith
Growth Check:
A boundary between two growth zones, not necessarily
annual (also see supernumerary)
Hyaline:
See translucent.
Nucleus:
The hypothetical or real origin of the otolith; synonymous
with focus or core
Opaque zone:
A zone that inhibits the passage of light. In transmitted light
opaque zones appear dark and in reflected light they appear
bright (white)
Radius:
A determined measurement from a focus to a specific point
Sagittal Plane:
The view of the otolith when lying flat, convex side up. Most
grinding takes place on the sagittal plane.
Supernumerary:
A growth mark or check not accepted for annual age
determination, also referred to as a growth check or false
annulus.
Translucent zone:
Previously known as the hyaline zone. A zone that allows
the passage of light. In transmitted light translucent zones
appear bright, in reflected light they appear dark.
Validation:
The confirmation of the temporal meaning of a growth
increment. Analogous to determining the accuracy of age
determination; used in reference to true age.
Verification:
Determining the precision (reproducibility) of age
determination, used in reference to the precision of estimated
age.
Zero band:
The first growth check outside the nucleus from where
continental age determination commences (~170µm radius
from centre).
ICES WKAREA Report 2009
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Figure 3.1. Illustrations of sagittal and frontal plane views of otoliths indicating the terminology.
The Age 0 ring is equivalent to the zero band referred to in this report. The anterior and posterior
regions of the otolith above are in accordance with the orientation of the eel (source: Christine
Gazeau, CEMAGREF).
Also useful: http://www.cmima.csic.es/aforo/index.jsp
ICES WKAREA Report 2009
4
| 8
Introduction
Eel age determination for Atlantic eel has long been problematic with much debate
on both the techniques and the interpretation with relatively few validation studies.
Validation is difficult given the terminal nature of ageing with otoliths and also the
relatively slow growth and long life cycle often involving different habitats. Ageing
using sagittal otoliths, rather than other structures such as scales and opercular
bones, appears to be the only viable option for eel and the extraction of eel otoliths
was described by Moriarty (1973).
The results obtained using different preparation methods may vary considerably
(Moriarty & Steinmetz, 1979; Moriarty, 1983; Berg, 1985; Vøllestad, 1985; Vøllestad &
Næsje, 1988; Fontenelle, 1991; Poole et al. 1996b) but few have been validated. The
ageing of slow growing eels and the occurrence of supernumerary zones has caused
much confusion (Dahl, 1967; Moriarty, 1972, 1983; Deelder, 1981; Poole et al. 1992)
although subsequently, the 'burning and cracking' method was validated in some
situations (Moriarty & Steinmetz, 1979; Moriarty, 1983; Vøllestad & Næsje, 1988;
Poole et al. 1996a). Burning and cracking was then recommended by an EIFAC eel age
workshop in 1987 as the best option for ageing eels (Vøllestad, Lecomte-Finiger &
Steinmetz, 1988), particularly for the slow growing and older specimens (e.g.
Vøllestad & Næsje, 1988). There have been many developments since 1988, both in
improved otolith preparation techniques, imaging and validations along with the use
of eels of known age and chemical marking of otoliths.
An initial session was held to commence the workshop looking at eels of known age
from Sweden, examples of otoliths from eels of known and unknown age prepared
by burning and cracking and by grinding and polishing and a range of other otoliths
including burnt and cracked glass eel otoliths for identifying the zero band where
continental phase ageing commences from.
Photo: Old Irish Eel
ICES WKAREA Report 2009
5
Otolith Preparation Techniques
5.1
Introduction
| 9
Ageing of fish from their calcified structures is the basis for much of the present day
fisheries management assessments and advice. Determination of age for Atlantic eel,
Anguilla anguilla and A. rostrata, has been especially problematic with much debate on
both the techniques and the interpretation with relatively few validation studies.
Validation is difficult given the terminal nature of ageing with otoliths and also the
relatively slow growth and long life cycle often involving different habitats. Ageing
using otoliths, rather than other bony structures or scales, appears to be the only
viable option for eel and the extraction of eel otoliths was described by Moriarty
(1973).
Age determination has largely depended on the use of sagittal otoliths since their use
for this purpose was first demonstrated by Ehrenbaum & Marukawa (1914). A wide
variety of preparation and staining techniques have been employed with varying
degrees of success and remarkably little validation (Vøllestad, Lecomte-Finiger &
Steinmetz, 1988). Christensen (1964) reported that eel otoliths could be successfully
prepared by burning in a flame and cracking to reveal the annuli on the broken face
of the otolith. The preparation of eel otoliths was described by Moriarty (1973) and
developed by Hu & Todd (1981). The results obtained using different preparation
methods vary considerably (Moriarty & Steinmetz, 1979; Moriarty, 1983; Berg, 1985;
Vøllestad, 1985; Vøllestad & Næsje, 1988; Fontenelle, 1991; Poole et al. 1996b) but few
have been validated. The ageing of slow growing eels and the occurrence of
supernumerary zones has caused much confusion (Dahl, 1967; Moriarty, 1972, 1983;
Deelder, 1981; Poole et al. 1992) although subsequently, the 'burning and cracking'
method has been validated in some situations (Moriarty & Steinmetz, 1979; Moriarty,
1983; Vøllestad & Næsje, 1988; Poole et al. 1996a). Burning and cracking was then
recommended as the best option for ageing eels (Vøllestad, Lecomte-Finiger &
Steinmetz, 1988), particularly for the slow growing and older specimens (e.g.
Vøllestad & Næsje, 1988). Svedäng et al. (1998) observed “The grinding method is as
useful and as valid as burning and cracking (Holmgren & Wickström, 1988; Vøllestad
& Jonsson, 1988).”
It is important to note that the preparation technique may vary depending on the
requirement. For example, preparation for gross ageing may differ from that for
scanning electron microscopy (SEM) or laser ablation. This report deals only with the
preparation for gross age determination.
5.2
Otolith preparation techniques – pre 1988
A comprehensive review of otolith preparation techniques was undertaken by EIFAC
in 1987 and published in 1988 (Vøllestad, Lecomte-Finiger & Steinmetz 1988). The
report discusses grinding and slicing, burning and cracking and whole otoliths ("in
toto"). The following is a brief summary of the main points:
a ) Grinding, polishing and slicing
Grinding and slicing otoliths was the most common preparation technique
used by almost all institutes, with their own modifications introduced to the
standard process. The convex side was usually ground although some labs
ground both sides. Slicing of otoliths to 0.2mm thickness was used in a number
of studies but was generally found to underestimate the age of eels.
ICES WKAREA Report 2009
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The use of Scanning Electron Microscopy, SEM, is good for examining external
structures and internal microstructure but is not appropriate for routine ageing
of large numbers of eels due to prohibitive preparation times and high costs.
Acetate peel was used for elvers and small eels less that 20cm. An acetate
impression was made from ground and etched otoliths. This technique was
used by Liew (1974) when discussing the annual formation of translucent and
opaque zones and their microstructure.
b ) Burning and cracking
Christensen (1964) first reported that eel otoliths could be successfully prepared
by burning in a flame and cracking to reveal the annuli on the broken face of
the otolith. The preparation of eel otoliths was described by Moriarty (1973)
and developed by Hu & Todd (1981) and has been used in a range of studies
involving larger eels. The otolith is burnt either before or after cracking (Note:
it is now recommended to cut the otolith before burning). It is important that
the flame is not too hot as incomplete, or patchy, incineration can occur.
A variety of mounting and viewing techniques were also described with
otoliths being mounted cut face up and immersed in a clearing fluid such as
cedarwood oil, cooking oil or a refracting medium. A more permanent mount
can be made by mounting the otolith in silicone sealant against a glass slide
(Hu & Todd, 1981).
c ) Whole otoliths
The "in toto" whole otolith method has been used since the early 20th century
(Ehrenbaum & Marukawa 1914). Otoliths can be cleared in 96% ethanol for up
to 48 hours before reading. It was concluded that this method generally
underestimates the age of older, slower growing eels.
5.3
Otolith preparation techniques – post 1988
Since 1988, significant advances have been made in both the preparation and the
viewing and image capture of otoliths. The following sections detail the main
techniques now used.
a ) Grinding and polishing
Six main protocols exist for the preparation of otoliths by grinding and
polishing, and are used in institutes in Belgium, Canada, France, Germany,
Italy, United Kingdom, Sweden and the USA (Table 5.1). The general principle
involves the mounting/embedding an otolith concave side down on a glass
slide. The bottom and sides of the otolith are affixed to the slide and the
adhesive is then lightly drawn over the top surface of the otolith which fills in
tiny crevices on the surface of the structure. The otolith is examined under a
dissecting microscope using a variety of light types including transmitted,
reflected and polarized. Annuli are rarely sufficiently visualized at this point
and as such the dorsal surface is lightly ground in the sagittal plane and then
polished using a variety of silicon carbide wet-dry sandpapers. From this point
onwards a variety of different techniques involving etching or staining is
employed to help improve visualization of annuli. It is these different
techniques which account for most of the variations between laboratories.
Full details of the Standard Operating Procedures (SOP’s) for each of these
methods can be found in Annex 3.
ICES WKAREA Report 2009
| 11
Table 5.1. Summary protocols for the preparation of otoliths by grinding and polishing for each
country.
FRANCE
JAPAN
SWEDEN
N.IRELAND
GERMANY
USA/
CANADA
Concave
side down
Transverse
Concave
side down
Concave side
down
Transverse
Concave
side down
Sagittal
grind
Transverse
Sagittal
grind
Sagittal grind
Transverse
Sagittal
grind
Embed
Epoxy resin
SpeciFix +
SpeciFix-20
curing
agent (5.2:1)
N/A
N/A
Clear mount
wax
N/A
Mount
Crystal
bond
Doublesided tape
(flip upside
down for a
“double
trim”)
Crystal
bond
Loctite Brand
super glue
Crystal bond
Epoxy resin
Grinding
Grinding
wheel (150
t/min, 1200
grit with
water until
otolith is
reached;
switch to
2400 grit.
Check
every 20
secs under
microscope
until you
reach the
nucleus
Grind with
“Discoplan
” using
70µm
grinding
paper to
“some
degree”
and then
switch to 13
µm
grinding
paper. Both
sides are
ground
Grind
manually
with 1200
grit until
centre and
edge are
visible;
water is
added
constantly
Grind
manually with
graded sand
paper in a
figure-8
pattern. Check
slide often to
see when centre
is exposed.
Grind
manually
with a series
of grinding
paper: 600,
800 and 1200
grit down to
0.1–0.2 mm
800 grit
paper
Polishing
A paste
with
aluminium
powder
1µm and
water.
Polish
manually.
Polishing
disk with
DPLubricant
Blue (wet)
and DPPaste
(diamond
paste) *very
specific
details for
the
polishing
machine.
3 µm (4000
grit) with
water
constantly
Jewellery
polishing cloth
Alumina
powder
slurry (0.3
μm, mixed 1 :
10 with
distilled
water) on the
plastic slide,
then cleaned
with distilled
water and on
a wet fibre
polish.
Aluminium
powder
Etching
5% EDTA
for 3 mins
1% HCl for
1 min. or 35% EDTA
for 3-5
mins.
1% HCl for
1 min. or 35% EDTA
for 3-5
mins.
N/A
N/A
5% EDTA
for 3 mins
Washing
Distilled
water
Distilled
water
N/A
N/A
N/A
N/A
Otolith
Alignmen
t
grind
grind
.
ICES WKAREA Report 2009
Staining
5%
Toluidine
blue
(smeared),
dry over
night
| 12
1%
Toluidine
blue
If HCl
stained 30120 mins
20 ml
neutral red
solution
(3.3g/L).
SIGMA* 1
N/A
N/A (crack
and burn is
done first to
highlight the
rings)
5%
Toluidine
blue
(smeared),
dry over
night
Distilled
water
N/A
N/A
N/A
If EDTA
stained 5-30
mins.
Rinse
Distilled
water
Distilled
water
(2 mins)
Recommendations:
•
Grinding and polishing typically useful for smaller/younger eels. Otoliths
from larger, older eels may be difficult to interpret in the outer bands due
to slow growth between annuli. There is also a danger of under-grinding
larger curved otoliths and hence arriving at the nucleus before the outer
annuli are exposed, leading to under-estimation of age. Therefore burning
and cracking is recommended for the older larger specimens
•
Care should be taken to modify protocols according to your set of otoliths
(e.g. grinding time, etching time, etc.)
•
Consideration should also be given to the long term storage envisaged for
mounted otoliths before choosing a mounting medium, e.g. experience
within the Working Group has found that some otoliths mounted using
super glue type media may detach from slides after a 2 year period. Similar
problems have not been noted with crystal bond or epoxy resin.
Summary statements:
•
The techniques are similar across protocols with preparatory methods also
being similar, and the major variations coming in ways to aid the
visualization of annuli such as etching and staining.
•
Some techniques appear to be quite laboratory-specific and also time
consuming.
b ) Slicing
Previous attempts at slicing led to serious under-estimates of age (Vøllestad,
Lecomte-Finiger & Steinmetz, 1988). This method has been recently attempted
by CEFAS (UK) (M. Etherton pers. comm.) and is based upon a variation on the
grinding and polishing theme of otolith preparation listed above. Otoliths are
embedded in black epoxy resin, a transverse section (0.2mm thick) is cut
through the nucleus that is perpendicular to the sulcus using an Accuton
diamond coated saw and viewed under reflected light. This method is still in
its infancy and as yet is not regarded as being suitable for reading eel otoliths.
Similar attempts were made using an otolith saw by the Swedish Board of
Fisheries (Wickström pers. comm.)
1
SIGMA-ALDRICH Product reference: N2889
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However, a similar US method for A. rostrata otoliths utilizes the same
preparatory methodology which has then been modified to include etching
using EDTA followed by staining using toluene blue (Stirratt, 2001) and this
was found to improve the method considerably.
c ) Burning and cracking
There have been relatively few studies of eel age and growth using burning
and cracking published since 1988. Poole has studied age of old slow growing
yellow (Poole et al., 1992) and silver (Poole et al. 1996a, b) eels from the west of
Ireland using traditional burning and cracking (Moriarty, 1973, 1983) mounted
in silicone (Hu & Todd, 1981). Matthews et al. (2001) aged eels from a large
study of the Erne catchment in NW Ireland and used silicone mounted otoliths
for back-calculated growth analysis. Poole (1994; Poole et al. 2004) also
successfully used burning and cracking to examine the otoliths of glass eel and
juvenile bootlace eel in order to confirm the "zero" age band (see Section 6.2).
Burning and cracking otoliths of such small eels is not an easy process but it
was useful for confirming the structures observed in the older eels.
Cutting the otolith through the nucleus before burning has been used in a
number of studies (Graynoth 1999; Richards, 1989; Todd, 1980). This reduces
the number of otoliths that shatter, or break unevenly, and improves the flat
viewing surface for mounting against the glass slide. This system was also
recently used successfully by Maes (unpublished) on otoliths from around
Europe.
For otoliths being prepared by burning and cracking for digital image capture
and growth analysis, pre-cutting produces a flat face that is easier to capture all
in focus, as being used in current Irish eel growth studies (O'Toole & Poole
unpublished).
Simon (2007) has employed a method involving burning before cutting and
grinding, adapted from Secor (1992). This method employs the burning stage as
a method of highlighting the bands, equivalent to staining, before preparing
the otolith using the grinding methodology. The drawback here is the extended
time required for grinding after burning and at times the otolith lacks
definition and contrast. The definition and contrast can be improved by
varying focus and light and reading from the actual otolith rather than the
image.
d ) Whole otolith clearing – "in toto"
The "in toto" whole otolith method has been used since the early 20th century
(Ehrenbaum & Marukawa, 1914) and is still used to age young eel populations
(Panfili et al. 1994a; Melià et al. 2006; Cullen & McCarthy, 2003).
The whole right and left otoliths are immersed into rosemary essential oil or
96% ethanol (in order to enhance the visualization of the growth marks) and
read under a microscope with reflected light against a dark background.
Other media used to immerse otoliths were: propandiol, camomile essential oil
or less so now, creosote oil.
Clearing otoliths whole has the advantage of being a quick method and is
efficient for ageing young eels.
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Clearing otoliths whole is not an appropriate method for slow growing eels
and it underestimates age of eels older than about 5 years old (Vøllestad &
Næsje, 1988; Panfili et al. 1994b). Therefore, it is recommended that clearing
otoliths should be used with caution.
5.4
Validation of Age Determination
Numerous efforts have been used in an attempt to validate the age assessments of
fish. Validating eel age is particularly difficult given their longevity and ability to
introduce intra-annular growth checks. A wide variation in eel length at age has also
been observed. The most powerful method providing direct evidence is to use
otoliths of known age by using, for example, stock fish that have lived or been reared
under natural conditions. Other methods of gaining direct evidence of the validity of
an age method is by mark-recapture methods, preferably accompanied by marking of
the otoliths, by for example tetracycline or alizarin (Figure 5.1). Other less direct
methods include mark-recapture estimates of growth compared to calculated growth
from otoliths for similar age groups, length frequency modes, examination of the
outer edge of the otolith during the season and comparison between different
methods.
5.4.1
Pre-1988 Validation
Included in the review by the EIFAC Working Party on Eel 1987 (Vøllestad, LecomteFiniger & Steinmetz 1988) for each preparation method was a section on age
validations, summarised here:
a ) Grinding and Polishing
Lecomte-Finiger (1985) confirmed that the formation of the opaque and
translucent zones in the south of France was annual by following the zones
through the season. Wickström (1987) found using stocked eels of known age
that otoliths roughly corresponded to known age, although problems could
occur with false checks in summer and late resumption of growth in spring.
Tulonen (1988) found, using eels of known age, that reliable estimates were
obtained up to 7 or 8 but interpretation of older eels was more unreliable.
Grinding both sides of the otolith had not been verified or validated by 1988.
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Alizarin marked in ‘97
& recaptured in ‘05
Figure 5.1. A. anguilla otolith (8 years) marked with Alizarin Complexone (upper left in reflected
light, upper right in transmitted light and lower in UV-light).
b ) Slicing
The method of slicing the otolith was tested on eels of known age by Moriarty
& Steinmetz (1979) and this was found to underestimate the age. There was a
difficulty in interpretation of double rings and thus the method does not
overcome the subjective nature of ring interpretation. Two Norwegian studies
(Haraldstad, 1984; Bergersen, Klemetsen & Sommerseth, 1987) both concluded
that slicing underestimated the age of eels. Berg (1985, 1986) validated this
technique using mark-recaptures up to 5 years after tagging to compare with
growth and was able to demonstrate false annuli.
c ) Burning and cracking
Several attempts have been made to validate the burning and cracking method
for eels. The ageing of eels of known age (two years) from ponds was
confirmed that the method gave good results (Rasmussen, in Vøllestad,
Lecomte-Finiger & Steinmetz 1988). Moriarty & Steinmetz (1979) showed that
the method can over-estimate the age of young eels but was the best method
for older eels. Interpretation of double and multiple rings can cause apparent
problems. Eels of known age (15 years) from a pond in the Netherlands were
studied by Vøllestad and Næsje (1988) and at a younger age by Moriarty &
Steinmetz (1979). About 26% of the eels were aged correctly and this could
have been higher as the sample may have been contaminated by additional eels
of unknown age. They concluded that burning and cracking was a good
method for eels with slow growth.
Mean annual growth increments (Moriarty 1983) were calculated for Irish eels
and these compared favourably with growth increments determined from
tagged eels from the same location.
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Dekker (1986, 1987) verified, using tetracycline labelled otoliths, that burning
and cracking worked well and the last annulus was correctly identified in over
90% of the otoliths and Aprahamian (1987) was able to recognise year classes of
stocked elvers using this method.
d ) Whole otoliths – in toto.
In faster growing eels, 80% of multiple readings gave differences of less than
one year and opaque and translucent zones were confirmed as annular
(Vøllestad, 1985, 1986). In slower growing eels, however, Vøllestad & Næsje
(1988) concluded the method was unsuitable for ageing slower growing eels.
5.4.2
Comparison between methods
Moriarty & Steinmetz (1979) compared five different techniques for ageing eels using
otoliths from eels of known age. There were considerable differences, 3 to 10 years,
between the different methods. Grinding and polishing gave the best results on
young specimens whereas burning and cracking gave the best result with the older
eels. Eels from the same pond were burnt and cracked and cleared in ethanol and the
difference was 4.5 years (Vøllestad & Næsje 1988). Other studies summarised in
Vøllestad, Lecomte-Finiger & Steinmetz (1988) showed similarities between methods
with burning and cracking giving comparable or older readings that in toto readings.
Cullen & McCarthy (2003) compared burning and cracking and clearing whole
otoliths in creosote and concluded that both methods yielded similar results. Clearing
may be appropriate for samples containing high numbers of small eels. Burning and
cracking was generally found to be a faster method than other preparation methods,
particularly for larger and older eels.
5.4.2.1 Grinding & polishing and burning & cracking (post-1988)
Four studies were undertaken prior to the workshop to compare otoliths from the
same eels prepared by the different methods, in order to compare the zero age band,
terminal age and common features between the otoliths.
Swedish eels of known age: otoliths of eels from Swedish lakes stocked with glass eel
of known age were prepared by grinding and staining with neutral red and by
cutting and burning and mounting in silicone. Figure 5.2 is of a five year old eel
showing good comparison between the two methods. The zero band is clearly visible.
A faint check is visible after the zero band due to the quarantine period. Figure 5.3 is
of an 8 year old eel which was identified as being 9 years by burning and cracking.
Clearly visible in the first year are two false checks due to stress related to strontium
marking of the otolith and PIT tagging before stocking. Figure 5.4 is of an 11 year old
eel and there was good agreement between the methods. A false check due to
quarantine is visible after the zero band.
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Figure 5.2. Otolith #30128 (known age – 5; captured in May) with the cut and burnt otolith
(transverse plane) (Ireland) on the left and the ground otolith (sagittal plane) (Sweden) on the
right. The arrow indicates the zero band and the ages were interpreted by the image creators.
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Figure 5.3. Otolith #30128 (known age – 8; captured in May) with the cut and burnt otolith
(transverse plane) (Ireland) on the left and the ground otolith (sagittal plane) (Sweden) on the
right. The arrow indicates the zero band, the circles indicate growth checks and the ages were
interpreted by the image creators.
Figure 5.4. Otolith #30128 (known age – 11; captured in May) with the cut and burnt otolith
(transverse plane) (Ireland) on the left and the ground otolith (sagittal plane) (Sweden) on the
right. The arrow indicates the zero band, the circle indicates a growth check due to quarantine
and the ages were interpreted by the image creators.
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Swedish West Coast Grinding: A pair of otoliths from wild eels from the Öresund
was ground, in the transverse plane and the other in the sagittal plane (Figure 5.5).
The eel was a female of 67.1cm length and had an estimated age of 16. The view of
the transverse plane is similar to the view achieved in burning and cracking.
Common banding was identified between the two views although some double
banding was also apparent. Indicated in the figure are the first annulus outside the
zero band and the 5th, 10th and 15th annuli.
This process of grinding in the transverse plane would be suitable for preparing
larger otoliths from eels of older age, similar to burning and cracking. It is, however,
a more laborious, technically more difficult process to mount the otoliths for
transverse grinding and consequently it is a slower procedure. It may have
application where older eels with otoliths marked by OTC or other chemicals need to
be examined, but the marks would be destroyed by burning.
Figure 5.5. An example of a Swedish west coast silver eel otolith that was ground and polished in
the transverse plane (left) and in the sagittal plane (right). The white dot indicates the zero band
and the white slashes indicate years 2, 5, 10 & 15.
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Lough Furnace, Ireland: Otoliths from yellow eels taken from Lough Furnace in
Ireland were aged using two methods, burning and cracking and grinding and
polishing, with one otolith from each otolith pair being prepared using one of the two
methods. Comparisons were then carried out between each otolith pair. Overall, the
results showed good comparison between the two methods used. Figure 5.6 shows a
pair of eel otoliths with the burnt and cracked otolith aged at 20 years, while the
corresponding ground and polished otolith was aged at 21 years.
Figure 5.6. Irish yellow eel otolith (#FuBr01-47) from L. Furnace with the cut and burnt otolith
(transverse plane) (Ireland) on the left and the ground otolith (sagittal plane) (N. Ireland) on the
right. The arrow indicates the zero band and the ages were interpreted by the image creators. The
ground otolith was not stained but viewed under polarised light. The image shown here is not as
clear as the original as polarised light was not used in the photograph.
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Lough Neagh, Northern Ireland: from silver eels taken from Lough Neagh in
Northern Ireland were aged using two methods, burning and cracking and grinding
and polishing. One otolith from each otolith pair was prepared using each method.
Comparisons were then carried out between each pair. Overall, the results showed
good comparison between the two methods used. Figure 5.7 shows a pair of eel
otoliths with the burnt and cracked otolith aged at 12 years, while the corresponding
ground and polished otolith was also aged at 12 years. This pair of otoliths also
shows a false check between the zero band and the annulus showing the end of the
first year’s growth (Figure 5.7). This is thought to be either a stocking check or due to
stress related to a quarantine period pre-stocking.
Figure 5.7. Irish yellow eel otolith (#NeaghBr03-06) from L. Neagh with the cut and burnt otolith
(transverse plane) (Ireland) on the left and the ground otolith (sagittal plane) (N. Ireland) on the
right. The arrow indicates the zero band and the ages were interpreted by the image creators. The
ground otolith was not stained but viewed under polarised light. The image shown here is not as
clear as polarised light was not used in the photograph.
5.4.3
Recent Validation (post-1988)
Validation of age determination by different preparation techniques should ideally be
undertaken using eels of known age, preferably from annual sampling from different
growth environment to cover inter and intra variation (Klumb et al. 2001).

Direct methods for ageing validation could be:
1.
Chemical marking like Oxytetracycline (OTC) (Figure 5.8 & 5.9),
Alizarin complexone (ALC), Strontium chloride (Figure 5.10),
preferable with glass eel (Chisnall & Kalish 1993; Dekker 1986; Cieri
& McCleave 2001, Simon & Dörner 2009).
2.
External color marking, for example Alcian blue applied with Pan Jet
(Chisnall & Kalish 1993; Poole & Reynolds 1996a).
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Indirect methods could be used with eel:
1.
PIT Tagging in conjunction with chemical marking (no need for back
calculation)
2.
PIT Tagging without chemical marking (back calculation needed)
3.
Coded wire tag (CWT) with eel >1g (Thomassen et al. 2000)
4.
Introduction of unmarked glass eel in an eel free waterbody
(Vøllestad & Næsje 1988; Wickström et al. 1996).
5.
Repeated sampling in the same year (marginal incremental method)
(Lecomte-Finiger, 1985; Oliveira 1996; Arai et al. 2000).
Caution: Back calculations should be used carefully so as not to violate basic
assumptions related to potential decoupling of otolith and body growth (Klumb et al
2001), particularly in the first year after the zero band (Poole et al., 2004). Do not use
marking and tagging methods that may disturb growth and/or induce false checks in
the otolith for validating growths. Validation with known age populations cannot be
directly extrapolated for areas with different growth potential (ecozone).
Figure 5.8. Otolith from an oxytetracycline hydrochloride marked glass eel (A. anguilla)
recaptured three years.
ICES WKAREA Report 2009
Figure 5.9. Otolith from OTC marked glass eel (A. rostrata) recaptured nine years later.
Figure 5.10. A. anguilla otolith marked with Strontium chloride.
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5.4.4
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Supporting Case Studies
5.4.4.1 Otolith Marking – Swedish and Canadian Studies
There are some direct and indirect marking experiments (still in progress) done with
A. anguilla and A. rostrata that could validate age estimation. In Sweden and Canada,
juvenile eels were marked with OTC or ALC and collected and sampled on an annual
basis to identify and describe growth increments over time in order to discriminate
between false and real annual checks. In general, our age reading corresponds to true
age but raises concerns about stress factors inducing abnormal checks, such as
waterfalls, dams, marking/handling procedures. Migration barriers could modify the
growth trajectory by investing more energy in migration instead of somatic growth
(Wickström, pers. comm.; Verreault, pers. comm.), as demonstrated in Figure 5.11.
For eels sampled early in the season (before growth has resumed) the last true annual
check is not yet visible or situated very close to the outer margin (Figure 5.12).
Figure 5.11. An otolith from A. rostrata, captured below a dam. The red lines indicate true annuli
for this 9 year old eel.
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Figure 5.12. An otolith from A. anguilla of known age (5y) with the last check close to the margin.
The red arrow indicates the last annulus and the black arrow indicates the mark due to Alizarin
marking and handling.
For otoliths with repeated narrow checks it might be difficult to detect and
discriminate between true and false checks, especially for the latter years in older
specimens (Figure 5.13). Readers must be cautious as handling and marking may
induce false checks, often weak but could still be misinterpreted as annual checks
(Figures 5.14 and 5.15). This leads to overestimation of age.
Figure 5.13. Otolith A. rostrata of known age (8y) with more than eight checks detected.
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Figure 5.14. A. anguilla otolith from marking experiments indicating weak false checks induced
by marking/handling procedures (Strontium treatment).
Figure 5.15. A. anguilla otolith from marking experiments indicating weak false checks induced
by marking/handling procedures (Alizarin treatment).
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On the contrary, we could also underestimate age in old eels where the last annual
checks cannot be clearly seen due to the minimal space between checks (Figure 5.16.)
For eels sampled during growth season, supernumerary checks could be seen and
misinterpreted as true annual checks (Figure 5.13).
Back-calculation done on PIT tagged eels (A. rostrata) trapped and measured for
length three times during its lifetime on the St. Lawrence River, allows us to infer
annual checks from the last validated annual check at the outer margin backwards to
the one corresponding to the first tagging event 11 years ago (Figure 5.17).
Figure 5.16. A. anguilla otolith of known age (19y) with less than 19 annual checks detected.
Figure 5.17. A. rostrata otolith with marking events (red arrows) allowing the identification of
the last eleven years.
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5.4.4.2 Coded Wire Tagging and Otolith Marking – German Study
For a stocking experiment, seven isolated lakes, without access to natural
recruitment, of <20 ha in size in the Federal State of Brandenburg (Germany) were
stocked with glass eels obtained from England in April and farm eels obtained from
commercial eel farms in May. Before stocking, all glass eels were either marked in a
bath with oxytetrazycline hydrochloride (OTC) or with alizarin red S (ARS) (Simon &
Dörner 2005). All farm eels were individually tagged with a sequential coded wire
tag (S-CWT) as described by Simon & Dörner (2005). One year after stocking,
monitoring of the stocked eels commenced and this was carried out every year until
2008. In May, all lakes were sampled. Sagittal otoliths from the sampled eels were
extracted, one was checked for the otolith marks and the other prepared by the
burning, cracking and grinding method described by Simon (2007). From the second
otolith, a back-calculation of growth was calculated using the adapted von
Bertalanffy growth curve (von Bertalanffy, 1957). The otoliths were examined to see if
the visible glass eel otolith and winter rings measurements corresponded with the
annual growth in the field (after Berg, 1988). In the Figure 5.18 you can see an otolith
from an eel captured in Lake Bohnenlander four years after stocking as glass eel.
Figure 5.18. Otolith from an eel captured in Lake Bohnenlander four years after stocking as glass
eel (black arrow is the zero band, white arrows are the annual rings)
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5.4.4.3 Mark-recapture Growth and back-calculated growth comparison – Irish Study
The age and growth of eels from three west of Ireland lakes were compared with
annual increases in length of eels from a mark-recapture study using unique alcian
blue tattoos (Poole & Reynolds 1996a). There was good agreement and the recapture
data compared favourably with the back-calculated growth rates thereby validating
the otolith analysis for eels in the Burrishoole system.
A subsequent recapture of an eel marked on 5/June/87 at 38.1 cm was recaptured in
the downstream fish traps as a silver eel on 27/September/2005 at 56.5 cm (Poole pers.
comm.). This eel grew on average 1.02 cm per year which compares favourably with
a mean female silver eel back-calculated growth increment of 1.4 cm per year.
5.4.4.4 Ageing and modelling – L. Neagh Study
Historical ageing studies utilizing burning and cracking methodology on Lough
Neagh silver eels concluded that the average age of escaping eel was 10-13 yrs for
male and 15-20 yrs for female, (Anonymous, 1969). More recently, Rosell et al (2005)
supported such estimates at 12yrs and 17yrs respectively via the use of otolith
grinding and polishing ageing techniques. Additionally, both estimates of age also
match with peak statistical relationships in the input to output time series, with
significant correlations at 18 years between glass eel input measure and total catch
(Allen et al., 2006) thereby validating the age determination by grinding and
polishing.
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Guidelines for Interpretation
6.1
Related Field Data
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The following field data, where possible should be recorded for each eel otolith and
be available along with the image (the image should be saved with the eel unique ID
number attached):
Unique Eel Identification Number
Location of Capture
Date of Capture
Length, and weight if available
Sex
Habitat type
Other features – e.g. wild, stocked, pre-grown in culture
6.1.1
Options for reading with/without a priori knowledge of vital information
During the workshop, discussion ensued about whether otolith readers should have a
priori knowledge of various aspects of vital information about the eel such as : site,
sampling date, length, weight, gender and life history details (e.g. life stage at time of
capture, or management history - quarantined and stocked). Since it is recommended
that age is estimated from each otolith by at least two readers, and preferably three
(See additional details about this in Chapter 7), there are therefore two main
approaches to providing vital information on the otoliths being processed :
6.2
•
Vital
information
fully
available
to
all
readers
In most cases, given the relationship between stress and metabolism and
the creation of annual and false check bands within otoliths, it is advisable
that the readers have vital information to assist with correct age
interpretation – this could save much time and effort if it prevents
erroneous interpretation.
•
Selective
vital
information
withheld
from
all
readers
There may be situations when a priori information might bias a reader’s
approach to age interpretation and a blind test is strongly recommended
(Nielson, 1992). If you have background knowledge of information likely
to influence your decision on age you should not partake in the blind test.
For example, when ageing otoliths of eels sampled in contaminated versus
reference sites, site information should be withheld and otoliths should be
processed randomly; otherwise, readers may bias their interpretation of
age/growth in accordance with their expectations of results.
Continental age – where to start? "Zero bands"
The ageing of the eel for the continental phase, has traditionally commenced from the
first clearly marked band outside the nucleus. This ‘zero’ band is assumed to be the
beginning of continental growth in the eel, and equates to the total length for the
glass eel (Moriarty, 1983; Poole et al., 2004). The next annulus is considered to be the
end of the first year of growth (Figure 6.1).
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Figure 6.1. Upper image: A burnt and cracked otolith (transverse plane) from A. anguilla
showing the location of the zero band and first winter annulus.
Lower image: A ground, polished and stained otolith (sagittal plane) from A.
anguilla showing the location of the zero band and first winter annulus.
This workshop focussed on the continental phase of growth. Growth in the oceanic
phase is not part of the terms of reference, and is not considered here. In addition,
‘elver’ and ‘elver checks’ are ambiguous terms that are not going to be applied here.
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Berg (1985), working far from the sea in Lake Constance, had difficulty in defining
the ‘zero’ age and chose the circumferential outline of the leptocephalus otolith as a
clearly visible starting point. Naismith and Knights (1988) burnt and cracked small
eel otoliths from the freshwater Thames River and described those without a growth
ring outside the ‘elver centre’ as 0+ fish. In a study by Poole et al. (2004), glass eels
were sampled from lower estuary to freshwater in the Burrishoole catchment over a
full season of arrival, migration and development. This permitted direct observation
of the stage at which the first dark band on the burnt otolith developed, coinciding
with the first winter and arrival/transition in the estuary, and hence the validation of
its significance as the zero age band in age determination.
Progressive changes in Burrishoole otoliths over time from November 1987 to August
1988 clearly demonstrated that the first dark band outside the nucleus is laid down
during the sojourn in the estuary between November and April (Figure 6.2). As such,
the band coincides with both the estuarine transition and the first winter in the
estuary and is a justified zero point for the origin of both age and growth
determination.
Figure 6.2. Images showing deposition of the zero band from November 1987 to August 1988 in
glass eels from the Burrishoole Catchment, Ireland. Otoliths were prepared using burning and
cracking in the transverse plane and show the location of the zero band and a radius of
approximately 170 um.
Additional points for consideration when identifying the zero band:
•
The nucleus in European eels is roughly spherical in nature, which aids its
identification in ground and cracked otoliths, as can be seen in Figure 6.3.
•
When ageing European eels, the radius from the centre point of the
nucleus to the zero band for burnt and cracked otoliths has an average
value of 170µm (Figure 6.2). For ground and polished otoliths, a diameter
value of 340µm is used to help define the location of the zero band (Figure
6.4).
•
The American eel, Anguilla rostrata, has average measurements of 120 µm
for the radius and 240 µm for the diameter.
•
For southern European eels (e.g. Portugal and Italy), some recruitment of
unpigmented glass eels occurs all year round and not only in peak periods
of recruitment from November to March (Gandolfi Hornyold, 1931; Petit &
Vilter, 1944; Bessa & Castro, 1994), so there may not be such an easily
defined start point that coincides with a winter zone as observed above.
The zero band radius/diameter measurement could therefore be more
useful in determining a starting point for measuring continental growth
(Figure 6.5).
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It is recommended that a study be conducted in Portugal and the Mediterranean area
on newly arriving glass eels and their progression through time, examining the
development of the eels and their otolith marking structures in order to clarify the
visible structures on the otoliths in these areas.
Figure 6.3. Sagittal plane of ground A. rostrata (3y), otolith showing the spherical nucleus and
the zero band.
Figure 6.4. Image of an A. anguilla sagittal plane otolith with an inserted circle of diameter 340um
indicating the location of the zero band (visible just inside the green circle) and a radius of 170
µm.
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Figure 6.5. Otolith from a Mediterranean eel showing a separation between the transition check
when the glass eel arrives and the first winter annulus. The transition check (150um) is analogous
to the zero band as shown in Figures 6.2 & 6.4.
6.3
Annual (winter) rings and false checks
Rings are deposited on the otoliths each year, alternating normally one opaque (in
summer) and one translucent (in winter) (Table 6.1). Winter checks are thin and
narrow because the accretion rate varies with growth of the fish, and during cold
months eel metabolism is slow. A year’s growth consists of both an opaque and
translucent zone.
Table 6.1: Classification of growth zones.
Ring type
Growth
Dimensional
Zoning
Reflected
light
Transmitted
light
appearance
Winter ring
slow (winter)
Thin and narrow
translucent
dark
bright
Summer ring
Fast (summer)
Thick and wide
opaque
bright
dark
During early attempts at eel age determination during the first decades of the 1900s,
scientists considered that every check corresponded to a winter ring. Later, Dahl
(1967) and other scientists found that is also possible that additional checks were
developed during one annual growth period. Moriarty (1975) found two additional
checks in one year. Therefore, an overestimation of eel age may occur and
consequently an underestimation of eel growth if each check is counted as an
annulus. The problem of such additional checks is that they might look like winter
rings and they were not developed consistently on each eel from a population
synchronously in each year from eels on one water body (Dahl 1967, Simon 2003).
Liew (1974) found similar additional checks in the American eel.
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A helpful way to discriminate between true annuli and false annuli is to count only
checks where they are:
•
clearly visible as a bold growth check to be considered an annulus,
•
in the case of sagittal plane ground otoliths the growth checks should be
visibly continuous around the otolith to be annuli
•
in the case of burnt and cracked otoliths, the growth checks should be clear
and continuous, at least down the inside edge of the otolith
•
false annuli are usually of lesser strength to the annulus, are discontinuous
and/or merge with other adjacent checks.
•
eel growth is highly variable and therefore it is difficult to predict ‘normal’
patterns of annual growth to facilitate the identification of false annuli.
Checks that appear too close to the neighbouring checks may be false
annuli and should be treated with caution. For additional information see
Graynoth (1999).
6.3.1
False annuli
Some possible reasons for false annual (winter) growth checks are given below with
some illustrated examples.
6.3.1.1 Stress related marks - Introduction
The otolith of the eel is typically composed of alternating translucent (or hyaline) and
opaque zones which are deposited with seasonal changes in fish growth (Casselman,
1992). The appearance of the zones was described in Table 6.1.
Metabolic activity has been identified as being linked with both otolith formation and
the creation of bands or checks within otoliths (Mosegaard et al, 1988; Wright, 1991)
who suggested that otolith growth in the first year might be more a reflection of
metabolic activity than of somatic growth whilst Lecomte-Finiger (1992) and Poole et
al. (2004) noted the unexplained increase in otolith size with development of pigment
but no increase in body size.
Translucent zones are deposited during slow growth periods in the winter when
feeding is suppressed (due to falling water temperatures and associated decrease in
metabolism) and are composed primarily of organic materials with lower amounts of
calcium carbonate, whilst opaque zones are deposited during fast summer growth
and are composed primarily of inorganic calcium carbonate (Tzeng et al., 1999).
Otoliths precipitate in the form of aragonite, but aberrant precipitation may result in
vaterite formation instead of aragonite (Gauldie, 1986). Vaterite otoliths are much
more translucent than aragonite. The proportion of vaterite to aragonite can vary in
abnormal otoliths from 20% to 80%. Within a pair of otoliths (sagittae for eel), one
may be a vaterite otolith while the other is a normal aragonite otolith. We recommend
avoiding the use of vaterite otoliths for age reading as growth checks do not appear,
or at least appear only partially in those otoliths.
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6.3.1.2 Impact of Stressors
Catadromous fish, such as eel, must endure a variety of environmental and
physiological stressors with resulting biochemical and physiological consequences.
Such stressors cause an internal hormonal response which induces a physiological
response in an attempt to adapt to that stimulus and as such can disturb the normal
physiological equilibrium, or homeostasis, of fish by forcing a shift in metabolic
activity.
Fish respond to stress in a variety of ways including the release of the stress hormone
cortisol which then induces a variety of physiological modifications ranging from
disturbance in osmoregulation to the inhibition of growth (Munck et al., 1984), effects
which are “recorded” in the deposition of daily rings within an otolith. Given this
relationship between stress/metabolism and the creation of bands within otoliths it is
advisable that a range of variables related to the eels’ life history are known and used
in conjunction with otolith reading by the first reader, whilst this information should
be withheld from the second reader as a pro-viso for quality assurance.
6.3.1.3 Glass eel/elver
Interpretation is characterised by a dark nucleus followed by a single first winter
check, referred to as a zero band (Poole et al. 2004). Unnatural stressors, such as
handling, quarantine periods (e.g. antibiotic treatment, addition of salt), can result in
the deposition of check marks between the zero band and the first annulus. Research
by Wickström, utilizing alizarin complexone and strontium chemical markers
(Wickström, pers. comm.), has indicated that the check marks observed in this way
correlate positively to both quarantine and stocking procedures (Figure 6.6).
6.3.1.4 Yellow eel
Yellow eel biology presents a number of challenges as this growth phase is noted to
be highly variable (Moriarty, 1983; Poole & Reynolds, 1996ab), resulting in periods of
slow and rapid growth reflected in the otolith transect. Numerous environmental
influences can lead to the formation of false/supernumerary checks through changes
in calcium and protein deposition rates due to events such as starvation, low water
temperature and sudden fluctuations in ambient water temperature, (Graynoth 1999).
Additionally, variations in the width of summer bands may be the result of migration
between habitats, during which the growth rate exhibits fluctuation (Jellyman 1997).
As a result, growth rate can be a useful determinant of individual behaviour as life
history trade-off theory supports the observation that eels subjected to stress or
increased activity exhibit reduced growth rates. Morphometrics such as eel length
and/or weight should be recorded and provided as the relationship between
individual eel length and the corresponding size of the relevant eel otolith has been
found to be significant, (Panfili et al, 1994). A record of gender would also be valuable
given that larger (normally female) eels tend to be older than their male counterparts.
(Moriarty & Dekker, 1997; Poole & Reynolds, 1996b; Tesch 2003).
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6.3.1.5 Silver Eel
The metamorphosis of yellow eel from a pre-pubescent stage into the maturing silver
eel stage is coupled to an observed decrease in growth rate (Tesch, 2003).
Subsequently, post maturation growth in the otolith is characterised by reduced
width of opaque zones, whilst additional check marks do not represent winter
growth periods but may be indicative of physiological or environmental stressors
such as silvering metamorphosis or unsuccessful migration attempts (Vøllestad, 1992;
Durif et al., 2006).
Awareness of such checks, and an understanding as to their formation, will enable a
more accurate representation of both growth in this period and overall age at
migration. Additionally, sexual dimorphism and the resulting difference in growth
rate means that knowledge of gender would be valuable for accurately assessing age
in silver eel individuals, (Holmgren & Mosegaard, 1996; Poole et al. 1996b, 2004;
Jessop et al, 2004).
6.3.1.6 Environment
Given the diverse nature of eel habitats, ranging from warm marine lagoons to high
latitude oligotrophic lakes (Tsukamoto, 2001; Tzeng et al., 2003; Harrod et al, 2005),
growth rates can vary considerably between individuals at both the inter and intracatchment level, depending upon temperature, food availability and the physicochemical properties of the habitat (Jellyman, 1997; Daverat & Tomas, 2006). Eels
residing in oligotrophic habitats typically display slower growth rates (Moriarty,
1988; Poole & Reynolds, 1996a, b) than those of mesotrophic (Aprahamian, 2000; Lin
et al, 2007), or eutrophic (enriched) habitats (Rosell, Evans & Allen, 2005; Simon,
2007). However, across the European distribution of eels, age and growth are known
to vary in relation to latitude, with eels’ resident at higher latitudes tending to grow
slower and be older than their southerly distributed counterparts (Vøllestad, 1992;
Panfilli et al., 1994ab; Daverat & Tomas, 2006; Jessop et al., 2008). All of the above
highlight the value of recording environmental data for its use in subsequent ageing
analyses.
6.3.1.7 Stocking and marking procedure
When eels are captured, held and transported for stocking, it can result in the
deposition of additional fine growth checks. Furthermore, several marking methods
for otoliths and eels can induce the creation of false checks (Berg 1986; Wickström
pers. comm.) such as treatments for OTC, ALC, strontium marking or tagging (Figure
6.6 & 6.7).
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Figure 6.6. Otolith from a Swedish eel captured in Lake Ymsen with two additional marking
related growth checks shown by blue arrows.
Figure 6.7. Otolith from an eel captured in Lake Bohnenlander four years after stocking as glass
eel with additional marking related check.
Figure legends:
Black arrow:
Blue arrow:
White arrows:
Zero band
Ring due to marking procedure with alizarin red S before
stocking.
True annual rings winter rings after stocking in the lake.
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6.3.1.8 Summer checks (false annuli)
High water temperatures and associated low oxygen concentrations in summer can
result in stress for eels producing periods of little or no growth, corresponding to the
deposition of a false, or false annuli within their otoliths (Figure 6.8; Tzeng et al. 1994;
Domingos et al. 2006). In addition, periods of cold or other unknown factors in the
wild can also lead to the deposition of checks, or false annuli, on otoliths.
Figure 6.8. Otolith from an unknown age eel captured in River Tiber with one double check in
winter and two different additional checks in summer.
Figure legends:
Black arrow: Zero band
Blue arrows: Double winter ring
Red arrow: False summer rings
White arrows: True annual rings winter rings
6.3.1.9 Quarantine and glass eel stocking
Quarantine periods are required in many countries before the stocking of non-native
eels into the wild. For example, a period of 10 weeks is required in Sweden. This
quarantine period has been shown to lead to the deposition of growth checks similar
to those described in Section 6.3.1.7.
6.3.1.9.1
Affect of culture
Stocked eel, pre-grown in culture for a number of months (e.g. 4-5 months in
Germany), appear to have features in the otolith that are more difficult to interpret
than those from eels stocked as glass eels (Figure 6.9), as they show several fast
featureless growth zones with growth checks deposited during the culture period.
These are not annual rings but most likely growth checks deposited as a result of
sorting, grading or feeding periodicity. An unawareness of this life history would
lead to an overestimation of real age (Figure 6.10).
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Figure 6.9. Otolith from an eel captured in Lake Bohnenlander four years after stocking as a glass
eel.
Figure 6.10. Otolith from an eel captured in Lake Bohnenlander four years after stocking as a pregrown eel.
Figure legends for above figures:
Black arrow:
Zero band
Red arrows:
False growth zones from the time in the eel farm.
Blue arrow:
Ring due to tagging procedure with coded wire tags before stocking.
White arrows:
True annual rings winter rings after stocking in the lake.
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Otolith reading calendar
For both A. anguilla & A. rostrata the following recommendations are made:
•
The age reference date is set as 1st of January.
•
Age is estimated based on the counts of winter rings.
•
The term ‘+’ growth can be used as an indication of additional growth but
this should not confuse the calendar age of the eel.
For yellow eels, it is recommended that attention be drawn to the timing of capture
and local conditions. Depending on the growing season of the capture location, the
appearance of winter annuli on the otolith will vary. In the early part of the year the
outer most winter annulus is generally not apparent until the new year’s summer
growth begins. This will vary between years and between locations. Therefore, an
additional year should be added to these eels sampled early in the year after January
1st in order to account for this undifferentiated annulus from the outer margin of the
otolith.
For silver eels, flexible interpretation in some local situations may be required. Silver
eel traditionally migrate as an annual migration “cohort”. This “cohort” receives its
age from the next January as the eels have completed their annual growth period and
we assume a putative annulus on the outer margin of the otolith.
In Northern European regions (e.g. Scandinavia) the silver eel run may cease
prematurely due to cold temperatures in the winter months and recommence in the
following spring, then both portions of the migration should be referenced to the
January in the middle of the run. Similar delayed runs can occur due to low water
and/or hydropower barriers that prevent migration.
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Accuracy and Precision
Previous works acknowledged the uncertainty of age estimation and its variability
(Svedäng et al. 1998) and previous groups (EIFAC, 1976) recommended comparative
readings. Svedäng et al. (1998) using otoliths of known and unknown age from fresh,
brackish and marine waters found that the accuracy of age estimation was uncertain
and deviations from the correct age were dependent on reader, locality and fish age.
Also while consistency within readers was high, a drift over time was noted.
7.1
Reference collection.
Reference collections are one key to both quality control and reduction of costs of age
determination (Campana, 2001). Before and during the meeting, pictures from
Ireland, UK, Sweden, Italy, Germany, Canada and the USA, with different methods
of preparation were exchanged. It was not feasible to undertake a full intercalibration before the workshop due to a number of factors, some relating to
inconsistencies with the available images.
Further work has to be done to implement a reference collection for eel based on a
representative sample of the whole range of latitude and habitats used by the eel.
7.2
Inter-calibration.
During the workshop, a small scale inter-calibration was carried out using 21
Swedish otoliths of known age. Twenty five readers inputted the ages onto the
software, some with prior knowledge of the samples, some without. A wide range of
interpretations were recorded for each otolith and the outputs were used to guide the
discussions for the report. The observed ages were also used in the training
workshop.
There is a need for a recurring co-ordinated inter-calibration programme in line with
PGCCDBS Guidelines (2008, 2009). This workshop clearly demonstrated the need for
accompanying metadata such as date of capture, geographic region, eel stage (yellow
or silver) and on possible treatments such as quarantine/farming, marking/tagging
etc. in order to improve the interpretation of age.
The main objective of an age reading inter-calibration exercise is to estimate precision
and relative/absolute bias in the readings from readers at different laboratories and to
ensure that these are still within acceptable levels. The aim is to decrease the
relative/absolute bias and to improve the precision (reduce the coefficient of variation
(c.v.)) of age determinations (their reproducibility) between age readers of the
different age reading laboratories.
Some of the participants at this workshop had positive experiences from similar intercalibration exercises for other species, e.g. herring, turbot, flounder and perch. We
have now noticed the need for, and therefore suggest that, a reference collection of
high quality digital pictures from Atlantic eels are produced and collected to
supplement the already existing collection from this workshop. Preferably this
collection should exceed a minimum of 100 otolith pictures representing as many
individuals. Five or more readers are preferably required for this exercise. If known
age eels are available they may certainly be included in the sample collection.
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Eels should represent both Atlantic species and their full geographic range, i.e. eels
from Scandinavia through to the Mediterranean for A. anguilla, and the Caribbean to
Newfoundland for A. rostrata. Both yellow and silver eels from wild and managed
stocks should be represented in this eel otolith picture data base.
The results from such an inter-calibration exercise are preferably analysed using the
“Tool for Age Reading Comparisons” (Eltink et al., 2000).
For practical reasons it is important to decide upon deadlines when all age readings
have to be entered to the database set up for this purpose (e.g. a SharePoint). It is
recommended that a follow-up workshop early in 2010 should be established on
inter-calibration, precision and accuracy.
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ICES WKAREA Report 2009
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Kolderveen, The Netherlands. Aquaculture and Fisheries Management, 19; 387-391.
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CM2008/ACOM: 15; 210pp.
Wickström, H. (1987). Growth of elvers stocked in some Swedish lakes. In Hal, halaszat és
természetesvizi környezet. XXIX Georgikon Napok Keszthely: 328-339.
Wickström, H., Westin, L. & Clevestam, P. (1996). The biological and economical yield from a
long-term eel stocking experiment. Ecology of Freshwater Fish, 5; 140-147.
Wright, P.J. (1991). The influence of metabolic rate on otolith increment width in Atlantic
salmon parr, Salmo salar L. Journal of Fish Biology, 38; 929-933.
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Annex 1: Agenda
Workshop agenda
Monday:
8:30 Word of welcome by the head of Cemagref of Bordeaux
9:00 Plenary session on methodology of otolith preparation. How many methods can
be accepted/recommended, what is scope of application of the methods (ex burning
and cracking for old eels, in toto for young eels, etching and dying for intermediate
etc) with reference to the previous work
10:30 coffee break
11:00 Methodology of otolith preparation. Work on subgroups
13:00 lunch
14:00 Methodology of otolith preparation. Work on subgroups
16:00 Plenary session
17:30 end of the day
Tuesday:
8:30 Plenary session on age estimation criteria, definition of age 0, and annual marks.
11:00 work on subgroups
13:00 Lunch
14:00 subgroups continued
16:00 Plenary session
17:30 end of the day
Wednesday:
8:30 Plenary session on age reading protocol.
11:00 work on subgroups, reader inter-calibration work
13:00 Lunch
14:00 work on subgroups, reader inter-calibration work
16:00 Plenary session
17:30 end of the day
Thursday: Collating of the report, circulation and discussion.
Morning: preparation and circulation of the report.
Late Session: 17.00 – 19.00hrs Discussion of report – could be completed on Friday
am.
Training for eel age estimation
ICES WKAREA Report 2009
Thursday:
9 am until 5pm
Etching dying
Burning cracking
Training for eel age estimation
Day 2 (Friday):
9am until 2pm
Etching dying (following)
Image acquisition
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ICES WKAREA Report 2009
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Annex 2: List of participants
Working Group on Age Reading of European and American Eel
WKAREA
20 - 24 April 2009
LIST OF PARTICIPANTS
Name
Address
Phone/Fax
Email
Jean-Christophe
Aymes
UMR INRA/UPPA Ecobiop
Phone +33 5
59515984
[email protected]
Quartier Ibarron
64310 St Pée-sur-Nivelle
Fax +33 5 59 545152
France
Carin Ångström
Swedish Board of Fisheries
Phone: +4617346477 [email protected]
Institute of Coastal Research
P.O. Box 109
SE-74222 Oregrund
Sweden
Gustavo Becerra
Jurado
Central Fisheries Board
Swords Business Campus
Phone +353 1
8842540
[email protected]
Swords Co. Dublin
Ireland
Kenneth Bodles
[email protected]
School of Biological Sciences,
Queens University Belfast,
Medical Biological Centre,
97 Lisburn Road, Belfast,
BT9 5PQ, N.I.
Fabrizio Capoccioni
Tor Vergata
Via Cracovia 1
00133 Rome
Phone +39
3470852870
[email protected]
Fax +39 0672595852
Italy
Jean Marie Caraguel
Société FISH-PASS
3 rue des Grands Champs
Phone +33 2 9977 32 [email protected]
11
ZA des 3 Pres
35890 Laille
France
Jennie Dahlberg
Swedish Board of Fisheries
[email protected]
Institute of Freshwater Research
Stångholmsvägen 2
SE-178 93 Drottningholm
Sweden
Francoise Daverat
Chair
CEMAGREF
Phone +33 557
Unité de Bordeaux, 50 av de Verdun 890806
[email protected]
F-33612 Cestas
France
Barbara Dekegel
University of Gent
KL Ledeganckstraat 35
B-9000 Gent
Belgium
[email protected]
ICES WKAREA Report 2009
Mélissa Eon
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[email protected]
CEMAGREF de Bordeaux
Unité ecosema, 50 av de Verdun
F-33612 Cestas
France
Mark Etherton
Centre for Environment, Fisheries & Phone 1502 524539
Aquaculture Science Lowestoft
Fax 1502 513865
Laboratory
mark.etherton@cef as.co.uk
Pakefield Road
NR33 0HT Lowestoft Suffolk
United Kingdom
Derek Evans
Fisheries + Aquatic Ecosystems
Branch, Agri-food and Biosciences
Institute for Northern Ireland,
Phone
+442890255551
[email protected]
Newforge Lane, Belfast,
BT95PQ, N.I.
Christine Gazeau
CEMAGREF
[email protected]
.fr
Unité de Bordeaux, 50 av de Verdun
F-33612 Cestas
France
Reinhold Hanel
[email protected]
Institute of Fisheries Ecology
Federal Research Institute of Rural
Areas, Forestry and Fisheries
Palmaille 9, 22767 Hamburg
Germany
Celine Ide
University of Gent
Phone +32 9 2645220 [email protected]
KL Ledeganckstraat 35
Fax +32 9 264 5344
B-9000 Gent
Belgium
Martha Jones
Cape Breton University
1250 Grand Lake Road
Sydney N.S. Canada B1P 6L2
Phone +1 902 563
1973
[email protected]
Fax +1 902 563 1880
Canada
Twan Leijzer
[email protected]
Wageningen IMARES
P.O. Box 68
NL-1970 AB Ijmuiden
Netherlands
Cristina Manzo
Tor Vergata
Phone +39 06
72595852
[email protected]
Leibniz-Institut für
Meereswissenschaften
Phone +49 431 600
4554
[email protected]
Düsternbrooker Weg 20
Fax +49 431 600 4553
Via Cracovia 1
00133 Rome
Italy
Lasse Marohn
D-24105 Kiel
Germany
Florian Nagel
Institute of Fisheries Ecology
[email protected]
Federal Research Institute of Rural
Areas, Forestry and Fisheries
Palmaille 9, 22767 Hamburg
Germany
Anne Odelström
Swedish Board of Fisheries
Institute of Coastal Research
P.O. Box 109
SE-74222 Ôregrund
Sweden
Phone +46 173 46469 [email protected]
ICES WKAREA Report 2009
José Manuel Oliveira
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National Institute for Biological
Resources INRB/IPIMAR, Av.
Brasília, 1449-006, Lisboa,
Phone
+351213027000
[email protected]
Fax +351213015948
Portugal
Ciar O’Toole
Marine Institute
Phone +353 98 42300 [email protected]
Furnace, Newport
Co. Mayo
Ireland
Phone + 353 98
42300
[email protected]
Leibniz-Institut für
Meereswissenschaften
Phone +49 431 600
4554
[email protected]
Düsternbrooker Weg 20
Fax +49 431 600 4554
Russell Poole
Marine Institute
Co-Chair
Furnace Newport,
Co. Mayo
Ireland
Enno Prigge
D-24105 Kiel
Germany
Elodie Réveillac
Institut du Littoral et de l
Environnement
Phone +33 5
46507641
2 rue Olympe de Gouges
Fax +33 5 46507663
[email protected]
17 000 La Rochelle
France
Dirk Schaerlaekens
Laboratory of Animal Diversity and Phone +32 16 324296 [email protected]
Systematics
Fax +32 16 324575
Deberiotstraat 32
bus 02439
3000 Leuven
Belgium
Joe Scutt-Philips
Centre for Environment, Fisheries & Phone
+441502524241
Aquaculture Science Lowestoft
Laboratory
[email protected]
Pakefield Road
NR33 0HT Lowestoft Suffolk
United Kingdom
Janek Simon
Institute of Inland Fisheries,
Potsdam
Phone +49 33201
40620
Im Königswald 2
Fax +49 33201 40640
[email protected]
[email protected]
14469 Potsdam
Germany
Guy Verreault
Ministère des Ressources
naturelles et Faune
186 rue Fraser
Phone +1 418 862
8649 ext. 226
[email protected]
Fax +1 418 862 8176
Rivière-du-Loup, Qc
G5R 1C8
Canada
Håkan Wickström
Swedish Board of Fisheries
Phone +4686990607 [email protected]
Co-Chair
Institute of Freshwater Research
Fax +4686990650
Stångholmsvägen 2
SE-178 93 Drottningholm
Sweden
ICES WKAREA Report 2009
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Annex 3: Individual Institute standard operating procedures for the
preparation of eel otoliths, by grinding and polishing
1. Grinding Technique for the Preparation of Adult Eel Otoliths. A. Oyama, Japan.
Working Process:
I. Embedding
II. Cutting
III. Grinding (upper side, side A)
IV. Grinding (lower side, side B)
V. Polishing
VI. Etching
VII. Staining
I. Embedding
1 ) Put the otolith on the side of “Multiclip”.
2 ) Mix “SpeciFix Resin” and “SpeciFix-20 Curing Agent” at the rate of 5.2 : 1
(in volume) using syringe.
3 ) Pour the mixed agent into the sample holder.
4 ) The agent is hardened 8 hours later, and take off the sample holder.
5 ) Put your sample on the slide with double-sided tape.
*I will call the upper side “side-A” and the lower side “side-B” from now on.
Side-A
Epoxy resin
Otolith
Multiclip
Glass slide
Side-B
II. Cutting
1 ) Cut the extra part of the resin of side-A.
Side-A
Side-B
2 ) Take off the sample from the slide, and turn it upside-down.
3 ) Cut the extra part of side-B.
ICES WKAREA Report 2009
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Side-B
Side-A
4 ) Take off the sample from the slide, and turn it upside-down again.
III. Grinding (side A, upper side)
1 ) Grind side-A with “Discoplan”. Use 70um whetstone to some degree and
after that, change to 13um.
2 ) Stop grinding when the ground surface reach's otolith edge.
*It is difficult to judge when we should change whetstone and should stop grinding.
Thus, this step needs experience. But I believe that you will be able to master after
some practice.
Otolith after the step of “side-A grinding”
IV. Grinding (side B, lower side)
1 ) Grind side-B in the same way. Use 70um whetstone to some degree and
after that, change to 13um.
2 ) Stop Grinding a little before you can see the core on the surface of otolith.
*As in side-A grinding, this step also needs some experience. The otolith of your
samples has very little core and we can not easily find it. And so, you should be
careful not to grind too much.
V. Polishing
1 ) Set the polishing disc (6um, not 1um) on the machine.
2 ) Make the disc wet sufficiently with “DP-Lubricant Blue”.
3 ) Take DP-Paste (diamond paste) 2-3cm and spread it on the disc.
4 ) Make acrylic sample holder wet with Lubricant, and set your sample on it.
5 ) Set the holder to Polishing machine (Polisher) using black pyramidshaped rubber.
6 ) Set the bottle of Lubricant to Polisher, and adjust drop speed. 2-3s/drop is
good.
7 ) Switch on.
8 ) Finish polishing about 30 minute later.
*Please use the machine of right side (we have two polishers). But when it is busy,
you can use left one.
*Use just one weight to polish the otolith.
*After finishing your work, please clean up the machine, sample holder, acrylic
rubber and so on.
Otolith after polishing
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VI. Etching
1 ) Etch the otolith with 1% HCl or 3-5%EDTA. Time is as follows.
1% HCl: 1minute
3-5%EDTA:
3- minutes
2 ) Wash the sample with water.
3 ) Make the sample dry with puffer. Don’t wipe out with paper.
*You can etch with each agent, but I don’t know which is better for your species.
Please examine on your own.
Otolith after etching
VII. Staining
1 ) Stain the otolith with 1% toluidine blue. Staining time in eels is as follows.
HCl etched otolith:
30-120 minutes
EDTA etched otolith: 5-30 minutes
*I don’t know whether toluidine blue concentration or staining time is suitable for
your species. So please examine on your own.
2 ) Wash the sample with water.
3 ) Make the sample dry with puffer. Don’t wipe out with paper.
(all process finished)
Otolith after staining - (complete sample)
2. Swedish SOP for Grinding and Polishing otoliths.
Slide preparation
The slide is marked with: area, fishing year, individual number, eel life stage.
Place the slide on a heating plate (95̊C) and melt the Crystal Bond on the surface.
Mount the otolith in the crystal bound in a convex position on the microscope slide.
The procedure must be handled in a ventilation (fume) cupboard.
Grind on a wheel beginning with 1200 grits silicon-carbide sanding papers,
increasing to 4000 grits until the centre and edge are visible. Add water constantly.
Polish with 4000 grit silicon-carbide sanding papers until surface is smooth. Add
water constantly.
Etching
20 – 40s in 1% HCl acid (time varies with the size of the otolith.)
Staining of eel otoliths
20 ml neutral red solution,( 3.3g /L). {SIGMA- ALDRICH Product reference: N2889
(,33% ) liquid, 3,3g/l.}
Add Sodium chloride 0.2 g and mix about 2 minutes
Add 0.1ml of Acetic acid (100%)
Stain for approximately 10 minutes (Can differ between samples),
Rinse in water for 2 minutes.
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3. Procedure for aging eel otoliths by grinding and polishing - AFBI, N. Ireland
CONTENTS
SECTION
TOPIC
1 & 2.
Scope and Field of Application
3.
References
4.
Principle of the Method
5.
Reagents
6.
Equipment
7.
Sampling procedure
8.
Operational Procedure
9.
Expression of Results
10.
Quality Assurance
11.
Reporting of Results
12.
Safety
1&2
Scope and Field Application
This procedure provides a safe and reliable method for processing eel otoliths
and assessing the age of the eel by counting the annuli illuminated via
polarized or transmitted light as a result of the grinding and polishing.
3
References
Novel method no reference available.
4
Principle of the Method
Grinding down an eel otolith from the convex side provides a flat surface
which is polished and viewed under polarized or transmitted light using a
low power stereo dissecting microscope.
5
Reagents
•
6
The only chemical used is Loctite Branded Super glue.
Equipment
Glass microscope slides, fine point forceps, mounted needles, slide container
box and Loctite Branded superglue, dissecting microscope.
7
Sampling Procedure
Not applicable.
8
Operational Procedure
•
Eel otoliths should be stored (in fish heads) in alcohol prior to mounting.
They are then removed from the heads, and dried for several day in cell
line containers prior to mounting.
•
Glass slides are labelled with appropriate code for the otolith.
•
A small drop of glue is placed on the middle of the right hand side of the
slide. The otolith is placed into this drop concave side down. Using the
mounted needle the glue is drawn around the otolith to ensure a tight seal
around it.
ICES WKAREA Report 2009
•
Prepared slides are normally left over night for the glue to harden.
•
The slide is hand ground slowly in a figure of 8 pattern using graded
sanding papers specifically for otolith work. Slide is checked every so often
to ensure that the grinding is in sufficient direction and force and that the
origin has not been removed.
•
When satisfied with the level of grinding the otolith is then polished using
jewellery papers and if needed jewellery cloth to remove any score lines.
The annuli are then read using the dissecting microscope.
9
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Expression of Results
Excel spreadsheet, several reads are required.
10
Quality Assurance
Continual examination of otolith during grinding to ensure precision. 10% of
otoliths re read by another reader and “second opinion” recorded.
11
Reporting of Results
Each result is recorded on project record sheet along with other sample
details. Results and other details are transferred to a specific project records
book and to the relevant file within EXCEL database.
12
Safety
•
Care should be taken when using the super glue as it will stick flesh
together. It can be removed using warm water.
4. Standard Operating Procedure for Burning, breaking and grinding of eel otoliths
Janek Simon, Institute for Inland Fisheries Ev.Potsdam-Sacrow 16 April 2009:
1 ) Scope and Purpose:
This procedure provides a safe and reliable method for processing and
preserving eel otoliths and assessing the age of the eel by counting the
annuli highlighted as a result of the burning, breaking and grinding
process.
2 ) References:
Secor, D. H., Dean, J. M., & Laban, E. H. (1992). Otolith removal and preparation for
microstructural examination. In: Otolith Microstructure Examination and Analysis, pp.
19–57. Ed. by D. K. Stevenson & S. E. Campana. Canadian Special Publication of Fisheries
and Aquatic Sciences 117, 126 pp.
Simon, J. (2007). Age, growth, and condition of European eel (Anguilla anguilla) from
six lakes in the River Havel system (Germany). ICES Journal of Marine Science 64: 1414–
1422.
3 ) Principle of the Method:
Burning the otolith highlights the winter bands so they can be
differentiated when examined by transmitted light. When the otolith is
broken through the transverse plane through the centre (nucleus), it
produces a flat surface for examination. Grinding the broken otolith
improves the flat surface for examination by transmitted light.
ICES WKAREA Report 2009
4 ) Reagents:
The chemicals used are 96% ethanol, distilled water, a clear wax with low
melting temperature (Mounting Wax Crystal Bond 590 Amber, Fa.
Buehler®) and alumina powder slurry (0.3 μm).
5 ) Equipment:
Small waterproof tubes (0.2 ml Cryo-tubes), clear plastic slide (c. 100 x 20
cm), candle, lighter, heating plate, glass microscope slides, slide container
box, line forceps, curved forceps, pen (edding 1 mm), self adhesive
grinding papers (600, 800, and 1200 grade) and self adhesive fibre polish
(Microcloth with adhesive, Fa. Buehler®).
6 ) Operational Procedure:
6.1 The eel otoliths, once removed from fish heads, were cleaned by
rubbing between the fingers, stored into labelled tubes with 96% ethanol.
Before burning the otoliths are left in a tube box in a dry place for several
days to dry.
6.2 Otoliths are removed from the tubes using a line forceps. The otolith is
held with this forceps across the centre and is moved several times
through the flame of the candle until they have darkened to a amber
brown colour. Care should be taken to burn the otolith enough that they
are easy to break but not so long that the otolith becomes fragile and easily
damaged.
6.3 The burnt otolith is put convex side down on the plastic slide and
broken with the pressure of one finger transversely through the centre.
6.4 A small piece of wax was placed on one end of a microscope slide and
melted on the heating plate. One of the otolith halves is picked up using
the curved forceps and embedded broken side down in the molten wax.
Note: The wax should be just soft enough to hold the otolith half upright.
After moving the microscope slide from the heating plate, the wax can be
moved with the finger nails around the otolith, so that the half otolith is
embedded fully in the wax. The other half of the otolith was embedded in
the same way on the other side of the microscope slide.
6.5 When the wax is cured (after several seconds) the otolith is ground
with a series of grinding papers (600, 800, and 1200 grade) down to 0.1–0.2
mm. Care should be taken to grind the otolith not to flat and not to deep.
6.6 Each otolith half was smoothed by polishing for 1 min. in alumina
powder slurry (0.3 μm, mixed 1:10 with distilled water) on the plastic slide,
then cleaned with distilled water and finished with a wet fibre polish.
6.7 Finally, after checking the correct thickness of the otolith under a light
microscope, it was labelled with a pen in the centre of the microscope slide
and stored in a slide container box.
6.8 The otoliths are read through the glass of the inverted microscope slide
under a light microscope using transmitted light and 125-fold
magnification.
6.9 To back-calculate growth, the distance between the winter rings of the
otoliths was measured under a light microscope with the help of an
eyepiece micrometer at 125-fold magnification.
Note: Small otoliths were not burnt and broken. There were embedded
convex side up in the clear wax and then ground (continuing by step 6.5).
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7 ) Advantages:
A preparation method that can be used by each laboratory (need no for
specialist equipment or machines)
Good for eels of ages from 1 to 15 years.
Permanent mount which preserves otoliths for long time periods.
8 ) Disadvantages:
The preparation method is time consuming (c. 25 min. for a couple of
otoliths).
Not a system for preparation of large numbers of otoliths (>1000/year and
person).
The preparation method is complicated by air temperatures over 30°C.
9 ) Safety:
Don’t store otoliths in formalin or in air.
Embedding and grinding of otoliths is complicated by air temperatures
over 30°C because the wax fails to cure adequately.
5. CEMAGREF protocol for Grinding, polishing and staining otoliths
Method
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Material
•
Silicon molds
•
Epoxy resin
•
Balance
Disposable pipette
•
Agitator
•
Plastic disposable Beaker.
•
Acetone
•
Wiping paper
•
Plastic tweezers
•
Histology slides
Technical
1 ) Prepare epoxy resin by mixing the component under an extractor fan,
respect the proportions and measure with a precision libra.
2 ) Mix well for 5 min and leave it for 10 other min to release the bubbles.
3 ) Place each right otolith on the bottom of a mould cavity, external side face
up.
4 ) Keep the reference of each otolith in each cavity.
5 ) Add drops of resin until the mould is filled up.
6 ) Remove the bubbles by gently moving the otolith.
7 ) Leave until the resin is hard.
8 ) Reference histology slides with the reference of the samples.
9 ) Remove the resin blocks with the embedded otoliths from the moulds.
10 ) Melt Crystal Bond glue on a heating plate at 150° Celsius.
11 ) Put some liquid "Crystal Bond" on the centre of a referenced slide and
quickly press the corresponding resin block upside down (the number
facing upwards) on it.
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Annex 4: Manual for the Ageing of Atlantic Eel (separate document)
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