Manual of Fish Sclerochronology-Ifremer PDF

Manual of fish
sclerochronology
Editors
J. Panfili, H. de Pontual, H. Troadec t,
P.J. Wright
In homage to H erve Troadec ,

researcher at the LASAA, trag ically ki \led
in an air crash on 29 August , 2001 .
Institut de recherche
pour le developpement
Manual of fish sclerochronology
Acknowledgement
Editors and authors would especially like to thank: Eric Dabas (fRO, Brest, France),
Louis Marec (fRO, Brest, France), Brest, France) and Bernard
(fRO, Brest, France) for their technh'al assistame, Olivier Duglmlay
mer, Brest, France) fw his technical assistance with the images and vid<!os, Richard
Miltner (CEFAS, Lowestoft, UK), Andre-w Newton (FRS, Aberdeen, UK) and Andreu'
(CEFAS, UK) their help in Ill. A. 3, DolliTes Montaner
the bibliographic references} Nathalte
technical assistance with video of
Bwdeaux, France) andJean-Lo1lh
France) their clJmments on Vll,
and Daniel Latmuile (lfremer, Bresl, France), Laic Antoine Nantes, France)
and Patrice Cayre URO, Paris, France) their comtant support and encourage-
ments. Finally, we would like to prliVided by all EFAN mem-
bers which Imdo!lbtedly imprliVed the scope
References
Book:
Panfili, Pontual H. (de)., Troadec H., Wright P.J. (eds), 2002. Manual of
fish sclerochronology. Brest, France: Ifremer-lRD coedition, 464 p.
Paper:
Troadec H., Benzinou A., 2002, Computer-assisted age estimation. In Man-
ual of fish sderochronology (Panfili ]., Ponrual H. (de), Troadec H.,
eds), pp. 199-241 Brest, France: lfremer-IRD (oedirion.
righrs No be reproduced, scored (0 r<.'cric:val or (rans~
mmed, in any form or mf"chanical, photocopying,

huJdc[5.
© Edirions Ifremer, 2002

ISBN
2
Contents
Preface 11
Foreward 14
Chapter I - Introduction
A. General introduction 19
H . de PoncuaJ, J. Panfili, PJ. Wright, H . Troadec
B. Historical 23
FJ. Meunier, J. PanfiJi
1. Osteichthyans 23
1.1. Scalimetry 23
1.2. Otolithometry 25
1.3. Skeletochronology 26
2. Chondrichthyans 27
Chapter 11 - Types of calcified structures
A. Otoliths 31
PJ. Wright,). Panfili, B. Morales-Nin, A.). Geffen
1. Description and function 31
l.l . Description 32
l.2.Function 35
2. Periodic increments 35
2.1. Primary increments 36
2.l.1. Primordium 38
2.l.2. First ring 38
2.2. Accessory growth centres 42
2.3. Seasonal and annual increments 43
2.4. Lunar-related structures 44
2.5. Structural discontinuities 46
2.6. Secondary growth zones 47
3. Regulation of incremental deposition 48
3.1. Exogenous influences on primary increment periodicity 48
3.2. Exogenous regulation of annual increment periodicity 50
3.3. Influences on accretion rate 52
3.4. Physiological regulation of otolith formation at the sacculus level 53
3.5. Hormonal regulation of otolith formation 56
B. Scales 58
F). Meunier
l. Description, diversity and function 58
2. Growth of scales and growth marks 62
3. Regulation of growth marks 64
C. Skeleton 65
F]. Meunier
1. Morphology of bones and their structural organisation 65
3
1.1. Constituents of bony tissue 66

1.1.1. Cells 66
1.1.2. Organic matrix 68
1 1.3. Mineral content 70
1.2. Spatial of bony tissue 71
1.2.1. lines 1
1.2.2. Vascularisation of bony tissues 72
1.3. Histophysiological significance of bony tissue
1.3.1. Bone growth 76
1.3.2. Bone remodelling 76
2. Periodic increments or "the skeleton as a flight-like recorder" 77
2.1. Growth marks 79
2.2. Determinism of the structure and
of growth marks 81
2.3. Some 83
3. of the incremental
3.1. Growth mark formation and ''',;'';:'VII,QlIlV
3.2. Calcium metabolism 86
3.3. Feeding 86
3.4. Reproduction 86
4, Conclusion 87
111- studies
A. Age estimation 91
B. Morales-Nin,]. Panfili
1, Criteria for the choice of calcified structures 92
2. Selection of increments 93
3. The process of age estimation 95
3.1. age estimation
3.2. Age group and assignation to age class 96
B. life history events 99
H. de Pomual, Wright, H. Mosegaard
1. Age at maturity 99
2. Lifespan (longevity) 100
3. Metamorphosis and settlement 100
4,M~~tion 103
C. Influence of shape and structure on the interpretation 105
]. Panfili, B. Morales-Nin
r ' .... ~."~ IV Validation and verification methods
A. Direct validation 114

PJ. ]. Panfili, A. Folkvord, H. Mosegaard, Meunier
1 Marking 114
1.1. Marking fish 114
1.2. Marking calcified structures 116
4
1.2.1. Fluorescent 116

1.2.1.1. 118
1.2.1.2. Whole-body immersion 118
1.2.1.3. Incorporation into food 121
1.2.1.4. Fluorescent 121
1.22. Temperature-induced marks in otoliths 122
1.2.3. marks in otoliths 123
12.4. Elemental otolith Sr and lanthanides) 124
2. Breeding and rearing 125
3. Data treatment 127
B. Semi-direct validation 129
J. Panfili, B. Morales-Nin
1. data? 1
129
1.2. Quantitative data 129
1.3. Examples of semi-direct validation studies 132
2. Problems of interpretation of otoliths 1
C. Indirectvalidation 13 5
B. J. Panfili
1. Comparison with length distributions 135
2. Methods using age information 1
D. Verification 138
B. Morales-Nin, J. Panfili
1. Different from one or several readers: bias 138
2. Different from several calcified structures 140
3. Trends in of growth 140
4. Accuracy and precision 141
Ch:~nh~r V - Some uses of individual data
A. Growth and 146

A. Folkvord, H.
1. Models of growth 146
1.1. Measures of 146
1.2. Allometry and age-independent growth models
1.3. Age-based growth models 1)
2. Back-calculation 1)4
2.1 Background and assumptions
2.2. Regression methods versus proportional methods 155
2.3. Starting in proportional methods: statistical
determination versus reference points 157
2.4. methods: SPH versus BPH 160
2.5. Non-linear BCFs 162
2.6. Problems of back-calculation 163
3. Recommendations 164
5
B. Ecological applications 167

H. Mosegaard, A. Folkvord, PJ. Wrighr
1. Recruitment studies 167
1.1 . Documentation of selective mortality 167
1.2. Bigger is better 171
1.2.l . Correlations of sizes at different ages and hierarchies 171
2. Life history timing 173
2.1. Birth date distributions 174
2.1 .1. Approaches to testing for size-dependent mortality
and hatch date 175
2.2. Ontogenetic transitions 176
2.3. Environmental changes 177
2.4. Spawning 177
3. Sampling selectivity 178
C. Demographic structures in stock assessment models 180
B. Mesnil
1. Age-structured models for stock assessments and management
advice 18 1
1.1. Single specie s, single fishery 181
l.2. Single species, multiple fleets 184
l.3. Multiple species, technical interactions 185
1.4. Multiple species, biological interactions 187
l.5. Spatial models 189
2. From age estimation to age-based models 189
2.l. Estimating the age composition of catches 189
2.2 . Case study: age-length keys in practice 191
3. Summary and critique 196
Chapter VI - Computer-assisted age estimation
H. Troadec, A. Benzinou
A. What is a computer-assisted age and growth estimation
(CAAGE) system? 202
1. Visual data processing 20 2
1.1. The digital image source 20 2
1.1.1. Digital image formation: 2D sampling 202
1.1.2. CS image acquisition 206
l.l.2.l. Polarisation microscopy 206
l.l .2.2. Image integration 208
1.1 .2.3. Image mosaics 208
l.2. Digital image processing 209
l.2.1. Contrast enhancement with point operations 210
l.2.2. Noise reduction with neighbourhood operations 211
l.2.2.l. CS image profile : a mUlti-component signal 213
l.2.2.2. Spatial convolution : from moving average
to Gaussian 2 14
6
1.2.2.3. Spectral filtering: the Fourier transform 216

1.2.2.4. Linear filtering of CS images 217
1.2.3. Edge detection by derivative operations 21 7
1.2.4. Structural information detection by morphological
ope~tions 220
1.2.5. The growth pattern: a limit to classical operators 222
1.3. CS quantitative analysis: from 10 to 3D 224
1.3.1. Growth zone counting: from 10 to 1.5 image profiles 224
1.3.2. 2D and 3D CS shapes 227
2. Cognitive information processing: are you reading or interpreting? 228
3. A little theory: visual perception and age estimation 231
B. How a computer can help in calcified structure analysis 234
1. What can you expect from a CAAGE? 234
1.1. An aid to quantification 234
1.2. Objectivity or standardisation? 236
1.3. Accuracy and precision 236
1.4. Productivity 238
1.5. Data and knowledge preservation 238
2. For what kind of study? 240
2.1. One-oH studies 240
2.2. Routine analysis 240
Chapter VII - Otolith microchemistry
H. de Poncual, A.J. Geffen

A. Introduction 245
B. Some basic chemical definitions and principles 246
1. Stable isotope mass fractionation 247
2_Radioactive decay 248
C. What are otoliths made of? 251
D. Otolith elemental uptake: how and where? 255
1. Between the external medium (water and food) and the blood plasma 255
2. From the blood plasma to the endolymph 256
3. From the endolymph to the otolith 257
4. Elements that are likely to vary depending on environmental
availability 258
E. Applications of otolith microchemistry 260
1. Patterns of migration and environmental history 260
1.1. Salinity 260
1.1.1. Migration behaviour as revealed by Sr/Ca ratio 260
1.1.2. Sr isotope composition 261
1.1.3. Stable isotopes 261
1.2. Temperature 263
1.2.1. Sr/Ca ratios as indicators of individual thermal history? 263
1.2.2. Stable isotopes 264
2. Metabolism and ontogenic events 267

2.l. Metamorphosis 269
2.2. Reproduction 270
3. Age estimation 271
3.1. 210Pbf'26Ra and 228Thf'28Ra disequilibria 271
3.2 . Atomic bomb radiocarbon chronometer 27 3
3.3. Other potential chronometers 276
4. Stock-population discrimination 276
5. Other applications 279
5.1. Habitat characterisation through elemental fingerprints 279
5.2. Mass marking 279
5.3. Analysis of fossil biogenic carbonate for paleoclimatology
and paleoecology 280
5.4. Pollution history and anthropogenic influences 280
F. What about other calcified structures? 284
G_ Microchemistry: methodological requirements and issues 286
1. Analytical techniques 286
1.1. Elemental composition 286
1.1.1. Surface analyses 289
1.1.2. Bulk analyses 290
l.2. Stable isotope measurement 290
1.3. Radionuclides 291
2. Data quality 293
2.l. Potential sources of uncertainty 293
2.1.1. Sampling 293
2.1.2. Fish storage 294
2.1.3. Otolith extraction 294
2.l.4. Otolith preparation 296
2.1.5. Composition measurement 296
2.1.6. Data analysis 299
2.2. Accuracy and precision of analytical tools 301
Chapter VIII - Preparation and observation techniques
A_Aid to decision trees 308

H . Troadec, H . de PontuaJ
B. Extraction and conservation of calcified structures 317
J. PanfiJi
1. Storage and preservation of whole fish 317
2. Scales 318
3. Otoliths 318
3.1. Extraction from large fish 319
3.1.1. Frontal head section 32 0
3.1.2. Transverse head section 320
3.1.3. Sagittal head section 322
3.1.4. Through the gills, ventral cranium section 323
3.2. Extraction from small fish 323
8
3.3. Otolith and conservation

3.3.1. Cleaning and handling
3.3.2. Storage and conservation
4. Other skeletal parts 328
4.1. Extraction 328
4.2. and conservation 328
C. Preparation of calcified structures 1
J Panfili, H. de Pontual
Scales 1
1.1. Direct observation 331
.2. Slide mounting 331
1.3. Cellulose acetate 332
2. Other structures
2.1. preparations
2.1.1. Whole calcified structures 333
2.1.2. otoliths 333
2.1.3. preparations for other structures 333
2.2. Embedding and Imr.r.,crn~j·I"n 334
2.2. . media 334
2.2.2. Embedding individual structures for sectioning 3
2.2.3. Embedding for incident-light observations
2.2.4. Multiple embedding of otoliths for sectioning 338
2.2.5. Impregnation of tissue
2.3. Spf'tlf1rlln 343
2.3.1. Sectioning individual structures
2.3.2. Multiple
2.3,3. Cryotome car""'."",
2.4. Mounting
2.4.1. Section mounting for subsequent of
2.4.2. Mounting otoliths from small fish 347
2.4.3. Slide of multiple otolith sections
2.5.
decalcification 351
2.6.1. Otolith aCid 351
tissue 1
352
353
2.8.1 Staining otoliths 35
2,8.2, Staining tissue 54
2,9. Some special considerations for 355
D. Observation 358
B. J Panfili
1. 358
358
358
Clearing media
1.1.3. Mounting media 359
9
Manual 01 fish sclerochronology
1.1.4. Illumination 359

1.1.4.1. Transmitted light 360
1.1.4.2. Reflected
1. 1.43. Profile projector
1.2. High-power magnification (compound microscopy)
1.2.1. Resolution
1.2.2. Immersion v. dry objectives
1.2.3. Illumination
1.2.3.1. Filters
1.23.2. Confocal microscopy 363
1.2.33. Transmitted v. reflected light
1.3. Polarised light
lA. Ultraviolet light
2. Electron microscopy
2.1. SEM 365
2.1.1. SEM characteristics
2.1.2. Sample mp,n,,,,,,'I[1
3. Microradiography
EJ. Meunier
E. Conservation of preparations 370
1. Conservation of calcified structure preparations 370

2. Scales 370
2.1. Unmounted scales 370
2.2. Mounted scales and scale impressions 371
3. Otoliths 371
3.1 Otolith collections 371
3.2. Whole and broken otoliths 371
3.3. Embedded otoliths and otolith thin sections 372
4. Other skeletal parts 372
Glossary 373
J. EJ. H. H. Troadec,
Wright, A.]. Geffen
References 385
Acronyms and chemical symbols
Author addresses 452
Index 454
Species, taxa and common names 461
10
Preface
of rhe age of individual members of populations of animals
improves of variaLion in srnlcture and abun-
dance. The age structure of the population can be and rates of
processes, such as and mortality, can be quan-
tified. This is particularly important when the dynamics of
populations and fisheries resources. In addition to
the total increase in mortality rate caused by It IS to
determine how the increased is distributed among age groups
in the population. population analyses are therefore
in the classical fish stock assessment models; i.e., the
mathematical tools thm worldwide form the basIS of scientific
management advice on
of age determination has
accive field in fisheries science. Ever since scieo-
age and of fish first planned to
in 1968 and then gathered five years
on
communications and science transfer have been an
component of this field of fisheries science. Over the past
regular imernational conferences and workshops have
been laboratory manuals prepared, and
reviews published, some of which presem excellent overviews of the his-
tory and development and current of various
the major research effort focused on
extended of time, age determination of fish
is still nor a matter. Wirhin international advisory
notably ICES, a need is often and for
ot tesring protocols for many Maintaining
within and among and laboratories is a con-
tinuOllS and seemingly process. In many very
unfortunate controversies over methodology have arisen and
lasted for many years, usually because several for the same
different results and none of these
some consider that

for some is still more an act than a science". The final
ofal! work should be to establish methods that
that can be used routinely
11
In Europe, the Union financially the concerred

action "The European Fish Network" (EFAN)
the four years, 99 scientists in Europe in the pro-
35 different universities and research insritutes
countries. EFAN aimed to conducr. and
coordinate collaborative research and and thereby ensure that
age determination becomes a reliable element of the assessments
underlying the scientific management of fisheries and environmental
resources. EFAN a series of five "cells" that contain
various topics: Methodologies and Information Process-
Informarion and Validation of and
Research and
In of all previous efforts and
has been no all-inclusive text
have and the various calcified structures used
tal hard strucrures, and oroliths. The editors and the authors, who
have all been active members have assembled here a compre-
hensive manual that is one of the most detailed reviews of the subject
done to date. These amhors are well-known authorities in the
have and have accumulated a wealth of
in the science and of age and growth determi-
nation of fish. and diversity of the manual are
apparent at a its combined rreatmenr of the theo-
retical and the
The manual addresses broad range of including the
ration of various types of calcified structures, and details the validation
of using and indirect
procedures. It includes an extensive review of the use of the par-
and and
An section considers
aspects of tbe every student who has ever delved inco the
has wished for its ultimate auromation. The review of the
rapidly developing and powerful field of chemical will be par-
ticularly valuable to those interested in both the and the par-
ticular. and examination are detailed from a
of view. Useful decision trees and quick and easy refer-
ence material for everything from structure storage, and
to contamination are pro-
vided. For the first time, a is presented that
includes for all types of calcified structures. Reference
material is extensive bur naturally is nor all-inclusive, the mass
of literature that has accumulated.
12
The breadth and depth of this coorribmion will make it

an imponanc reference manual for years to come. The
technical, and information assembled here ensures
that it will be a well-leafed reference manual within easy reach and
valued on the laboracory bench and office desk of
routinely or just inceresred in this
fisheries science and rprnn,r.,r.
The rev iewers
John M. Casselman Er/end Moksness

Glenora Fisheries Station I nsti lUfe of Marine Research
41 Fish Hatchery Lane FJyjdevigen Marine
R.R. 4, Picron Research Station
Ooratio KOK 2TO, Canada N-4817 Norway
13
Foreword
Sclerochronology, the study of calcified structures to reconstruct the
past history of living organisms, is central to fish biology and fisheries
management. Whilst there have been many publications, symposia
and reviews in this field these have all been limited in scope. This man-
ual aims to provide an overview of the current theoretical and practi-
cal aspects of sclerochronological studies. Such a review is timely since
there have been tremendous research and technological developments
in rhis field during the past decades. By providing informarion on the
nature of calcified structures, the uses of these structures for fish
research and the methodology for preparation and examination, the
manual attempts to provide a thorough guide to researchers, techni-
cians and students new to the field as well as those interested in
expanding their range of expertise.
Originally, the manual was conceived by the LASAA (Laboratoire de
sclerochronologie des animaux aquatiques), a joint laborarory between
the French research institutes Ifremer Onstitut fran~ais de recherche pour
l'exploitation de la mer) and IRD (institut de recherche pour le
developpement). It was first presented in 1996 at the European Fish
Ageing Network (EFAN) preparatory meeting in Ghent, Belgium. EFAN,
a FAIR Programme Concerted Action sponsored by the Eutopean Com-
mission, was established to promote exchanges, collaborative research
and training in sclerochronological studies. By means of this network it
was possible ro expand the authorship and with it the aims and scope of
the manual. This expansion and production of the project was further
facilitated by a FAIR accompanying measure to EFAN, ManAgeFish,
which additionally enabled the development of the manual 's multime-
dia version and meetings between authors. Work on the manual began
in 1999 and the book was reviewed during author meetings in 2000
(University of Balears, Palma de Mal/orca, Spain) and 2001 (Museum
national d'histoire naturelle, Paris, France).
The manual is presented on two media, a book and a multimedia DVD-
version. Each chapter is written by a group of leading workers in their
field. Whilst the authors are from European institutes, their experi-
ence includes both temperate and tropical regimes as well as marine
and freshwater habitats. As a consequence the manual is extensively
illustrated with examples from around the world and the techniques
described should be relevant ro fish from all regions, and therefore to
researchers working in any region or ecosystem.
Whilst the multimedia version derives from the book it greatly ben-
efits from the addition of videos and the inreractivity permitted by
digital media. This DVD version is thus supplemented by (1) a set of
14
Manual 01 fish <rl"Yn('hrr,nnlno\!
42 videos which illustrates the main technical procedures, interac-

tive and animated and (3) a mode based on aims and
constraints (decision which presents an alternative to a classical
reading,
The number of references in the field of sclerochronolo-
gy is enormous and as such the manual does not atrempt to present an
exhaustive bibliography, However, there is an extensive list of refer-
ences from earlier journals and some grey literature, in order
the reader. a list of acronyms, an index and a
can be found at the end of the which is
aimed at those who are unfamiliar with the field,
The work of the editors has been greatly aided by a technical review
of the structure and by Hugh Allen Cordoba, at
the of 2001. The scientific content of the manual has been
reviewed by Professors Casselman of the Canadian Ontario Min-
istry of Narural Resources and Erlend Moksness from the '"'r.rn,'~'''
the 200 L After accredita-
tion by the editors, the version has been translated in French
by the French authors of the book.
The editor"
15
16
Introduction
Cha r I
Introduction
17
18
Introduction
A. General introduction
H. de H. Troadec
the term is derived from the Greek

roots skH~ros "hard", khronos "time" and logos "reason". this
science aims to reconstruct the past from the
of their calcified structures Its scope covers problems of age
estimation as well as the estimation of the time and duration of life
events. Its methods are based on the of various types of
that provide temporal chemical
and/or
Data on the age and growth of fishes are essential for the understand-
of vital traits and populations (e.g. U"'~f-'«U,
mor-
of population demographic structure and its
to age based stock assessment). The ecological and
paleoecological of such data include the of
responses of to environmental pressures, whether natural
or anthropogenic pollution, coastal
zone Given the current poor state of many
resources, the demand for reliable sclerochronological data for deci-
sion-making related to managemem and sustainable
tion of resources is
As with the cs of some invertebrates molluscs or
hermatypic it has long been observed that those of fish show
roughly periodic structural patterns LA. I) that are related to vari-
rate induced both envirorunental
ic) and facrors such as events
Summerfelt & Hall, 1987). has a great deal in com-
mon with dendrochronology, i.e. tree-ring science. The latter has
Calcified structures have the potential to grow the life of

the fish and act as a permanent record whose definition varies from
one structure to another in relation to its biomineralisation
processes and functional role. Three major types of mineralised parrs
in the division of scJe-
LA.I): which
19
The assessment of individual age and through CS

to be much more informative and than statisti-
cal methods used at the level analysis of
guency data) or alternative individual methods such as metabolic
menr accumulation. In consequence, has as
IAl
a discipline of invaluable informacion to several
calcified structures fields of particularly in fishery sciences and marine
that can be utilised
To achieve this status, it had still has) to answer a number of very
for sclerochronological
studies, and the three main basic many of which were still are) far from trivial:
types of structure (otolith,
scale, skeleton) (modified
from Pantili, 1993).
Scale
Supra·occipital Opercular
Fin ray
Skeleton bones
What does each CS record? ----~
As permanent CS constituce more or less

individual biological archives which need to be decoded to extract che
relevant and useful information. Visual present various kinds
of which on both rhe CS and the scale of observa-
tion. For instance, while seasonal patterns may be observed on every
cs, daily increments are observable on otolichs ac
cation. Bones and scales act to different extents as reservoirs of cakium
and phosphorus salts and thus and
cesses. Such have to be taken into account when
ing che information provided these structures. Disruptions in
growth patterns are observed and correspond to life events
20
Introduction
whose idemity is sometimes anything bur obvious. Moreover, infor-

mation acquisition and imerpretation are often complicated by the
great plasticity of cs observable at various scales (individuals, popula-
tions, species). However, at the individual level, the imerpretation of
incremems in CS can lead ro a true chronological record of life evems
and growth.
What are the methods employed to reveal the information required?
Data provided by cs an alysis result from an acquisition process in

which methodology plays a pivotal role. Many methods of exrracrion,
preparation and observation have been developed in the course of the
20th cemury, the choice of which depends fundamentally on the CS ro
be studied as well as on the level of information required (type and
temporal precision).
Are the data obtained accurate and precise?
Data quality is a key issue in all sclerochro nological studies. Age esti-
mates that are neither accurate (i .e. close ro the true value, which is
essentially not known at least for wild fish) nor precise (i.e. presenting
large disagreements between repeated measurements) would be of poor
value for subsequent use.
Validation studies that aim to verify the presumed periodicity of a
given signal are essential basis for sclerochronological studies. They are
the only way to test the technology and the resulting accuracy of age
estimation . It is al so essential ro assess data precision, i.e. variability
between repeated interpretations (readings) of a given CS (either
between or within readers) for revealing the most appropriate schemes
for reading and interpretation. Recourse ro computer vision techniques
assists the reader at various levels of CS processing. Such techniques
increase reading precision, provide unequalled measurement capabili-
ty and allow interpretation data to be conserved.
Are the chemical patterns recorded informative

and what do they reflect?
The idea that CS possess chemical tags or "chemo-prints" was recog-

nised in the 1960s, opening another research field which is rapidly
expanding thanks ro the development of ever more sophisticated and
sensitive analytical tools. The range of applications is potentially
enormous, which explains the amount of research effort recently devot-
ed ro the field (more than 400 articles have been published during the
past decade). The discipline also has ro face questions of data quality,
21
which is particularly difficult to assess, as well as those of interpreta-

tion of complex chemical signals that are under both environmental
and physiological control.
How does this work?
Despite a rather long hisrory and development, sclerochronology is far

from being an exact science and, in many cases the decoding schemes
for structures and chemistry remain incomplete and debatable, espe-
cially in some species. On-going experimental work in the laboratory
and mesocosms provide strong sources of suppOrt for the validation of
hypotheses drawn from field data. Ultimately, however, better under-
standing of the biomineralisation processes and regulation mechanisms
is essential ro the full interpretation of signals in cs.
The literature on fish cs is enormous: for instance the ASFA (Aquatic

Sciences and Fisheries Abstracts) database contains more than 2300
references on otoliths from 1978 to 2000. Although some specific
ropics have been dealt with in book reviews or symposium proceedings
(e.g. Bagenal, 1974; Prince & Pulos, 1983; Casselman, 1987; Sum-
merfelt & Hall, 1987 ; Bagliniere et aI., 1992; Smi th, 1992; Stevenson
& Campana, 1992; Secor et aI., 1995a; Fossum et aI., 2000), none of
these offers an overview of the current standing of methods, practices
and applications of sclerochronology for new workers in the field or
other interesred end-users. This book is intended as an attempt ro fill
this gap.
The first part of the book deals with rhe basis of sclerochronological
studies by providing descriptions of cs, their increments and the reg -
ulation of the incremental deposition process. The rationale behind
sclerochronological studies and the uses of cs are mentioned in order
ro guide the reader for furure analysis . A detailed description of the
validation process is provided, as this is one of the most important
steps in sclerochronology. Some uses of the data are then described in
order ro outline the most important applications of cs in research and
fisheries assessment. As most laborarories involved in sclerochronolo-
gy are now equipped with image analysis systems with capabilities
ranging from fully interactive ro auromated data di g italisation, the
basic principles of image processing of cs are also described. The book
then provides a review of orolith microchemistry, which is a rapidly
developing research field in sclerochronology. Finally, detailed descrip-
tions of the numerous techniques for preparation and observation of
cs are provided in order to help the reader ro progress in this field .
22
Introduction
B. Historical
FJ. Meunier, J. Panfili
Estimates of the age of individual living organisms are needed in

many types of studies. The methods developed to collect age informa-
tion may be very old, as in the case of dendrochronology, i.e. estimat-
ing the age of trees from their annual growth rings. In fact , knowledge
in this field has existed for many centuries, as was noted by Leonard
de Vinci and by Montaigne (1580-1581) in accounts of his travels in
Italy: " . .. the Artist ... told me that all the trees show as many rings
as they have years ... The part of the trunk oriented towards the north
is narrower, shows the tighter rings ... ; so whatever rhe wood that we
show him, he prides oneself on being able to age the tree . .. ". Mean-
while, the Swedish monk Hederstrom (1759) was the first to propose
in an 18th century treatise that vertebral rings in fish could be
counted to estimate their age. However, the first serious attempts for
estimating the age of Vertebrates were not made until the end of the
19th century. Normally, the ages of animals were estimated on the
basis of repeated features in a calcified organ or a hard tissue, for which
reason rhe various methods were summarized by the term "scle-
rochronology". Within this field, more precise words were developed
to characrerize specialized approaches (scalimecry, otolithometry,
skeletochronology).
Studies of cyclic patterns recorded in the CS of fishes are very numer-
ous, and they cannot all be included here. However, a cerrain number
of references concerning the theoretical aspects of this field of research
are useful, as they highlight advances in our understanding of the
growth of hard parts and their ability to record time (BagenaJ, 1974;
Bagenal & Tesch, 1978; Summerfelt & Hall, 1987; Bagliniere eta/.,
1992). These stud ies can be craced back ro the beginning of the 20th
century but the ideas and underlying backgtound for this science were
laid down even earlier.
We rhus initially deal with works which gradually developed into the
methodologies usually used today for bony fishes (Osteichthyans). We
then consider the history of more recent work which has permitted the
development of similar approaches in cartilaginoLls fishes (Chon-
drichthyans).
1. Osteichthyans
1.1. Scalimetry
Leeuwenhoek (1696), and then Reaumur (1716), suggested that con-
centric peaks (i.e. circuli) on the surface of a fish scale corresponded to
23
the various stages of Kunrzman (1824), later challenged the

validity of the circuli as indicators of annual growth. Several
authors concenrrated on the relationship between cirmli and
1833-1 1841). (1861), fol-
periphery of the scale

observed in carp that the growth of the scale was more in
of food abundance and when food was scarce, the scale
circuli were narrower. He then proposed the use of these structures as
means of the average age of fish and practical
based on carp.
of some reservations (Brown, 1903; ichthyolo-
gists at the mm of the 20th century developed a method of individ-
ual age estimation in fishes from the variations in the arrange-
ments of the circular peaks and other ornamentations on the surface of
elasmoid scales (Waiter, 1901; Thomson, 1 1905;
1912, inter alia).
1908, 1910) discovered and described the
marks of reproduction marks") on the scales of salmon and
the foundations of the of back-calculation to follow
the annual growth of fish. Dahl (191 analysed the causes of the false
marks and with Lea (191 he gave the first applications of
scale measurement of successive sizes in individual
Conscious that the use of the scale in the age estimation rested on
rather than on well esrablished Masterman (191
on salmon in which he demonstrated the
the cogency of his by mor-
and statistical observations. In fact, what he
question of the validation of
salmon. these pioneer

on scales were published in
fisheries.
The growth of the scale surface is indefinite and is
in certain areas and clearly-defined seasons, with variations in
the external medium. It known that, poor seasons, the inter-
val separating the circttfi is narrower andior that new cirCIIli are formed
that do not match the first & 1992); this nar-
or discordance is very clear under transmitted and was
first named annuli. From the of these annuli was born the
measurement of scales: the age estimation of fish and estimates of
individual growth 1907,1911; 1 Viberr&
1
24
Introduction
Although in its early stages at the of the 20th

century, scalimetry was of results on
many In for the of imerest for fisheries.
However, Waiter (1901), Brown (1903), Dahl (1911) and
showed that serious causes of error could appear with the occurrence
of false marks or false annuli, because scales may also record various
biological evems and stresses (physiological wounds,
whether
over, in certain the annuli are difficult to
estimation very delicate or Other
then turned to mher CS, moJiths and/or bones, which
also record the events that affect
1.2. Otolithometry
estimation that is very
used in Teleost 1992). This branch of
science was also at the end of the 19rh century,
with rhe work of Reibisch (1899) The
concretion of the inner ear, with is nor
srricdy an elemem of the bLlt its nature
it similar as far as sclerochronology is concerned.
Originally, as with scales, otOliths have been used to seasonal
and annual growth cycles, and there is now an abundant literature on
the Their observation as whole parts or after is
accurate age estimations (Viberr &
the discovery Pannella (l971) of
mems inside the otOlith structure has up new fields of inves-
were enabled to reconstruct the stages in
of individuals, most of rhe time for larvae and
niles, Otolith microstructure is thus a very sensitive recorder that
covers a wide rime scale. the used to reach the
finest scale can be More at
the of rhe 19805, it was discovered that otoliths also incor-
chemical elements from their environment (biOtic and
via processes. Chemical of otolith con-
tents offered new methods of research and areas of
and the reconstruction of life history traits, Here also
are and still under The num-
ber of scudies of otolith works is and symposia devoted
solely to otolith research and its have already occurred
the past ten years (Secor et ai., 1995a; Fossum et at., 2000).
oroliths have been used for age estimation of
fish and are also valuable in Other fields than the study and
growth (Fossum et at, 2000).
25
1.3. Skeletochronology
If the developmenc of fish skeletochronology began a few years after
the first steps in scale measurement, the first applications appeared, in
fact, in the middle of the 18th century, with the work of Hederstrom
(1759). This author, generally ignored, was shocked by the fabulous
longevities usually ascribed at that time to common fish like pike
(more than 200 years, see also Casselman, 1974). Having observed
that the vertebrae of several species showed rings, he suggested that
they could offer an index of age. The enumeration of these rings on the
vertebrae of several species (pike, cod, perch, eel, bream) gave him, for
the age of these animals, values close to those accepted today. On the
basis of his results, Hedersrrom created the foundations of stock
management techniques, certainly awkward, but probably the first in
the field of aquaculture. Published in Swedish this work remained
unknown for many years and it was necessary to wait uncil 1904 to
find studies based on bones, in connection with scales and otoliths
(Heincke, 1904, 1908; Cunningham, 1905). An endorsemenc must
be made on the work of Clerk (1927), the first modern author to
develop skeletochronology. He made a comparative study of the
"growth periodicity of bones" in Osteichthyans, Amphibians and
Mammals. He insisted much on the fact that growth is a complex phe-
nomenon under the control of external factors (such as the influence of
the environment and the climate) as well as internal factors. He rec-
ognized that cyclic structures appear in the periostic bone and showed
that certain bones, through the abundance of their periostic formations,
are more suitable than others for the analysis of the cyclic growth on the
one hand, and that the processes of rebuilding involves the destruction
of these structures on the other.
The range of the tools for skeletochronological studies grew rapidly,
from hard spines ro soft fin rays, endoskeletal parrs of pectoral fins,
deithra, supra-occipital, opercular, vertebrae. Menon (1950) drew up
an exhaustive list species by species. Authors usually employ either
whole parts or slices, exploiting the differences in transparency of the
osseous layers deposited during the seasons: opaque zones during the
growing season, rranslucenc annuli during slow growth. When the
annulus is reduced to a narrow line of annual growth cessation, a sim-
ple histological method earlier developed for Tetrapods (Klevezal &
Kleinenberg, 1967; Smirina, 1974; Pascal & Castanet, 1978, inter
alia), based on staining with haemaroxyline, facilitates the observa-
tion of these chromophilic rings (Meunier et aI., 1979; Boet, 1981;
Meunier & Pascal, 1981). The use of bones for age estimation is nev-
ertheless limited to Osteichthyans species, in which scalimetry and
otolithometry give doubtful results. Access to the skeleton is often
more difficult than the simple removal of scales, or even of otoliths.
26
Introduction
2. Chondrichthyans
The first works on sclerochronology for Chondrichthyans are much

more recent (by at least a half century) than those relating to Oste-
ichthyaos. Sharks and skates lacking both an osseous skeleton and
otoliths (reduced only to fine calcium carbonate granules), it was neces-
sary to use other tissues to record skeletal growth marks. Generally
speaking, specialists use the vertebral bodies, of which the cartilagi-
nous structure is fairly well known (Ridewood, 1921 ; Moss, 1977;
Hoening & Walsh, 1982) or spines of the dorsal fins, when these exist
(Squaliformes and Heterodontiformes), and whose morphogenesis has
also been studied on several occasions (Markert, 1896; Goodrich,
1907; Peyer, 1957; Holden & Meadows, 1962; Maisey, 1979). Growth
marks were also described on the teeth (Tanaka, 1990) but these did
not give rise to skeletochronological applications.
Since the work of Ridewood (1921), it has been known that in many
Chondrichthyans, the vertebral bodies presenc concencric rings of cal-
careous cartilage separated by rings from slighdy or uncalcified carti-
lage, giving the vertebrae differenc properties of transparency to light.
These rings are more or less visible at the surface of the vertebral cones.
This regular alternation of calcified and less calcified secrors probably
corresponds to more or less regular cyclic phenomena. Haskell (1949)
was the first to clearly formulate the assumption of a close connection
between these vettebral rings and the growth of the animals. and who
proposed to use vertebral sections to estimate age. The first effeCtive
practical use for the age estimation for a skate was published by
Ishiyama (1951) and for sharks by Parker & Stott (1965). These first
studies were followed by new applications, particularly in the past twO
decades.
A second approach ro age estimation in Chondrichthyans is based on the
study of the spines of the dorsal fins. However, this concerns only taxa
which possess these spines (i.e. Heterodonciformes and certain Squali-
formes). The spines of the dorsal fins have a structure similar to that of
the teeth and cutaneous denticules. They consist of dentine organised
around a long cavi ty and covered with enamel or emaihld (Markert,
1896; Maisey, 1979). Unlike teeth and the cutaneous dencicules, which
are subjecr to replacement, these spines are capable of growing indefi-
nitely, with rhythmic discontinuities. If those are synchronized with a
seasonal rate/rhythm they can be used for age estimation (Kaganovskaia,
1933; Bonham et al., 1949; Holden & Meadows, 1962).
With the developmenc of skelerochronological studies for Chon-
drichthyans, the same difficulty in autOmating reading techniques as
for Osteichthyans arises. Some studies on vertebral bodies have been
27
of variations in
on the basis
are the most prom However, this
that the growth target criteria of the skelecal structures are
well established to be llsed as a suppOrt for automation
1990).
28
Types of calcrtied structure s
Chapter 11
Types of calcified
structures
29
30
Types of calcified structures
Calcified structures have different

in order to
otoliths, scales and skeletal tissue for studies it is
to understand the nature and formation of increments
within these different cs. In this we describe the
morphology and structure for the different cs. we review the
current of accretion processes and how these are influ-
enced by internal and external factors.
A. Otoliths
p. Panfili, B. Morales-Nin, A. Geffen
and function
The inner ear, which is found in all functions both

as an system that detects sound waves and a vestibular sys-
tem which detects linear and angular the
organisms to maintain balance, In the inner ear is a paired struc-
ture embedded in the cranium on either side of the head dose to the
midbrain. Each ear is a sacs and ducts
filled with endolymph, a fluid with viscous
ItA.l). The gross anatomy of these labyrinths and the structure of the
organs are known from many fish
(Lowenstein, 1971). Teleosts have three semi-circular canals
arranged orthogonally to each which detect angular accelera-
tions, The canals open into a series of interconnected cham-
bers or otic sacs that contain sensory the that detects
both linear accelerations and sound.
In there are three such otic sacs, each containing a
calcareous structure, an that acts as a stimu-
lating the kinocilia Chair" cells) of the macula, The three otic sacs are
the which contain the lapililtS and
!tAl), Each otOlith is fixed over
into which sensory cilia
the otolithic membrane (on-
zone that covers the sensory
exhibits a reticulated or
comb zone, which consists of very
loose networks of fibres sensory and non-sensory of
the macula. The zone extends from the otOlith surface to the
of the sensory hairs and its function is probably that of
31
mechanoreception . The lumen of the entire system is filled with

endolymph. In species of Ostariophysi the swimbladder is used to
enhance audirory stimulation of the inner ear (Popper & Fay, 1993).
Figure lI.A.l Asteriscus

Position of the otoliths
within the inner ear
of Teleo st fish (modified Sagitta Semi-circular
from Secor et al., 1992). canals
a) Dorsal view of the
vestibular apparatus
Lapi//us
in a typical Teleost species.
_ '+-:Y--Brain
The top of the cranium
is cut away (frontal section).
b) Otoliths within the
labyrinth systems of typical
Teleost and Ostariophysean
fishes.
Ast = asteriscus;
lag = lagena (vestibule); a
Lap = fapil/us;
Sac = sacculus (vestibule);
Sag = sagitta;
Utr = utriculus (vestibule).
Lag
Sag
Sag
Typicat Teleost Cyprinoid
1.1. Description
The oroliths of the three Otic sacs differ in size and shape (fig. II.A.2).
Differences in orolith shape tend ro reflect phylogeny and develop-
ment, although there is considerable inter- and intra-specific variation
(fig. II.A. 3). Inter-specific differences in shape appear ro be due to
both genetic and environmental influences (Lombarre & L1eonarr,
1993; Nolf, 1995; Torres et at., 2000). Due ro their inter-specific vari-
ation in shape, otOliths have been found ro be useful in taxonomy
(Hecht, 1979), as well as permitting the study of food webs from par-
tially digested remains (Surer & Morel, 1996; Olsson & North, 1997;
Watanabe & Sairo, 1998; Alonso et at., 1999, inter alia). Similarly,
otoliths from archaeological and paleontological finds have also been
32
used in the reconstruction and

Otolith
variations in popularions and stocks
\""'-,'J"~" et a/., 1989; Castonguay et a/., 1991; Campana & Cas-
FriedJand & Reddin,
\I;f),AiaJ"",i~ nirnbaria
S, sagitta;
A, asteriscus,
bar 300 (from
Tomas Paniili,
of various otolith
shapes from
(photo
bar 10 mm,
33
In most species the sagitta is the largest otolith and is most often used
in age estimation. However, the asterisCtls is the largest otolith in
Ostariophysean species (Adams, 1940). Most studies of otolith forma-
tion have focused on the sagitta and sacculus. In the li terature, the term
"otolith" is often used to describe anyone of the three pairs, generally
the sagitta, bur it is important to define this in any study.
Otoliths are generally lateraiJy compressed and left-right symmetri-
cal, except in flatfish and catfish. Details of the terminology utilised
in describing otolith morphology are given in figure II.A.4a. An
otolith has three planes of orientation, following those of the fish;
sagi[[al, frontal and transverse (fig. II.A.4b). This orientation must be
defined carefully in describing any otolith preparation, and reference
should always be made to the standard terminology (e.g. transverse,
saginal. frontal sections). The proximal face of the sagitta has a groove,
the sulcltS amsticus (fig. II.A.4 a, b), which allows contact with a sen-
sory epithelium (macula) of the sacculus (Dunkelberger et aI., 1980;
Fay, 1980; Pla[[ & Popper, 1981). A typical sagitta is elliptical on its
sagi[[al plane, is compressed in its internal-external axis, with a con-
vex proximal face and a concave distal face, and a main axis of growth
oriented in the antero-posterior direction (fig. II.A.4). However, in
several epipelagic and pelagic fish such as tunas, Istioforids, dolphin-
fish, Cyprinidae and deep water species dorsal and ventral sides of the
otoliths are asymmetrical, displaying a butterfly shape.
Posterodorsal Anterodorsal
Figure II.AA dome
A cross section through Dorsal dome Growth
dome Antirostrum marks
a typical sagitta illustrating
the component parts \ I
(modified from Pannella,
19801.
a) Internal and external
faces of a typical sagitta.
b) The three planes Postcaudal /
of orientation of a typical trough
sagitta. Core
Posteroventral ./'
dome Neuron insertion Sulcus acusticus Primordium
area
a Internal face External face
Sulcus acusticus
Sagittal plane
Frontal plane
b Transverse plane
34
Ocoliths are formed extracellularly from the crystallisation of the

aragonite form of calcium carbonate OntO an organic matrix template
composed largely of a keratin-like protein, otolin, which is rich in
aspartate and glutamate residues (Degens et at. , 1969; Watabe et at.,
1982; Morales-Nin, 1987a). The ocolith grows or accretes by the addi-
tion of concentric layers of proteins and calcium carbonate, resulting
in a structure somewhat comparable to that of an onion (chap. IILe).
1.2. Function
Fish labyrinths are involved in the maintenance of equilibrium and
have nervous cells that are sensitive ro pressure, gravity, angular move-
ment and sound vibration (Grasse, 1958; Lowenstein, 1971; Blacker,
1974). Tbe pars superior of the labyrinth (semi-circular canals and
utriculus) deals with posrural information whilst the pars ill/erior (sac-
culus and lagena) is the sound receptor (fig. II. A. 1). Teleost otoliths are
similar to, but larger than, the otoconia of other Vertebrates. The
ocoliths are involved in mechanoreception , acting as electromechani-
cal sound and displacement transducers rhat convert shear forces into
electrical impulses by distorting the kinocilia of the nervous end-
organ or maCttla in the fish inner ear (Poppet & Hoxter, 1981). Rela-
tive motion between the sensory epithelium and the otolith bends the
ciliary bundles and stimulates the eighth cranial nerve. Tbe otoliths
add mass co the gelatinous layer of rhe tbree otic sacs, increasing their
sensitivity to gravitational and otber linear acceleration forces (Ross &
Pore, 1984). The sNlms amsticm of the otolith bas a direct relationship
with the macula of the vestibular epithelium , which is directly con-
nected with the auditory nerve (Grasse, 1958). The receptor systems
are rather different for Ostariophysean species , in which the internal
ear is in contact wi th the swim bladder thtough a complex of bones
known as the Weber complex (Grasse, 1958).
It has been hypothesised that sound reaches the fish ear via two dif-
ferent pathways. Because the fish 's body is approximately the same den-
sity of water, it moves with the water in response to an impinging sound
field. The otolith, however, is denser than the rest of the body, and so
moves with a different amplitude and phase from the sensory macula and
the body. Thus, the sound source directly stimulates the inner ear. In
addition, because the swim bladder contains gas less dense than the
body, the walls of the swimbladder vibrate . This produces indirect
stimulation through otolith displacement (Poppet & Lu, 2000).
2. Periodic increments
Otoliths exhibit a range of incremental structures that are often

formed regularly over time scales ranging from sub-diurnal to annual.
Unlike skeletal calcium, which may be mobilized for homeostasis
35
1 ocoliths do not appear [0 be subject co mineral

resorption except under extreme suess (Mugiya & 1989).
otoliths appear to be suitable for age estimation.
Fish age estimation on visible in otolith The
growth patterns of most interest are at four levels of resolution:
increments, permining a resolution of days;
- seasonal zones, a resolution of several months or a
season;
- annual a resolution of years;
discontinuities in (he otolith (ultra)structure, which correspond to
various stresses that were not regular the life his-
tory of (he individual.
The mechanisms which (hese visible panerns are slightly dif-
although at the level are (he result of varia-
tions in the relative calcium and protein content of the increments or
zones 1
2.1. Primary increments

increments are formed from the successive of a
mineral-rich and a matrix-rich, mineral-deficient around a core
et aI., Morales-Nin, Mugiya, 1987; Zhang &
Runham,
that form these increments. A review of otolith
nr<'c~,nr~,d at che first international on otolith research pro-
posed the terms L- and D-zones (Kalish et al., 1 for the mineral-
and matrix-rich These terms refer to the
appearance of the L- and D-zones appearing light and
dark when viewed under rransmirced light ILA.5a,
b). The difference in the chemical of the rwo zones also
leads to their different appearance under scanning electron
following acid The L-zone is rich in calcium carbonate and
appears elevared in SEM whereas the D-zone is richer in and
poorer in calcium and appears like a in SEM d, el.
This terminology will therefore be used in the following review.
Pannel!a (1971; 1 first discovered incremems in otolirhs
and [hat they were deposired daily. The literarure on
daily increments has led many researchers to infer that lflcre-
mems can be assumed [0 be formed such an assump-
[ion is invalid for a number of reasons. Otolith incremem
may not be or be discernible in
McGurk, AI-Hossaini &
Inter-observer have shown that otolith structures are
often interpreted different readers & Mok-
sness, 1991). increments may not be
some rime after 1987).
Sub-daily increments and discontinuities in the increment record may

also occur (Campana & Neilson, 1985). The daily deposition of incre-
ments generally appears to cease in the adult and/or juvenile life his-
tory stages of long-lived fish (Pannella, 1971, 1980). In some cases
thi s apparent cessation in the daily periodicity might be related to the
formation of very narrow growth increments below the detection limit
of the light mi croscope (Morales-Nin, 1988; Moral es-Nin & Ralston,
1990). However, ultrastructural investigations have also demon-
strated that primary increments are not deposited daily in some
species (Volk et ?tf., 1995). Clearly then , the interpretation of
microstructural growth patterns in wiJd fish requires an understand-
ing of the physiological process and reg ulation of otolith accretion and
of the environmental factors that inf1uence them (Campana & N eil-
son, 1985). For otolith primary increments to be of Llse in age estima-
tion, the processes involved in their regulation must either be syn-
chronized to cyclical environmental events or possess an endogenous
circadian rhythm, entrained to a diel environmental cycle (Geffen ,
1987). In addition, increment formation must be independ e nt of
Figure II.A.5 - A transverse thin sec tion of the otolith of Vinciguerria nimbaria (PhotichthyidaeJ. The primary increments composed
of L- and D-zones are clearly visible.
a) Detail of the core area under tran smission light microscopy. Scale bar = 10 ~m (photo J. Panfili).
b) Detail of the adult growth area under transmi ssion light microscopy. Scale bar = 10 ~m (photo J. Tomas).
c) Detail of the core area after aCid etching under SEM. Scale bar= 10 ~m (photo L. Marec & E. DabasJ.
d) Detail of the adult growth area after acid etching under SEM. Scale bar= 10 ~m (photo J. Tomas).
e) Detail of the primary increments after acid etching, under SEM. The L-zone is rich in aragonite crystals whereas the D-zone
corresponds to deep grooves. One primary increment IS equal to 1 L-zone + 1 D·zone. The figure shows one complete L-zone
and 2 complete D-zones. Scale bar = 1 ~m (photo L. Marec & E. Daba sJ.
37
somatic growth. Experiments have shown that otoliths continue to

accrete even when somatic growth has naturally ceased (Brothers,
1981; Wright et al, 1990; Mugiya & Tanaka , 1992) or has been arti-
ficially restricted (Mosegaard et al, 1988). This continuity may be
related ro differences between the growth of sensory systems such as
the inner ear and other parts of the body.
Primary increments are only visible at high magnification (light
microscopy or electron microscopy). They vary in size from less than
1 ~m to 12 ~m (PanneiJa, 1974). The width of the D-zone is always
less than 1 pm (around 200 to 500 nm) whereas the width of the L-
zone is more variable (from around 0.4 ~m to 10 ~m). However,
because otoliths do nOt grow uniformly the increment width will also
depend on the radius along which it is measured and how the otolith
is secrioned (chap. III.C). Sub-daily increments may be laid down par-
ticularly during periods of fast growth. These structures can generally
be differentiated from daily increments because they tend to be less
well-defined and distinct than daily increments (Campana, 1992).
2.1.l. Primordium
The otolith develops from one or more partially calcified primordia
exocytosed by epithelium cells in the inner ear (Mann et al, 1983).
These cores have been termed primordial granules and they are the
primary or initial components of the primordium. In sagittae the gran-
ules may be composed of vaterite, whereas the rest of the primordiuTlZ is
typically aragonite. Examples of these different types of primordium are
shown in figure II.A.6. The priTlZordium can be either circular, elon-
gated or multiple, depending on the species. In the case of multiple
primordia these coalesce to form the core of the otolith (fig. II.A.6).
The term "nuclew" has also been used to describe the core region of the
otolith, although this term is not recommended since it has also been
used to describe a much larger central area of the otolith (see glossary).
2.l.2. First ring

Otoliths develop in the later part of the egg stage in fish. For some
time after fotmation, the otoliths grow continuously and mostly
without obvious incremental accretion. The time at which incremen-
tal deposition begins differs from species to species. Tbis point in time
is often (conveniently) marked by a distinctive feature, usually a
prominent check (fig. II.A.6d, e). Th e re is considerable confusion
about the terminology used to name this feature, as well as about its
biological significance and timing. This first increment may not have
the same physiological basis in all species. However, there are practi-
cal advantages in standardising counting procedures using this as a
reference point (Neilson & Geen, 1982).
38
Types of calcrtied structures
a d
Figure II.A.6
Examples of otolith centres a) Aphia minuta, b) Goryphaena
hippurus, c) Sa/mo sa/ar (photos B. Morales-Nin, compound
light microscope) and d), e) Vinciguerria nimbaria (photos l.
Marec & E. Dabas, SEM).
c
In several species increments have been noreci which are mosr likely (Q
have been formed before harching (fig. ILA.7). Ir has nor been clemon-
s([arecl conclusively wherher or nor rhese increments are ([ue s([ucrures
or merely optical artefacrs . The opaciry of pre-harch increments is
rarher different from posr-harch primary increments (fig. !I.A.7).
These srruCtures are mosr apparent in young larvae wirh small otoliths.
Hatching is nor really a developmental srage in fish larvae, since a sin-
gle batch of siblings may harch ar different srages of morphological
development. In many species the firsr increment or otolith check may
39
be formed on the day of hatching, and may be properly termed a hatch

ring. However, in other species , the first increment may be formed in
association with a particular developmental stage, irrespective of
whether the embryo has hatched or nor. For example, in Soiea soiea the
increment that is formed when the mourh opens is more prominent
than any preceding increments, and this increment is used as the refer-
ence point for counting (Lagardere, 1989). In Citlpea harengtts, the first
prominent increment is formed welt after hatching and rowards the
end of the yolk-sac stage (Geffen, 1982). In both species the timing of
this increment varies, and depends on developmental rates. In juvenile
oroliths of the Siluriforme species HopiosternulIl iittoraie and Megaiechis
thoracata, the hatch check is welt differentiated (Ponron et al., 2001)
(fig. II.A. 7). In these species the hatched larvae are welt developed and
the ocoliths appear CO exhibit some increments before the hacch check
(fig. II.A.7). In a number of other species, the transicion co exogenous
feeding is represented by a prominent incremenr. Examples of first
incremenrs ciced in che literature are presenred in cable II.A.l.
Figure II.A.7
Examples of otolith hatch
check in Mega/echis
thoracata. The black arrows
indicate the hatch check
and the white arrow
the multiple primordia core s.
The hatch check has been
localised after a validation
experiment. Some
increments are also present
inside the check
and then before birth .
Scale bar = 50 ~m
(photo D. Ponton).
40
Table II.A.l. - References on otolith structures associated with life history events. Otolith structures
refer to those found in sagittae except those denoted with an asterisk, which were lapilli (from Wright
et a/. , 1998),
Species Term used Alternative Rela ted life his tory Reference
b~ author terminology event (timing)
Ammoc/ytes Yolk -sac absorpti o n chec k Yolk-sac absor pti o n (Wr ight , ( 993)
mC/ri ml), Second ar y g tow th cen tre Accessoty Meta mo rph os is & (Wt ig ht , 1993)
/lrimordia settlem en t
Ang/li!la First ring Fitst check End of yolk-sac phase (Lecomte-Finige t, 1992)
allgllllla Deep g tove Firsr inges ri on (Lecomte-Fini ger, (99 2)
Trans ir ion ring Ttans i tion ri ng E nd of metamo tp hosis ILe 'o mte-Finig et, ( 992)
Chromis C heck Settlement chec k Ti me of set tie me nt (Tho rtolcl & Mili cich , 19 90)
alripeClortllis*
Clllpea Fitst heavy ting Fitst chec k End of yolk-sac ph ase (Geffen , 1982)
harengm H atch chec k Fitst ch ec k End of yo lk-sac p hase (Moksness, 1992)
Hatch check Fi tst chec k Between hatch and (Hoe i, 1997)
yolk-sac absotp ti on
Engralffis Check ring First chec k End of yolk-sac p hase (Palometa et a/. , (988)
er/crelsiro!liS
Gadns lfIorh"a Nucl ea r check Firsr chec k H atchi ng (Bolz & Lo ug h, 1983)
Yo lk-sac check E nd o f yo lk-sac (Bo lz & Lough , 1983 )
H atch chec k Firs t chec k Tim e of hatching (Ca m pana , 198 9 )
C hec k Fitst check Hatching (Ge ffe n & Nash , 1995)
HalirhQl!res Hatch check First check H atchi ng (Kishiro & Nakazono, 199 1)
telJllispinis
HoploJlf:mlllll H atch check First check Ha tC hing (Po o.ron el a!. , 200 1)
littorale"
Melanogranllll/IJ Nu clea t chec k First chec k H archi o.g (Bo lz & Loug h , 1983)
aeg!ejin1fJ* Yo lk-sac check E nd of yolk -sac phase (Bolz & Loug h, 1983)
Hatch check (two) Hatching (Campana, ( 989)
Merin(cillS Accessory primorclia Accessory Metam orphosis & (Mo ral es-N io & Alde berr ,
1Jl£'rill(cim prill/wC/ia se ttlement 1997 )
!VI IryostornllS Accesso ry primordia Accessoty Metamor p hosis (eye (Too le et al., 1993 )
jJacifi(J(s primordia mig rati on ro se rtl ement)
M yc tophidae Accessory primordia Accessory Ttansfo rming larvae (Linkowski,1 99 1)
(55 speci es) primordia
OnrorhYllchm H atching chec k Firsr check Hatching (Volk et al., 1984)
kda Seawa ter rraosfer ting Ttan siri on ti ng Seawater tra nsfe t (Yo lk et al.. 1984)
Onrorhynchm Tran sit ion zo ne T rans ition zone Seawater transfet (Yolkelal., 1995)
!,orhllsrba Emer,l!ence check Emergence chec k Emetgence from borrom (M o rrensen & Carl s, 1995 )
OI/(QrhynrhllS Hatch ring First check Hatching (Matshall & Parker, 198 2)
nnka First feeding chec k C rirical period (Marsh all & Parket, (982)
OreorhrOllliJ H atc hi ng ch eck First ch eck One day afrer hatching (Zhang & Runham , 199 2a)
niJotia/ l
Plmroflerf<s Accessory Accessory D uring aod (J earld d aI., 1993)
ameriranm g row th centres primordia after metamorp hos is
Chang ing habitat
Seconda ry Accessory M eramo tphos is (Sogard , (991 )
g ro w th cen tre primordia
PletfrOntrtes Accessory primordia Accessory Meramor p hos is (AI-Hossaini Cl cd. , 1989)
platessa primordia
Accesso ry primordia Accessory Setrlement (Karakiri & Wes ternhagen,
jJrimorC/ia 1989 )
41
Pol/achim virem Hatch check Fitst check After hatching (Ca mpana, 1989)
PomacentrllS Check Settlement check Tim e of settle ment (Thorrold & Mili cic h, 1990)
coeIUli,.*
Sco/Jhlha/1Il1lJ Fitst heavy ring Fitst check End of yolk-sac phase (Geffen , 1982)
maximllJ
Sebas/eJ jord<1ni Extrusion check First check Parruririon (Ralsron el aI., 1996)
So/eo so/ea Hatch check First check Hatching (Lagardere, 1989)
Mouth opening check First feeding (L1gardere, 1989)
Yolk-sac exhaustion check Sratved larvae (Lagardere, 1989)
Theragra Check Check Physiological changes (Nish im Llra , 1993)
chalcugramlll<l
TrachJYhynchlls Accessory pril/l(frdia Accessory Migrmion (Massut ; el al. , 1995 )
IrachyrhymhtlS primordu,
Viflt"igllerr;a Hatch check First check Hatching (Tomas & Panfili , 2000)
7limbariCl
Salmonid ocolirhs display a number of prominenr incremenrs, each

relared ro a differenr developmenral evenr. The earliesr incremenrs
which surround rhe enrire ser of primordia coincide wirh vascularisa-
rion of rhe yolk sac and rhe developmenr of red blood celJs. There is a
prominenr incremenr which marks harching , and anorher prominenr
in c remenr which marks emergence from rhe subsrracum. Under
harchery condirions, checks which correspond ro firsr feeding have
also been observed. For ecological srudies, age esrimarions are based
on coums from rhe emergence mark.
2.2. Accessory growth centres

During rh e larval phase mosr orolirhs conrinue ro accrere around rhe
primordium. However, in rhe orolirhs of many species addirional planes
of growrh are formed ar larer d evelopmemal srages and from rhese new
series of incremenrs emanare. These new planes in orolirh grow rh
resulr from rhe developmenr of accessory growrh cenrres. Accessory
growrh cenrres are parriculariy common in rhe largesr orolirh (sagitta)
of mosr species and rh e lapillltS of Cyprinicls or asterisms of Osrario-
physeans. Accessory growrh cenrres are ofren referred ro as accessory
primordia. However, rhe rerm accessory g rowrh cenrre is preferred, in
order ro avoid confusion wirh primordia which conrain mulriple pri-
mordial granules. Figure II .A.Sa, b shows examples of accessory pri-
mordia in rwo marine species.
Since orolirh shape influences sensiriviry co sound frequencies (Popper
& Hoxrer, 1981), rh e formarion of accessory growrh cenrres may be
relared ro a rransirion in physiology, habirar or behaviour. These srruc-
cures are found in many species rhar undergo a marked habirar change
ar rhe rransirion from rhe larval ro rhe juvenile srage. Examples of
rhese srrucrures in juvenile fish are shown in rable ILA.l. Accessory
42
primordia can also be found on the oroliths of adults. For example, they
ate very common on the distal face of the asterisC/l.S of CoiosJoma ma'Top-
011utm,where they appea r as auronomous srrucrures which g row with
the orolith, also showing seasonal incremenrs (fig. II.A. 8c, d, e) .
2.3. Seasonal and annual increments

Seaso nal incre m e nr s, also termed seasona l zones, m ark s, rings or
ctnlluii (see g lossary), are often di stingui shable on oroliths. They are
often visible in rrop ical as well as temperate species. These zon es ca n
be visible in both wb ole (unrreated) orol iths and/or afte r some form of
preparation (cbap. VIII). The two main types of seasonal zo nes have
-
c
b
Figure II.A.8 . Various examples of access ory primordia.
a) Coryphaena hippurus (photo B. Morales·Ninl.
b) Light micrograph of Me rluccius merluccius otolith . Scale
bar = 400 ~m (photo B. Morales·Nin).
c) d) e) Colossoma macropomum. Ac cessory primordia (stars)
deposited on the distal face (arrows) of the sectio ned and
stained otolith. The proximal face is above. The chromophilic
zones correspond to seasonal increments. Scale bar = 500 pm
(pho tos J. Panfili).
e
43
different opacities. Under transmitted light the opaque zone is dark

and the translucent zone is bright, and under refleered light the
opaque zone is bright and the translucent zone is dark (fig. II.A.9). In
addition ro their macroscopic appearance tbe twO seasonal zones differ
with respect ro the width of primary increments, the thickness and
size of aragonite crystals (Morales-Nin, 1987a), the frequency of
growth discontinuities and organic layers (Mugiya et aI., 1985), the
ratio of calcium carbonate ro protein matrix (Casselman, 1974, 1982,
1987; Mugiya, 1984), and elemental ratios (Casselman, 1982, 1983;
Kalish, 1989, 1991a). It is the combination of these facrors that leads
ro differences in the optical densities of the two zones. Seasonal zones
can reach a few hundred microns in width and are therefore visible ro
the naked eye or at low magnifications (lax - 40x). The difference in
the organic matrix content of the two zones can be underl ined after
burning which turns the organic matrix a rich opaque brown , or after
staining, which colours the chromophilic organic zones (fig. I1.A.l0
and chap. VIII).
Annual increments, also termed annual marks, rings or annuli, are
often interpreted when taking into account the succession of several
seasonal increments. Most temperate and many tropical species
exhibit annual increments, usually comprising opaque and translu-
cent zones. However, in some tropical species biannual growth incre-
ments have been reported, probably related ro multi-annual changes
Figure II.A.9
in environmental and hydrological facrors (Yosef & Casselman, 1995).
Whole otolith of a plaice,
Pleuronectes platessa
(47 cm TU, showing opaque
2.4. lunar-related structures
(0) and translucent (T) zones A common fearure of the oroliths of juvenile and adult fish is a pat-
viewed under transmitted
tern of thick increments separated by numerous less prominent incre-
light (a) and under reflected
light against dark ments. This pattern is usually repeating and has been hypothesised ro
background (b)' The opaque reflect lunat cycles. The best examples of lunar patterns are seen in rhe
zones are dark in
transmitted light and bright oroliths of juvenile flatfish, but they have been described in a wide
in reflected light, range of species. Presumed lunar patrern s have been described in
and inverse for translucent
zones. Scale bar = 2 mm
(photos J. Panfili).
44
a c
b d
Figure IIAlO - Enhancement of the otolith zones after staining. A, anterior; D, dorsal; P, posterior; V, ventral (photos J. Panfili).
a) Sagittal section of an otolith (asteriscus) of Colossoma macropomum (Serrasalmidae, 63 cm SUo Central and dorsal areas
observed in reflected light against a dark background. Scale bar = 1 mm.
b) Sagittal section of the Co/ossoma macropomum otolith (al after aCid etching (EDTA) and staining with toluidine blue. Central
and dorsal areas observed in reflected light against a dark background. The growth zones are enhanced. Scale bar = 1 mm.
c) Sagittal section of an otolith of Angui/la angui/la (Anguillidae, 55 cm TLl observed in reflected light against a dark background.
Scale bar = 500 ~m.
dl Sagittal section of the Angui/la angui/la otolith (cl observed in reflected light against a dark background, after acid etching and
staining with toluidine blue. Scale bar = 500 ~m.
bathypelagic fish otoliths as well, bringing into question the actual

cause of the patterns and what cyclical (physiological or behavioural)
processes they may reflect.
The terminology used ro refer to the presumed lunar pattern varies
(Pannella, 1980). "Lunar partern" usually refers to sets or sequences of
increments, each beginning with a prominent, high-contrast, incre-
ment which is most often termed a "check" (discontinuity; see 2.5)
regardless of rhe cause of formation. The check is followed by a ser of
lower-contrast increments. The widths of the L-zone of the check and
subsequent increments are usually uniform. Several authors have
made use of these repeating features for age determination, on the
assumption that each sequence tepresents a 14-day lunar cycle. Fewer
have attempted to discover the source of the environmental signal that
45
imprints the distinctive parrern. Campana (1987) desc ribed alternat-

ing patterns of high and low contrast increments, each containing
either approximately seven or 14 increments. The formation of these
patterns corresponded well ro variations in tidal height and rhe lunar
cycle. Geffen & Nash (1995) showed that the pattern in Pleuronectes
plate.rsa contained seven increments, and that the discontinuity which
separated the normal increments coincided with dates half-way
between spring and neap tides. Linkowski (1996) has also described a
clear lunar pattern in the primary increment growth of four species of
the genus Hygophum in the North Atlantic.
2.5. Structural discontinuities

Structural discontinuities, also known as checks, are breaks within the
regular arrangement of the primary increments (Pannella, 1980; Cam-
pana & Neilson, 1985; Gauldie, 1987; Morales-Nin, 1987a; Gauldie
& Nelson, 1988). These may interrupt the succession of seasonal or
daily increments in a cyclic or acyclic way. They can be distinguished
under high or sometimes low magnification, usually after some prepa-
ration. The discontinuiry affecrs the growth pattern or the direction of
growrh. They generally appear after acid etching as deep grooves in
the otolirh surface and under the microscope they appear as wide D-
zones (fig. II.A.1la). The organic matrix is usually abundant in these
discontinuities (fig. II.A.l1b) (Morales-Nin, 1986b), which may be
why they are often stainable after acid etching by certain histological
dyes (Pannella, 1980). A discontinuity preceded by incremenrs of
decreasing width might correspond to a seasonal ring (fig. II.A.1lc).
Discontinuities are typical of all species and are probably induced by
disturbances or stresses suffered by individuals in their biotope. Pan-
nella (1980) proposed a classification for disconrinuities (checks)
according to their structures and presumed causes, although withour
much justification. However, research has demonstrared thar cerrain
discontinuities are related ro developmental events, such as the change
from pelagic to demersal life, or settlement in coral reef species. For
example, rhythmic growth patterns and checks in Merlltccim capensis,
M. paradoxus and Genyptertts cape11Jis were found to be related ro activ-
ity patterns and different life strategies (Morales-Nin, 1987 b). As a
discontinuity represents an interruption in growth of unknown dura-
tion, its interpretation can be ambiguous and may pose problems in
making estimates of daily age (Campana & Neilson , 1985). When the
duration of formation of a discontinuity is known, for example with a
winter stress check, such structures can be useful for estimating
annual age.
46
Types of calcified structure s
a
b
Figure II.A.]] . Discontinuitles found within otoliths.

al Discontinuities (arrowsl in the regular arrangement of otolith
primary increments of Vinciguerria nimbaria (Photichthyidael
(photo J Panfili).
bJ Scanning elec tron micrograph of a demineralised otolith of
Dicentrarchus labrax showing the organic matrix and the
tran sversal groups of fibres co rresponding to discontinuities.
Scale bar ~ ] ~m (photo B. Morales-Ninl.
cl Scanning electron micrograph of a Dicentrarchus labrax
demineralised otolith showing the decreasing increment width
and the structural discontinuity corresponding to a seasonal
increment Scale bar = 10 ~m (photo B. Morales-NinJ.
c
2.6. Secondary growth zones

In many species rhe esrimarion of annual incremems is made difficulr
by rhe presence of non-periodic "secondary " zones. This rerm applies
co a range of non-seasonal zones characrerised by differem opaciries
and rhickness_ The rwo major rypes of secondary zones are fal se and
splir rings. False rings appear as rranslucem zones wirhin an opaque
zone . They are parricularly common in rhe firsr year of ocolirh growrh
and in many cases are easily confused wirh rhe firsr annual incremem
(fig. II.A.12). For insran ce , d epending on rhe spawning period, age-O
Trachums mediterralleus presems four rypes of orolirhs rhar diffe r in rhe
presence, number or appearance of false rings (Karlou Riga, 2000).
Splir rings appear as double srrucrures, almosr as rhough rhey were
composed of rwo unusually rhin rranslucem bands separared by a very
rhin opaque band. In some species rhe annual incremem is composed
of mulriple rings , wirh a narrow well-defined rranslucem zone fol-
lowed by some very opaq ue marerial (fig. ILA.13).
47
Problems in distinguishing between secondary and true seasonal zona-

rions is a major cause of age-reading errors. There are currently no
objeCtive criteria for identifying secondary structures, despite exten-
sive reviews of this problem. Lirtle is known about rhe causes of these
secondary structures, alrhough a number of factors including temper-
arure, food intake and developmental transitions have been implicated
in their formation.
Secondary growth structures are also present at the primary increment
level in species with peculiar life histories, such as Myctophids and
relared species which start their life in rhe upper layers of the warer
column and lmer make diel migrations between deep water during the
day and the surface at night. Secondary related daily increments have
been described in rhree species of tropical Myctophids (Gartner, 1991)
and in Vincigtterria nimbaria (Tomas & Panfi li, 2000). These secondary
increments appear ro be sub-daily incremenrs. Due to the possible
variation in incremental structures discussed above, it is important to
describe all structures recorded carefully according to rhe standard ter-
minology (see glossary).
3. Regulation of incremental deposition
3.1. Exogenous influences on primary increment periodicity

Several studies have examined the relationship between increment for-
mation and specific environmental facrors and a number of possible
synchtonising factors have been proposed. Pannella (1980) suggesred
that increment periodicity may be related w the number of peaks in
feeding activiry. Feeding frequency has been reported as influencing
increment periodicity in some species, for example, Oncorhynchtts
tschawytscha (Neilson & Geen, 1982) and Pleuronectes platessa (Al-Hos-
saini & Pitcher, 1988), bur not in others such as Lepomis macrochir
(Tauberr & Coble, 1977), Oncorhynchtts nerka (Marshall & Parker,
1982), Platichthys stellattls (Campana, 1983) and Salmo salar (Wright
et aI., 1991). Moreover, starved fish often continue to deposit daily
increments (Taubert & Coble, 1977; Marshall & Parker, 1982; Cam-
pana, 1983; Wright et cd., 1990). There thus appears to be little evi-
dence to suppOrt Pannella's hypOthesis of a relationship between incre-
ment periodicity and peaks in feeding activity.
Owlith growth is sensitive to temperature in a number of species
(Brothers, 1981; Mosegaard et aI., 1988) and Brothers suggested thar
remperature fluctuations are a major influence on increment forma-
tion in temperate stream fish. Thermally-induced marks on otoliths
demonstrate how strong and sudden remperature variarions may dis-
rupt otolith growth (Volk et aI., 1994). Gauldie & Nelson (1990a)
proposed a carbonic anhydrase-regulated sysrem for owlith formation.
48
IIAI2
ring (Frl) in the sagitta
of a I·year old whiting,
zone
of an annulus is also shown
for comparison,
Scale bar 0, I mm
(photo PJ Wright),
a b
Figure ILAJ 3
False rings,
Scale bar I mm
Morales·NinJ.
Sagittal otolith
of Trachurus trachurus Such a chemical sysrem would have eemperacure as ies main exrernai
capensis showing
the multiple zones comrolling facro(. [he role of [he marrix in orolith
in the nuclear area, formarion is no[ well and [his also be relared ro
b) Multiple growth zones
in a Trachurus trachurus various external cues
capensis otolith, appear [0 be necessary for incremenr formanon
& 1977) and Fundu/w hete-
& Neilson (1985)
uanSitlOnS may be mediated
age, as appear ro be essemial for daily incremem
deposition in rhe larval bur nor the juvenile stages of nota-
Im (Campana, Many fish exhibit incre-
menrs with rhyrhmic and a similar Structure and rhickness
to those found in shallow-warer fish, rhe absence of lighr and
daily 1987, Lombarte &
1990; Morales-Nin et at.,
small variations in tidal currents the
of plankrophagous prey may act as a daily
49
In a review of environmental manipulation experiments Campana &

Neilson (1985) suggested that the endogenous circadian rhythm con-
trolling orolith accretion was entrained to photoperiod, but could be
masked by sub-daily temperarure cycles or feeding patrerns. If incre-
ment periodicity is controlled by an endogenous circadian rhyrhm
then increment deposition would be expected to continue in the
absence of entraining stimuli, although the absence of an entraining
stimulus would be expected to eventually lead to a divergence from a
daily deposition rate . Several studies have shown a continued daily
increment deposition rate in the absence of one potential entraining
stimulus such as light-dark transition. Constant daily rates of incre-
ment formation have been reported in juvenile fish held under con-
stant light (Campana, 1984), darkness (Radtke & Dean, 1982) and in
the absence of cyclical variations in light, temperature or feeding fre-
quency (Wright et af., 1991). However, environmental manipulation
experiments do not provide unambiguous experimental evidence of an
endogenously regulared cycle of increment formation, since fish may
have an endogenous feeding rhythm. Moreover, no study has demon-
strated a divergence from a single increment per day, as mighr be pre-
dicred when there is no entraining stimulus, although this may reflecr
rhe short « 30 days) duration of rhese experiments.
Support for light-dark transitions as a sign for entrainment has come
from ultrasttuctural and radio-labelling experiments. Tanaka et af.
(1981) demonsrrated that in Tilapia ni/o/ica, the otder offormation of
the L- and D-zones was dependent on phoropetiod, as a teversal of rhe
light-dark cycle was found to induce a tevetsal in the order of the two
layers. Using radiolabelled calcium (4SCa) to study in vivo orolith cal-
cification in CaraJSiUJ aura/us, Mugiya and coworkers (1981) found an
apparent diel cycle in calcificarion that was associated with phorope-
riod. However, these experiments were flawed because no considera-
tion was given ro the possible effecrs of isoropic equilibration on 4SCa
incorporation. Nevettheless, later in vivo experiments, involving juve-
nile Sa/mo sa/ar subjected ro an isoropic equilibration period, did
demonstrate that orolith calcification was entrained ro dark-light
transitions (Wtight e/ af., 1992). Radiolabelling experiments have
also demonstrated die! cycles of both calcification and organic matrix
formation, associated with photoperiod, within isolated samlfae
(Mugiya, 1987).
3.2. Exogenous regulation of annual increment periodicity

At present the regulation of annual increment formation in otoliths is
not well understood, although it is commonly assumed that seasonal
zones are related to seasonality in somatic growth and environmental
factots. One view is that seasonal variarion in orolith formation is
related to cyclical physiological changes in the fish such as the onset
50
that occurs
IS
these takes the form of correlations in the tlm~

of the different processes and is too weak and insufficient
to allow either ro be The formation of the zones
in relation ro is controversial, given that in sev~
eral the opaque zone coincides with the time of year when fish
are reprodunively while in others the formation of
translucent zones has been related to and How~
ever, zone formation is
fish
have elevated
calcium concentrations, this rakes the form of protein-bound
calcium which wiJ I not affen calcium ioo levels in rhe endolymph
(Kalish, 1991a). As yet 00 studies have been
in order to between calcium and componenrs in
and otolith formation.
The season of formarion of opaque and translucent zones may
during development and in relation to distribution. In
Gadus mw/m?! fcom the Norch for the opaque zone forms
earlier rowards the southern extremes of this range and
later furthet nOf[h. Within each stOck younger
down the opaque zone up to fout months before older
fish. occurs when the translucent zone is well into the pro~
cess of formation. The in opaqlle~zone formation
Vianet ?!t (1989)
rhe summer in the Mediterranean bur in winter in Northern

waters. The time of translucent-zone formation in Sebmtes
entomelcts from the U.S. PaCific coast has been found to vary with sex,
area and year In tbis a link
between temperature and translucent zone formation is apparent
alchough mher SL1ch as food or
nutrient content of the prey, may also be important. Further evidence
of zone formation was found for several of
Acanthurids from eastern Australia (Choat & of
fish showed that rhe formation of opaque zones
in water temperature in the summer.
5]
Manual ollish sclerochronology
3.3. Influences on accretion rate

Temperacure can enhance owllth accretion
somaflC is affected
although high temperatures can also have a
mem width
Ralsron &
tinue to aCCfete even when somatic
(Wright, 1
resuicred rtsu Its in slow-
growing individuals relatively otoliths. In order to
this phenomenon, Secor & Dean (1989) that orolith
accretion may be determined the interaction of (WO components:
the daily of mcrement which continues even
during of no somatic growth, and an component
that varies with somatic growth. a number of
srudies have found that the increase in orolirh accrecion race wich tem-
perature is much more similar to the trend in mecabolic
rare chan co the optimum curve of somatic rate
a/.,1987; eta!., Hoff&Fuiman, J993).
studies by (1991a) and Yamamoro have shown
that individual differences in increment width correlated with
mecabolic rare rather than somatic et a!. (988)
an
ocoiirh
were held,
metabolism relationships for fish.
individual in oxygen consumption and increment size indi-
cates that orolith accretion responds more to a
in temperature than in metabolic rate (Wright et al., 200J).
The action the metabolic response associ-
also appears to have an influence on otolith
accretion rate 1999). The process accre-
tion rare thus appears to be relared [() components of the metabolic
rare, Given the influence of these components on otolith accretion
rare, periods of smrvation would only be to lead [() a gradual
decline in incremenr widths, Evidence for SLlch a response has been
found in a number of studies & 985;
Eckmann & J 987; Molony & Choat, 1990; Umezawa &
1991; Bradford & 1 & Runham,
52
of calcified structures
3.4. Physiological regulation of otolith formation at the sacculus level

Wilbur (1980) that biomineralisation systems had three
properties in common:
all systems involve the transpon of ions and provide
concentrations of ions (i.e. which exceed the solubillty
surface. This enables the formation of
of
alkaline pH must be maintained so that, once
mineralisation can
formation is often
The observation that otolith increments are {,()!Tlnr.<"(1
rich and a mineral-deficiem zone (L- and sug-
gests that one or more of the above must vary The
deposition of mineral-rich zones may therefore (i) be related to a diur-
nal limitation in (involving either a
of the calcium and carbonate ion concentration at
the otolith surface or a decline in or involve the
organic matrix. An with the mineralisation of mollusc shells
suggests that either the insoluble matrix red
mineral deficient zone) acts as a barrier to or
biting within the soluble
matrix are secreted on to the mineral-deficient
1981; Wilbur & 1983). These fJu,~nJ1C
have been considered for otOliths the past three decades.
The otolith is from the fluid of the sac of
the inner ear. Otolith calcium carbonate is in the form of twinned
although abnormal of cal-
1990,
et at, Otolith calcification is rate-limited by
the number of nucleation sites the nsoluble matrix
Mann et at., 1983) as well as by
tion will therefore be the ultimate determinant of the rate of otolith

1990). This matrix is
et
Payan et aI., 1999). As in mollusc

of
(\Xfright, 1991
in the rate of may therefore the
rate of mineralisation. The less sol uble otOlith matrix is of
a protein et al., 1 The matrix is denser in
phase and its amino acid
with age (l\forales-Nin,
53
Invesrigarions of isolared samllae have indicared rhar acrive, regulared

ionic rransporr occurs rh rough rhe epirhelia (fig. II.A.14b) .
Endolymph calcium ion concenrrarion is influe nced by inrracellular
aerive rransporr rhar is sensirive ro plasma calcium concenrrarion.
Similarly, proron secrerion rh rough rhe saccullts is driven by an energy-
dependenr (Na-ATPase) mechanism rhar is sensirive ro plasma pH
(Payan et al., 1999). Changes in plasma ion concenrrarion would
rherefore be expeered ro have a direer effecr on rhar of rhe endolymph.
However, rhe precise mechanism by which plasma calcium and pH
induce changes in rhe physico-chemical co ndirions ar rhe orolirh sur-
face is nor clear. This is because rhe sensory kinocilia barhed by rh e
endolymph are sensirive ro changes in Ca 2 + concemrarions well below
rhe solubility produer needed for calcification (Mugiya, 1987; Wright
et et!., 1992). The seasonal variation in free Ca 2 + ions in rhe endolymph
of rainbow troU[ ranges from 65.4 % of rotal calcium levels during fasr
growth ro 79.1 % during slow growth (Mugi ya, 1966), which proba-
bly represems the range over which Ca 2 + can vary withour causing
physiolog ical malfunction of the neural mechani sms of the mamlCl
(Gauld ie, 1990). It is thus necessary ro explain how ion levels are ele-
vated at the otolith surface above the background concentrations
found in the endolymph . Proximo-distal gradients of ion concentra-
tion have been detected in the endolymph, a condition which will
favour the biomineralisation process (fig. II.A.14b) (Payan et a!.,
1999). Calcareous spherules have been observed in close association
with the orolith surface of a number of fish species and these may be
involved in elevating ion concentration ar the otolith surface (Dale,
1976; Wright, 1990). These spherules are formed and secreted from
the otolithic membrane and are transporred ro the surface of rhe
orolirh within rhe fibrous sub-cupular mesh work (Dale, 1976 ;
Wright, 1990). Diurnal rhythmiciry in orolirh calcification may be
mediared by a diel variarion in plasma chemisrry, as Mugiya (1984)
and Wright et a!. (1992) found a parallel diel decline in orolirh ca lci-
ficarion and roral and free plasma calcium concentrarion. Mugiya
(984) also found a seasonal reversal in rhe rhyrhm of orolirh calcifica-
tion associated with a reversal in [he diurnal plasma calcium cycle.
However, similar cycles in plasma and endolymph composirion in
PlellYonectes plCltessCl (Edeyer et ClI., 2000) were not associated wirh
changes in rhe ionic gradients within rhe endolymph (Payan et a/.,
1999). Neverrheless, Wrighr et al. (1992) found rha[ an induced
depress ion in plasma calcium led ro a net loss of calci um from [he
mineralising oroli[h increment, which indicates rha[ calcium ion con-
centrarion a[ rhe oroli[h surface is sensirive ro plasma concentration.
While [here may be a periodical ionic limi[arion ro oroli[h calcifica-
[ion, however, this alone can nor explain reports of a diel variation in
matrix secretion (Mugiya, 1987; Wrigh[ et a!., 1990) or rhe formation
54
of matrix-rich (Warabe et al.,

1987). The distribution of matrix and mineral in the otolith
appears to occur in two The first is associated with the twin-
plane of the basic 1995).
is a
(Smith,
of the matrix-mineral
association appears in the form of the dense band of fibres chat cor-
in size and orienracion to the D-zone of the IOcre-
et at., 1 This observa-
tion is consisrem with the diel variation in insoluble matrix protein
indicated radio-labelling 1987). The (WO
ro
Intermediate area
GC
'":!.' NE
.!:1
'"
I/)
SI HC ::>
(J Tot Ca Concentrations
'"
.r::;
'"
::lE
~
0.. SC
IIA 14 - Schematic representation of the saccular epithelium (transverse section of sacculus) of the inner ear of a Teleost
the hypothetical model of elemental transport across the epithelium (modified from Pisam et al .. 1998; Payan et 01., 1999)
a) Map of the cell distribution within the saccular epithelium. The macula consists of half cells (HC), which are In contact with
nerve endmgs (NE), cells (SC) and, at its granular cells (GC). It is surrounded by a "meshwork area" con-
taining large (U). "patches area" contains (SI).
b) of elemental transport across the saccular Note that overall movement of W results in net
of W.
c) Schematic of chemical concentrations in the and distal zones. The Y-axis shows the concentrations
while the X-axis the proximal~istal otolith axis. Proteins total calcium (loICa) and HCD) concentration were dlrect-
Iy measured, whereas Ca 2+ and pH concentrations were estimated.
55
Manual of fish sclerocnronology
a for growth and the second to stabilize the

otherwise soluble (Wright et a!., 1992) and
unstable morph (Mann et at., Gauldie &
It is therefore necessary to consider the regulation of both ion concen-
trations and matrix in the ofL- and D-
zones. Given the correlation between otolith calcification and plasma
ion concentration, the concentration of certain ions in the may
have a direct effect on cellular secretions of matrix or may covary with
some other signalling factor. In addition, the calcification neurosecre-
tory in the macula has a cycle which is related to the
of daily increments & Nelson, 1990b; et
al., 2000). A number of recent studies have identified the function of
the different within the saccular epithelium and the
to otolith (Payan et a!., 1
et aI., ILA.1 The secretory cells are
located in the macular area, Within the endolymph,
are more concentrated in the proximal while total
in the distal ItA.
In summary, the evidence to date indicates that the formation of the
calcium carbonate-rich L-zone is influenced by intracellular active
transport of calcium ions (Mugiya, and protons the sai'-
mllts (Payan et al., 1997; et aI., 1999) which in turn are sensi-
tive to calcium concentration and pH et al., 1992;
Payan et al., 1997). Secretion of the matrix varies diur-
with a during the formation of D-zone
et al., 2000). Production of the marrix and
the soluble inhibitor mllst also have a role in
of mineral accretion the formation of the L-zone.
3.5. Hormonal regulation of otolith formation
under endocrine control

either directly or via meraboJic influences
Mosegaard et aI., 1988). Growth hormone (SU-!) may also be
has been found to a reduction in
(Mugiya. 1990) and orolith demineralisation
and otolith mineralisation in fish can be
injections of extract (Simkiss, Such hor-
monal could influence both ion transport and matrix pro-
duction in the samdtts. \'Vright at. (l that as
calcium concentration is
mones, die! in the concentrarion of these hormones
may be responsible for the periodic decline in otolith calci-
fication. carbonate in molluscs involves neu-
56
ral control (Zylstra et aI., 1978). Neural control of calcium concentra-

tion in the JacclI/m a explanation for the direcc
tracking of seasonal and toml calcium levels of the blood
the 1995).
suggests the involvement of the
Endocrine secretion displays a circadian
animals and through the intermed of
metabolic rate, controls most physiological processes
IU"UHHJlU'p:;,l'Ld, studies have demonsEr<Hed diurnal
variations in the levels of several hormones in the plasma of Teleosts

(Matty, These include et al, 1981) a hor-
mone known to influence skeleral growth and calcification in rainbow
trout (la Roche et al ,
B. Scales
F.]. Meunier
1. Description, diversity and function
Fish scales display a high degree of polymorphism (Goodrich, 1907;

Van Ooscen, 1957; Berrin, 1958; Whicear, 1986). Forming in che
upper parr of che dermis (Fig. ILB.la), chey are mineralised e1emenrs
chac belong ro che superficial skeleron. This book essenrially deals
wich che Teleoscs, so we shall oniy discuss che scales of ch is caxa. The
celeoscean scales belong ro rhe elasmoid rype like rhe scales of rhe
Amiidae, che Coelacanrhidae and rhe Dipnoi (Goodrich, 1907; Kerr,
1952; 1955; Cascanec et aI., 1975; Meunier, 1980; 1984a; Meunier &
Zylberberg, 1998). Some orher "primirive" Osceichrhyan caxa, such as
che gars and che bichirs, have ganoid scales, whose suucrure di ffers
from char of elasmoid scales (Goodrich, 1907; Kerr, 1952; Francillon-
Vieillor et al. , 1990; Zylberberg et aI., 1992). These are chick and jux-
raposed scales wich a surface of polysrrarified hypermineralised sub-
srance, rhe ganolne, which has an epidermal origin (Meunier et ai.,
1987; Sire et al. , 1987). The bony basal plare of chese ganoid scales
shows growrh marks, probably wicb an annual cycle, bur chey have
never been used for ageing srudies excepr in paleo-ichchyological work
(Thomson & McCune, 1984). The elasmoid scales are usually regarded
as being of dermal origin (Zylberberg et ai., 1992) whereas ganoid
scales are of epidermo-mesodermal origin (Sire et ai., 1987).
Two forms of elasmoid scales have been described (fig. 1LB.2): crenoid
ancl cycloid, according ro wherher or nor chey possess more or less
minure spines on rheir poscerior margin (Goodrich, 1907; Berrin,
1958). The general srrucrure of chese scales, cycloid and ccenoid, is rhe
same. The spines of crenoid scales show various morphologies
(Robercs, 1993) bur chis has no consequences for rhe cyclical growrh
marks. Whacever rhe cype of elasmoid scales (cycloid or crenoid), che
superficial layer in mosc species displays concemric ridges which are
frequendy crossed by radial groves, rhe radii.
Elasmoid scales can be divided inro rwo main regions (fig. 1LB.lb):
che anrerior (or covered area) and che poscerior (or covering area). They
are deeply inserred inro che dermis and enclosed in a scale-pocker
(Sire, 1988). The scale-pocker, which is associaced exclusively wich
elasmoid scales, is che dermal space which houses rhe scale. le is clearly
delimiced from rhe subjacenr stratllm co1lZPact/J1IZ by rhe "scale-pocker
lining" a bilayered sheer of fibroblasc-like cells (Whicear et al, 1980).
Elasmoid scales are imbricare, chin lamellar places (Berrin, 1944)
rhac consisr of rwo layers (Berrin, 1958; Zylberberg et al, 1992)
58
Se
Ra . :<~--- Mu
o
________ ' - v - '
Fa Ar
CA CP
Figure II.B.l - a) Antero-posterior section in the tegument of a Teleost fish showing the imbricate scales obliquely inserted in the
dermis. Ep = epidermis; D = dermis; Mu = muscles; S = stratum compaclum; Se : scale. b) Superficial view of a typical elas-
moid scale (Cichlidael; CA = anterior area; CP : posterior area; Ra = radius. cl Diagram 01 a longitudinal section of an elasmold
sc ale (adapted from Sire, 1985). Ar: posterior; Ce = external laye r; Ci: circulus; Co: bony layer; CM: Mandl's corpuscules;
Ep= epidermis; Fa = attach fibers; Fm: mineralising front; Le: limiting layer; Pb: basal plate. dl Cross section in the margin
of an elasmoid scale showing the scleroblasts of the episquamma (upper), the hyposquamma (lower) and of the margin (left).
Ci = circulus; Co: bony layer; El: elasmoblast; Pb = basa l plate; SM = marginal scleroblast; SS: superficial scleroblast (pho-
tos F.J. Meunier).
Figure II.B.2 - Superficial Side of elasmoid scales (SEM). al Cycloid scale of Coregonus lavaretus (Salmonidae, Salmoniformes).
Scale bar: 500 ~m. b) Ctenoid scale of Microchirus azevia (Soleidae, Pleuronectiforme). (the anterror part of the scales IS on
the left). Sc ale bar: 250 ~m (photos F.J. Meunier)
59
I1.B.l a thin ornameoced "the external

which overlies a thick lamellar, mineralised basal with
a charaneristic structure called isopedin, which is
as a derived bony tissue 1987 -1 In the poste-
rior region of the scale which is overlaid by the
layer over/ies the external layer et at., 1979). In some
fibrils inserted in the external of the
layer and fasten on to the basal me m-
ILB.lc); chese are called fibres
the cohesion between the scale and
hprh"ro & Meunier, 1981;
in two main spatial networks either as a double-

structure (fig. usual! y in che taxa of the
or as an "plywood" II.B.3b) which
characterises derived groups (Meunier & 1
Generally the diameter of these
lie parallel to each other in a stratum, is greater (50 nm to 150
than the diameter of the fibres bone
nm) (Meunier, 1987 -1 Another type of collagenous fibrils, the
TC fibres which are orthogonal to the strata of che basal has been
described in several taxa, ly in Cyprinid and Characiforme
fishes (Zylberberg & 1982; & 1
II.B.3· Freeze-fractured scales (SEM); al friderici (Anostomidae, Characiformes). Scale bar = 100 ~m. b) Macrourus
(Macrouridae, Gadiformes). Scale bar = ~m (photos F.J. Meunierl.
60
In Elasmoid scales, the external layer and the limiting layer are regu-
larly mineralised, whereas mineralisation of the basal plate is incom-
plete (Meunier, 1984a,b; Zylberberg et at., 1992). At the location of
the radii the superficial layer lacks mineralisation (Sire & Meunier,
1981; Meunier, 1984b; Zylberberg et at., 1992). The specific organi-
sation of the collagenous component of the organic matrix (plywood-
like structure) results in an original mineralisation route: Mandl's cor-
puscles (Baudelot, 1873; Schonborner et at., 1981; Zylberberg et at.,
1992). The diameter of the fibrils is significantly greater than that of
Figure II.B.4 . Mineralising front of the basal plate (SEMl. al Scale of fsox lucius; the surface of mineralising front is smooth. Scale
bar ~ 50 ~m. bl Detail of Mandl's corpuscles in a scale of Astronotus ocellatus. Scale same as cl. cl Fusing Mandl's corpuscles
in a scale of Ophicephalus striatus. Scale bar = 50 ~m. dl Detail of the mineralising front of the basal plate In a scale of Hoplias
almara, The mineraliSIng front is rough (numerous cracks) because of the presence of le fibres. Scale same as c) (photos F.J.
Meunierl.
61
seems to induce the specific

which have a char-
develop ahead of the
then fuse with each other
front which appears
marrix is removed
are pre-
sent, TC fibres also occur in the mineralisation process of rhe basal
plate & 1982; Zylberberg et al., 1992,
front is rough
an imernal reservoir of
et at., 1989) which can be
physiological circumstances (Mugiya &
Persson, such as in adult salmon
sition to maturity (Persson et al., 1998; Kacem &
Among the function of elasmoid there are
of (he and hydrodynamic functions Burdak, 1979). An
important of elasmoid scales is their ability to regenerate.
Teleost fish lose a certain of their scales in the course of
their life Fouda, When a
"_,",Arl?'" is usually undamaged and it remains as
an empty space in which a new scale 1

Bereiter-Hahn & Zylberberg, 1993). The rpP'Pn,'mJ
its
the marks
1987) so that the new ones are unusable for age
estimation. The number of scales grows with age, which
may make it difficult (0 COunt the growth marks. For this reason, i ( is
necessary to sample an number of scales in order to age fish
by means of
2. Growth of scales and marks
Two processes are involved in the

external increases (Q the of marginal scleroblasrs
ILB.ld) which are osteoblast-like 2) the basal plate thick-
ens with the of new strate!
scleroblasts (forming the hyposquama), the eIasmoblasrs which under-
lie (he deep surface of the scale.
It is generally believed that the external layer does not thicken during
the growth of fish, at least in the anterior area of the scale (Zylberberg
et al., 1992). However in some cases, this layer does show
which appears as thin inc:rementallines 1997) bur have
never been used as an indication of external cyclical phenomenons.

Moreover, they do not obscure previously deposited circuli and do not
interfere with the ability to interpret age.
Cyclical growth marks have been described only on the surface of the
scales and they can be studied using classical light microscopy tech-
niques or, if necessary, with SEM . Cyclical events such as seasonal
metabolic slowing, spawning maturation , ete., induce morphological
modifications of the ornamentations, especially the ridges or circttli
which become much narrower and , then form an annulus. Frequently,
when the scales grow again during the spring, the new circuli are more
or less discordant on the ones which had been deposited during the
previous year. In other circumstances, the annulus may be strength-
ened by a marginal process of erosion. This is the case in migratory
Salmonids (Crichron, 1935 ; Van Someren, 1937; Richard & Bagjin-
ieee, 1990) (fig. II.B.5) and menhaden (June & Roithmayr, 1960), for
example. So far, however, no study has demonstrated that cyclical cli-
matic variations can also have repercussions on the histological
Figure II.B.5
Surface of scales of a male organisation of the basal plate. The older the fish, the more numerous
salmon showing erosion are the strata in the basal plates of the scales. However, studying the
areas. a) The whole scale.
Scale bar = 500 ~m. number of strata in the basal plate failed ro age coelacantbs (Sire, per-
b) Detail of the erosion sonal communication), whereas the growth marks of the superficial
cavities. Scale bar = 5 ~m.
AA = anterior area; layer gave an interpretation of age (Hureau & Ozouf, 1977).
PA = posterior area
(photos A. Kacem).
63
On the other marks are obvious in the bony basal

scales, They show the same characteristics as the
growth marks of the bony skeleton, i.e. a wide area of rapid
and a narrow area of slow growth which may be
in bichir scales (Meunier, 1980). For this they will be discussed
with annual bone marks.
3 ... ..,"'U ...... UII marks
In our opinion, the various factors involved in the regulation of scale

marks are the same as chose skeleron marks.
may both show annual bur, to the best of our
no accurate infra-annual (lunar or
marks) has been described, unlike in (he case of the otoliths (see
We therefore consider the regulation of scale marks in
the following which is devoted to skeleron growth marks (see
II.C3).
64
C. Skeleton
FJ. Meunier
1. Morphology of bones and their structural organisation
In Ostei chth ya ns, as in Tetrapod s, the term "bone " can refer to various
concepts:
- an ana to mical organ such as a vertebra, opercull/m, or spin y fin ra y;
- a ti ss ue, i. e. the bone tiss ue which m akes up a ll bone ;
- chemical co nstituents, i.e. the organic mac romo lecules and min eral
crysrallites cha racteri sti c o f bone tissu es . To avoid a ny co nfus ion,
Petersen (19 30) d efined four successive levels of inregrat ion of bone
corresponding to four di fferent modes of investiga tion (tab. n .Cl , see
also Francillon-Vieillot et at., 1990). It is essenrially the first and sec-
ond levels of integ ration which are involved in stu d ies of age es ti ma-
tion . But fo t a better unders tanding of the histop hysiology of bon e in
re la tion to the vari o us cycl ical and n on -cycl ical co nst ra ints that
d e termine b o ne stru c ture , we also briefly describe the third and
fourth leve ls of integra t ion.
Table II.C.l. - Levels of integration of bone (modified from Petersen , 1930; Francillon-Vieillot et al.,
1990).
Order of Approximate Strucmres Examples of Related biological
Strucmre scale characteristic techniques used problems
of the order
First otder lm-Imm Anatomical level DisscCllon, X- ray, sawing; Co mpara tive anatomy
0/ in.tegra.! ion Observation: naked eye, and mo rphol ogy of th e
Bo ne motp hology, binoc ular m,ctoscopy skeleton;
vascular orieotation, Overall grow rh
' rabcmlae
Second orde r 1 mm -lOO flm Histologica.l level Hi srology, hisrochemis try, Cl ass iucarion or rissues,
of integration mi cro X-tay, labell ing if! l'il'l! ; grow rh and remode lling
Ori enta rion , size, nu mbe r O pri cal mJcroscopy, SEM processes;
of 'rabeodae, vascul ar ca nals; Bone hi srophys iol ogy
srru([lIte of ex trace ll u lar
marrix
Thi rd order Cytological Le·vel
100 flm-I flm Ce lls; CY lology, c),wc hem istry
of i n tegration Hig h power of bone cells; ph osphocalClC
Derails of cells, ex rracellular of lig hr mi croscopy, metabolism at ce llula r
matri x: orientati on, polarizin,l! microscopy, level. Irra.5 rrucrure
_ _ __ __ _ __ _ _a_m_o-'u_n_' ,'-o_r"' ga_'_lis- ",_tl_·o_n___ -'S_E_M...:.,_·I_L _M_ _ _ _ _ _ _ of bone marr ix
fo urth orde r 1 pm -tO nm ,"I olecn/ar levei TEM , X- ray di ffracti on , l llrras u uc tural
of integration microprobe, re larionships
chemi ca l and biophysica l bi ochemica l and molec ul ar between ce ll organ el les and
orga nisation of organic and rec hni ques exttacell ul il r marrix;
minetal compOne nts bi och em isrry of bone
matrix and bo ne
mi nera lisat ion
65
1.1. Constituents of bony tissue

as in Mammals, bony tissues are made up of an
mineral constituents and various types of cell (Fran-
cillon-Vieillot et al, 1990; Meunier & Fran,;ois, 1992a). The organic
matrix is a network of more or less orienred fibrils embed-
ded in various complex molecules such as the (Glim-
The mineral constituent consists essemially of
which uces thac deposit directly on rhe
""'ClIlJlD fibrils. The mineralisation of tissues in Oste-
ichthyans is an process, i.e. there is an interactIOn between
fibrils and mineral (see and chap.
ILC 1 1.3).
1.1.1. Cells
The bony cells (scleroblasrs of 1890) are of three types: 1)
the surfaces and which
the and mineral consti tuems of 2) the osteo-
cytes, which are embedded in the bony substance and which a
function in tissue; 3) the osteoclasrs, whose main role is
the destruction of bone (Francillon-Vieillot et at., 1990; Sire et al.,
1990; RicqU:s et a/., 1991; Meunier & 1992a).
The osteoblasts first (he proteic macromolecu essen-
collagen, in from of the bone surface. are then progres-
surrounded by the 05teo-
cytes. also contribute to the mineralisation of the matrix
\'\.!hen osteoblasts are numerous on the
external surface of a they form a sort of thick the
periost. When lie on the surface of vascular caviries
endost (Francillon-Vieillor et al., 1990).
The osteocytes are more or less cells with numer-
ous branched cytoplasmic extensions in the extracellular matrix
II.C.la) (Stepban, However, characteristic of many
Teleosts is the lack of osteocyres in the bones,
evolved groups such as the
1859; Blanc,
e! al., 1
23600 known Teleost
believed
that (be lack of osteocytes in acellular bone is a result of the with-
drawal of the osteoblasts ahead of the matrix front soon
bony substance (Moss, 1 1987).
primary bone (see chap. ItCl is
extensions ongmatmg from superficial
1987; Meunier
66
& Huysseune, 1992; Hughes et al. , 1994). These cytoplasmic exten-

sions may be more or less ramified in the bony tissues. As far as skele-
tochronology is concerned, the cellular nature of bony tissue, i.e. cel-
lular bone versus acellular bone, is of no great importance for the
seasonal rhythmic growth of bones, and many acellular bone species
Figure II.C.l. a) Cellular bone (ground section, transmitted natural light) in the frontal of Arius proops (Silurilormes) showing typ-
ical star-shaped osteocytes (arrows) with numerous cytoplasmic processes; Scale bar = 50 ~m). b) Acellular bone in the supraoc-
cipital (cross section, Masson's trichrome staining) of Trachurus trachurus (Carangidae); pb = primary bone, sb = secondary
bone, vc = vascular cavity or canal. Scale bar = I 00 ~m (photos F.J. Meunier).
Figure II.C.2 - a) Acellular bone (ground section, transmitted natural light) in a dorsal fin ray 01 Lethrinus nebulosus (Perciformes,
Lethrinidae) showing numerous osteoblastic canaliculi in the primary bone. Secondary bone is limited by cementing reversal lines
(arrow) and is devoid of canaliculi. b) Microradiography of the same section showing the hypermineralised cementing lines
(arrows). Secondary bone is less mineralised than primary bone; ot = osteon; pb = primary bone, sb = secondary bone, vc = vas-
cular cavity or canal. Scale bar = I 00 ~m (photos F.J. Meunier).
67
which show growth marks are aged on the basis of the study of their
bones (Casselman, 1974; Meunier et al., 1979; Johnson & Saloman,
1984, inter alia).
The osteoclasts, whose existence was denied for several decades (Blanc,
1953; Moss, 1963) have been now generally recognised and accepted
(L6pez, ]973; Meunier, 1983; Glowacki et al., 1986) in both cellular
and acellular bone. But unlike mammalian bone, in which they are
typically multicellular, they are frequently unicellular in fish bone
(Sire et ctl., 1990). The osteocJasts destroy bony tissues, forming a
depression on the bone surface: Howship's laCtlna. When several How-
ship's lacunae merge, the osteoclasts may create fairly wide cavities.
After some time, these cavities may be more or less filled with new tis-
sue known as "secondary" bone (see chap. rr.c.1.3.2). Thi s process of
erosion and reconstruction is calJed bone remodelling (Enlow, 1963).
If the remodelling of bone is very developed it may contribute to the
disappearance of growth marks and, thus leild ro underestimates of
age. For this reason , it is essential to know how to recognise the pro-
cess of bone remodelling (see below) for age estimation studies by
means of bone histology.
1.1.2. Organic matrix

The main parr of rhe organi c matrix is formed by the collagenous
fibrils; in mammalian bone it is thought that collagenous fibril s repre-
sent 23 co 32% of the dry weight (Casselman, 1974), depending on
the degree of minerali sat ion, the collagenous fibril s being lower when
bone is hypermineralised (Herring, 1972). These fibrils are embedded
in complex proteoglycans that play various poorly understood roles.
The collagenous fibrils can be divided into twO main categories: the
intrinsic fibrils that constitute the fram e of bone and the extrinsic
fibrils which have an anchoring function for various struCtures (gener-
ally ligaments and tendons) in bone. Sharpey 's fibres are extrinsic col-
lag enous fjbrils (Francillon-Vieillot et al., 1990; Meunier & Fran~ois,
1992a); they are made of closely-packed coJJagen fibres (= bundl es)
which insert more or less perpendicular into th e collagenous frame of
bone (fig. II.C.3a). These Sharpey's fibres when present warrant that
bone is primary (see below).
The collagenous intrinsic fibrils make up a three-dimensional net -
work, whose organisation is well known and which corresponds to
specific biologica l constraints. Three spatial model s have bee n
described for collagenous arrangement in bone: "woven-fibre bone
matrix" , "parallel-fibre bone m a tri x" and "iameilar bone matrix"
(Francillon-Vieillot et etf., 1990; Ricql cs et etf., 1991). First described
in mammalian bone, these three sparial arrangements of collagenous
matrix have been also recognised in fish bone (Meunier & Fran ~o is,
1992a ; M eunier & Huysseune , 1992) Collagenous fibrils look like
68
ray (cross paracrysralline sttuccures and as sllch they deviate

ground transmitted
polarised light) of Cyprinus This pwperty is used to study the orientation of the
(Ostariophysii, in bony tissues,
vm'.mc.,,,, They bright
orthogonal to the bone The woven-fibre bone is formed of loosely coarse
surface, fibres of differenr sizes; are distribuced withouc any ordered spa-
bJ Acellular bone
tial arrangement, Under light, woven-fibre bone looks dark
in cross-section, \X7ben present, the osteocytes (in cellular are
(Perciformes, randomly disrribuced and are rather round, and elon-
Lethrinidae) a number of ramified processes that tun in
lamellar secondary
pb bone, very fine tunnels called canalictt/i, The poor of
sb = bone, woven-fibre bone is characteristic of a pro-
Scale bar ~m
(photos F,J, Meumer), cess of as rakes bone (Casraner et a/. ,
1992;Castanetetal,1993).
bone", the col-
and all have the same on-
according co the orientation of the section, The osreocyres

are flanened and randomly distributed and rheir
tions preferentially run in che same direccion as che
cell, Parallel-fibre bone is as
woven and lamellar bone as far as ics
is concerned (Casranet et al.,
Lamellar bone is tbe most
thin (or ta~lzettae
[ils lie in
69
L 1. 3. Mineral content
The mineralisation of bone is a phenomenon (Glimcher,
1998). In this of rhe topic.
The mineral content consists of calcium whIch forms crys-
mllires close to hydroxyapatite fish bone
mineral also contains a amount of carbonate as well as very
small amounts of several other components such as
and ions: Si, Ba, et at.,
et at., 1992). While it used to be believed that an amorphic
deposition 1972), it is now assetted
that the Hamorphic" is in face made of minute
lites (Glimcher, 1998). However, the high level of whitlockite in sev-
eral shows that the crystallisation of calcium
(see a Iso Baud, &
The mineral are formed short! y after the

matrix. The surface of the which is mineralising,
forms the front of mineralisation. The between the of
the matrix and its mineralisation may
especially in the basal plate of scales (see above).
sues, the diameters of the fibrils are
bone collagenous fibrils. This
the
70
Types 01 calcified structures
which are mineral concretions that ahead of the

from of the basal before with it (Schon-
bbrner et al., 1981).
The bone matrix controls rhe orientation of the
atite which are oriented along the
In such cases, mineralisation is
1968). The converse situation is mineralisation characterised
a radiating of independent of the
nous fibrils which leads co mineralised bur this is
rare (FranciHon-VieiUor et a!.,
There are (WO ways of of mineral in bone: the
rate of mineral and (be of mineral. The tate of mineral, wbich
is obtained the incineration bone, a assess-
mem of the mineral constituents in bone; values usually lie between
60 and 70% of rhe (Moss & 1963;
1974; Kacem et
af., 2000). The
measuring local variations in mineral levels (bat may be

The of mineralisation of bone is
per unit volume i.e.
tbe tew values avail-
able lie berween 0.8 and 135 see 1983).
Tbe of mineralisation seems (0 be in acellular than in
cellular bone (Meunier, 1
1.2. Spatial organisation of bony tissue

of cell types, of the marrix and its spacial
and of (he mineral componenr
allow the characterization of bony tissues et Cl/. ,
et et!., 1991; Meunier &
tain otber accurate structural measurements may ofter
dara that will be useful in and
information, which ate hidden in the bony tissue. Sucb data include
the lines and the vascular network of bone.
1.2.1
can show a number of
lines in the extracellular matrix which mayor may not
very narrow and can be
and Periodic Acid Schiff. They are
under transmined or dark in reflected light on unsrained ground
senions. There are (WO kinds of lines: the lines"
and the "reversal lines" The lines ot arrested lines
71
(AGL) (in French: "Iignes d'arret de croissance"; LAC) are concordam

with the bone lamellae (fig. II.C.4a). In carp, the only fish in which
their ulcras(fuccure is known, they are approximately 2.5 pm thick
and they consist of thin fibrils, 5 nm in diameter (Castanet, 1981) .
.. .
........
.
, "
. ,
Figure Il.e.4
a) Four annuli (arrows) in a scale (cross ground section, micro-
radiography) of Polypterus bichir (Polypteridae). The minerali-
sation of these annuli is the same as that of dentine (d) but obvi-
ously less than the superficial ganoin (g). Scale bar = I 00 ~m_
bl Reversal cementing lines (thick arrows I in a dorsal spiny ray
.
(cross ground section, microradiography) of Katsuwomus
pe/amis (Perciformes, Thunnidael; also note the numerous
osteocytes (thin black and white arrows). bp = basal plate, pb
= primary bone, sb = secondary bone. Scale bar = I 00 ~m
(photos F.J. Meunier).
The second type of cememing lines, the reversal lines (in French "lignes
de resorption") emphasize small discorclances in successive bone
deposits (fig. II.C.2b, 4b). The reversal lines represem a break in the
appositional growth of bones (Cascanet et aI., 1993). They are delicately
crenellated and hypermineralised. They separate a deposit of secondary
bone from another older one (see below) (Amprino & Engstrom, 1952).
This reversal line acts as a cemem or paste between the tWO bony areas.
In bony fish these cememing lines are 1 pm thick and consist of a dou-
ble edge of opaque material (Casta net, 1981). The crenellated aspect of
the reversal lines is a result of the osteoclastic activity that precedes the
deposit of secondary bone (Matrajt et aI., 1964).
1.2.2. Vascularisation of bony tissues

Bony tissue includes empty spaces that house blood vessels, adipose
cells and fibrocytes. These spaces are called vascular canals or vascular
cavities, depending on their shape (Francillon-Vieillot et aI., 1990;
72
Ricqles et af. , 1991). One of the funcrion s of the vascular canals is a

trophic role for bon e since they provide m erabolites ro the surround-
ing bony tiss ues. They form a vascular network which is more or less
developed, depending on the raxon. When vascular areas are present,
it is kn own as vascular bone (fig. II.CSa); when bony ti ssue lacks vas-
cul ar strucrures, it is call ed avascu lar bon e (fig. II.CSb) (Francillon-
Vi e illor et af. , 1990; Meunier & Fran~ois, 1992a). In spongy bo ne, the
total volume of the cavities is hig her than bony substance volume (fig .
II.CSa). Generally speaking, the vascular canals are regarded as be ing
more developed when the metabolic activity of the fish is high, as for
example in rhe tunas (Amprino & Godina, 1956).
Compac t bone is generally relatively poor in vascular areas wh ereas it
is characterised by spong y bone. When present, thi s tends to be rather
loca li zed in the inner reg ion of bones and is surrounded by a more or
less thick compact bone (MeLlnier & Fran~ois, 1992a).
Figure II.C.5 - a) Spongy bone in the supra-occipital (cross ground section, microradiography) of Pomadasys hasta (Perciforme s,
Haemulidae) showing erosion cavities (ec); Sc ale bar = 50 ~m. b) Ava sc ular bone in the dorsal spiny ray (cross ground section,
transmitted natural light) of Cyprmus carpio (Ostariophysii, Cyprinidae) showing two annuli !thick arrows) and flattened osteo cytes
(thin arrows) characteristic of pseudo-lamellar bone. Sca le bar = 50 ~m (photos F.J. Meunier).
73
In elasmoid vascular canals are scarce. transverse

holes ("canalicules 1873) have been described
in the f>ncrp'rI{)T cross in cerrain taxa:
for where they are 30-
among other strllcrures, vessels and nerve

are true vascular canals but do not
growth marks,
to characterise different modes of
the
ray of the tittortlle which grows
and in thickness. We can define three types of vascular canals
et al., 1990):
- "radial vascular canals", which link the inner parr of the bone (even-
tually the medullar cavity) to its ourer
" vascular
tal axis of the ray;
"c i rcular" vascular more or less concentric and to the
surface of the bone.
It is clear that in any bone we can describe the
sation of the vascular network with rhe association of these different
primary vascular types (see for
There are twO types of vascular canals: the
cular canals, i.e. rhey form ar rhe same rime as rhe of
bone. The osteoblasts secrete components around the vessels of
the so that are incorporated into the bony tissue.
It is ly believed that the more numerous the vascular
canals or cavities, the more acrive the metabolism of the ani-
mal (Ricqles et at., The second type consists of the
vascular which result from the of the osteo-
clasts. These cells act as rrepans, tunnel-shape cavities that
are invaded by On the walls of these vascular
endosteal osteoblasts often substance which makes
up the from the older bone
reversal rp,'I")pnr. When these sec-
vascular canals are
axis of a are called osteons
1.3. Histophysiological significance of bony tissue

A study of bone the various classes of Vertebrates
fossil taxa) shows that the basal constituents (cells,
all at the same point
ical the Ordovician
there do nor exist any
74
Figure II.C.6 - Hoplosternum lit/orale. Cross section of pectoral spiny rays (microradiography) (photos F.J. Meunier).
a) an experimental bred spec imen; showing the superficial odontods (white arrows) and one area of hypervascu larised bone
(black arrow) corresponding to the first spawning season. Scale bar = I mm. b) One wild female showing four hyperminerali sed
annuli and the poorly vascularised bone; Scale bar = I mm. c) One wild male showing two hypervascularised zone s (a sterisk).
Scale bar = I mm. mc = medullar cavity.
evolurionary rrends in bone srru crure rhat are directly re lated to the
evolution of vertebrate taxa, bur tather to rhe constraints linked to
physiological speciali zation . The classification of bony types which
follows fro m the various association between the presence o r absence
of osteocytes, arrangements of collagenous fibril s, the presence or
absence of vasc ular areas etc., has no evolutionary signifi cance. On th e
contrary, it has various types of physiological sig nificance which are
expressed in Osreichthyans as well as in R eptiles and Mammal s (Fran-
cillon-Vieillor et a/., 1990 ; Ricqles eta/. , 199 1) and whi ch are related
to epigen etic consrraints such as seasonal g rowth , m etaboli c intensi ty
and breeding.
75
Manual of fish sc lerochronology
1. 3.1 . Bone growth

Bone growrh is an apposirional process rhat results from the acriviry
of the osteoblasts, which are locared in the periosteal membrane (see
above). Perios t produces primary bone which last for the life of rhe fish
or can be partially destroyed, ro be rep laced by new bone, which is
rhen considered as secondary bone (but see below). The s([ucrure of
primary growing bony tissue depends on rhe rate of osteogenesis; i.e.
wherher ir is young or adulr, on the one hand , or a fi sh wirh a hig h o r
a low metabolic rate, on rhe other. Whatever rhe factors rh at influence
the rare may be, the osreoblasts are under the influen ce of seasonal
rhyrhms .
The m easurement of periosteal os reogenesis is based on vital labeIling
techniques (Meunier & Boivin, 1974; 1978; Beamish & McFarlane,
1983; Babaluk & Craig, 1990; Boujard & Meunier, 1991; Trebaol et
a!. , 1991). There are very few srudi es of rh e rate of periosteal g rowrh.
Depending on the leng th of rhe fish, irs age and physiological srage,
periosreal osseOLlS apposi rion ranges between 0.1 co 20 pm/day (Sim-
mons et a!. , 1970; Casselman , 197 4 ; Boujard & Meunier, 1991; Meu-
nier & Fran"ois, 1992b, illter alia).
1.3.2. Bone remodelling

ln chaprer H .C.l.1, which deal t wirh rhe srudy of elementary compo-
nents of bony rissues in Osteichrhyans , we saw thar it was necessa ry to
discriminare primary fr om seco nd a ry bony ri ssue. Primary bone is
new bone thar is deposi red by periosric cell , i.e. osreoblasr, activiry,
without previous bone destrucrion. These ce lls are also res ponsible for
increases in bone rhi ck ness by adding new peripheral layers. On the
contrary, secondary bone, which is deposired by endos real osteoblasts,
follow s an erosive process thar is the resulr of osreoclast acriviry (fi g.
II.C.3b, Sa). These osteoclast cells, frequently unicellular in fish bone
(see above, Sire et a!., 1990), induce vascular cavities and /or vascular
canals which may be progressively fi Iled by new bony rissue. Thi s sec-
ondary bone is always separared from rhe primary one by a specific
cementing line named "reversal ce menting line " which has rhe same
hi scological properties as rhe res ring lin es, excepr for their irreg ular
appearance (see before). This phenomenon is called bone remodelling
and eve n if primary and secondary bony rissues show the same basic
consrituents the y are of quire different p hysiologicaJ sig nifi ca nce
(Francillon- Vi ei jjot et a!., 1990; Meuni er & Fran"ois , 1992b).
Remod ejjing is not very developed in most O steichthyan raxa, so bony
ri ssues can usually be regard ed as relarively permanent rhcoug hour the
life of individual fish. On rhe other hand, runa and related species show
a bundant remodelling in rheir skeleron, at leasr in the axial skeleron
(Amprino & Godina, 1956; Poplin et a!. , 1976), superficial bones such
76
as fin rays being relarively spared (Meunier, unpubl. observarions). Age

esrimarion can possibly be done by smdying owlirh s (Srequen et CIf.,
1995; hoh & Tsuji, 1996, interalta). Moreover, body gwwrh in fish is
regarded as un ceas ing (DU[ra, 1994; Goss, 1994). Th e bony skelewn
gwws rhroug hom life, eve n rhough afrer firsr reproduccion gw wrh
slows down signifi camly (Bwwn, 1957). The ske lewn record s, via spe-
cific sr[U crL![es, i.e. skeleral growrh marks, rhe rhyrhm of growrh as
well as rhe evems which affecr ie. Therefore, cenain skel ecal elemems
can ofren be used as a record of age as soon as rhe bony rissues show
reperirive srru cmres, rhe growr h marks, and if rh ese s([uc(Ures are syn-
chronised by a cyclical seasonal facroL
Ce rc ain Teleosr families (Carangidae, Drepanid ae) or individual
species (belonglOg w rhe Gad idae , for example) may develop hyperos-
rosis, i.e. abnormally swollen bones (Desse et af., 1981; Dri esc h, 1994,
inter alta). In rh ese bones, growrh (primary bone apposi rion) seem s w
have been srimulared and rem ode lling is generall y very abunda m
(Des se et al., 1981; Meunier & Desse, 1994; Meunier & Zy lberbe rg,
1998). So rhey are nor ar all suirable for ageing smdies.
Figure 11. C. 7
A caudal vertebra of
a Teleo stean fish showing
seven growth marks (annulI)
on the centrum surface
(hollow arrow). The arrow
points the head of the fish.
(HA ~ haemal arch;
NA ~ neural arch;
VB ~ vertebral body).
(After Laerm, 1976),
2. Periodic increments or "the skeleton as a flight-like recorder"
Fiar bone does nor need e laborare spec ial processing for skel e-
wchronological smdy (fig. II.C.7, 8a), unlike '"long" bones like spiny
ray s, which musr be processed us ing bisrological rechniques (fig.
II.C.6, 8b, 9). N ormally, mild cleaning and observarion in a clearing
77
medium for easy discrimination of

growth marks on flat bone et at., 1991;
Lecomre et at., 1 imer lQ bones the
material is observed either on a surface after the undeminer-
alised bone has been secrioned (Deelder & Willemse, Beamish
& 1951; Marzolf, 1
1975; Olatunde, 1979; Loubens & Pan-
or on srained frozen sections on dem i neralised
of these technical con-
of accurate of
the events which have occurred the lifetime of
the fish (Casmnet et at., 1992, 1993),
rhomboidalis the growth marks shown
Opercular (reflected
on black etc.), are the "zones" and "anrmlj"
Scale bar [heir use for is very similar to scalimerry. Authors who have
b) Cyprinu5
ground section dorsal made use of skeletochronology have very little attention to the
ray (transmitted light). of the structural suppOrt of the marks.
bar 1 mm.
(photos F.j, Meunier).
78
Figure II.C .9
Five AGL (arrested growth
line s) (hollow arrows).
Cross section of a spiny ray
(haematoxylin e) of
Plagioscion sQuamoslssimus
(Sciaemdae). Bone is
ace llular and show. arround
the medullar cavity (mc),
secondary bone with some
secondary osteons (Os ).
separated from primary
bone by a reversal
cementing line (RI).
Scale bar = 200 ~m
(photo F.J Meumer).
However, knowledge of their hisrological strUC(Llre is esse nrial ro the

understanding of the relation ships between the skeleron and th e
genetic and epigenetic facrors of growth (Castanet et a L., 1977; Cas-
tanet et aL., 1993).
Table II.C.2 . Formation of the growth marks (modified from Castanet et al., 1993).
High metabolism period Low metabolism period
Tem pe rate: spring-sum mer Temperare: winte r
Tropical: wer season Tropical : d ry season
Young ----------------- Adulr Young ------------------_ A_d_u_l_r _ __
Fasr-growing bone Woven bone Pseud o-lam ellar bone Pseudo-lamellar bone Lamellar bone
Interm ed iary Woven bone Pseudo-lamellar bone Anntdm wirh
g rowi ng bone Lamellar bone AGL
Slow g rowing bone Pseudo-l amellar bone Lamellar bone AGL AGL
2.1. Growth marks

Growth m arks that are use ful for agei ng are only found in primary
bone. This is why the inregrity of this type of bone is essential for age
estimation studies, because remodelling destroys this particular infor-
mation (Casta net et aL., 1992; Castaner et aL. , 1993). For example, it is
pracrically impossible ro es timate the age of tuna by their verrebrae
because these undergo an imporranr remodelling process that destroys
79
primary bony areas. Moreover, growth marks have specific properties,

under light microscopy, which are used co distinguish them in age
estimation scudies. There are three categories of growth marks in
bone: "zones", "annuli" and "arrested growth lines" (AGL).
The "zone" is the thickest growth mark. It appears dark under trans-
mitted light and bright in refleered light (fig. II.C8b). When bone is
vascularised, vascular canals and cavities are more numerous in zone
marks than in annuli. In fast-growing young animals, opaque layers,
i.e. fast-growing layers (zones), are made of woven fibre bone wirh iso-
diametric osteocytes randomly distributed. In adults, the bone growth
decreases because of the competition of reproduerive metabolism, the
woven fibre bone is replaced by parallel-fibre bone or, eventually by
lamellar bone, both with more or less flattened cell lawnae (fig.
II.C5b) (Castanet et a!., 1992; Castanet et a!., 1993).
Annttli are clearly less thick than zones. Generally speaking, in bony
fishes the annttlllS consists of one or more bony lamellae which are weakly
hypermineralised in comparison with the mineralisation of the fast-
growing bone of the zone (Meunier, 1988; Castanet et a!., 1993, inter
alia) (fig. II.C4a, 6). Moreover, the density of osteocytes is slightly
lower in the anntllm (Casta net et a!., 1977; Castanet et a!. , 1993). Annuli
Ot slow-growing layers correspond ro slow osteogenesis . So, they are
normally narrower than the adjacent bones (i.e. zones) and they are made
of lamellar bone. They always appear more translucent than the zones,
i.e. white in transmitted light and dark in reflected light.
The "arrested growth lines" are cementing lines which are concordant
with the bony layers (see above). In any species the anmllu5 is lined on
its peripheral side by an AGL, sometimes by twO (or more) AGL AGL are
rest lines (Casranet, 1981; Ricqles et a!., 1991), i.e. they mark a tempo-
rary cessation of local osteogenesis (Castanet et a!., 1992; Castanet et a!.,
1993). They generally are more translucent and more refringent than
other marks. So they appear as the brightest struccutes when bone is
observed with polarised light. They also are most chromophilic (with
Haematoxylin, PAS stains) and frequenrly hypermineralised. In adults,
they may frequently be the only sign of the cold season.
The combination of a fast-growing zone and an annultts represents the
growth of one year. The zone corresponds to a period of fast growth,
i.e. bone deposited during the period of high metabolism. Contrary,
the a nl1t1111S marks the slowing growth of skeleton when the metabolic
aerivity is falling. It is now well known that bone growth marks are
more or less distinct according the growth dynamic of the various
skeletal elements (Castanet et a!., 1992; Castanet et CI/., 1993). So the
se/eered bones will be those which have the highest number of marks
and the clearest ones. Moreover, there will be little or no remodelling
(fig. II.ClOb,e).
80
a) Pectoral
localisation of
level for the sections, Scale
bar = mm, b) Drawing of
ground section showmg
three annuli, the medular
cavity (mcl. has
the pari
annulus, Scale
bar 1 mm.
c·e) Lethrinus nebulosus
(modified from Meunier et 1-
ai" 1979), c) Dorsal spiny
localisation
suitable level
for the sections Scale bar
= 10 mm.
d) DraWing of a ground
section showing one
annulus,
e) of ground
fourteen
annuli, an obvious
of the six last ones after
first spawning. Resorption
has partly destroyed
the first annulus,
(d,e; scale bar = 500 ~m).
= apex, Os osteon.
area secondary
bone,
2.2. Determinism of the structure and spatial organisation of

marks
as
(0
years, even more than

a (en wry inter
The growth rate of bone is
raner et &tl., 1993),

which lead to bone
81
growdl processes and with chose of che ditTereor skeletal ele-

meors. According to bone structure
and che aspect of marks from CO !f!
the same bone and between different bones of the same individuaL
This fact is very important for the choice of suitable bones for
studies.
Simdarly, accord to the evolution of the rate
the structure marks and their sequence will
from birth to dearh. Until sexual when
annuli or AGL are well wide zones of fast-
will become closer and closer during adulthood
10e) (Castanet
et ,11., 1993).
no more growth marks are recorded
guences for age determination if
Finally, individual and
also lead ro differences m
of bone
as in 1ctrapods see Casranet &
for the the variations of the histological characteristics between
marks are linked to various internal factors
(issue hormonal sexual genet-
ical seasonal alremances, food
The of
iorernal determinism that orders the skeletal growth

the external facrors take if are themselves
submitted to 1992, I They
check the width of the various
by the effects of the
Moreover, any cyclical anomaly of an external can in
turn generate more or less fine structural differences in the struc-
ture and, so, creates "false growth
marks". If cenain supernumerary marks are accidental
after a food a dryness, a marked
from normal events of life:
("birch mark") (Lecomte et at., 1 recruit-
menr (surnumerary (Meunier et at., 1
marks"), annual adult (Compean
1980). These supernumerary matks appear as an
alltmlttJ or AGL, the characteristic of which are similar to
that of normal amm!ttJ and AGL. Such supernumerary
as birth marks, are also known in Chondrichthyans (Branstteter &
1987, Seki et aI., Wintner& I
82
2.3. Some examples

The carp fish of the remperare
whose skeleron consists of vascularised cellular espe-
in rhe spiny rays of the dorsal and anal fins (Stephan, t
Meunier & 1981). Several bones show annual marks:
the mandible of the lower (Castaner £If.
et cd., 1 the (English, 1952;
the dotsal and anal rays (Casraner et at.,
1981). The rays are most suitable for
these animals. During rhe wimer, there is no bony on rhe spiny
ray and before rhe rem[fl of merabolic acrivity an anrmlm is deposited.
This consists of one or twO bony iamell£te and at least one line.
then and continues sum-
mer and early aUrLlmn wirh the of a sllccession of
bony imnell£te (Mellnier & 1981). The ,;mnulllJ is weakl y
mineralised 1981) and the Ca/P ratio of the mineral is the
data).
often show
scales in these where the otoli rhs do nor

case is thar of the Atipa a South
American catfish (Callichthyidae) which lives in coasral swamps. This
fish builds a nest with grass and bubbles for At rhe same
season a new area of

and so on. By rhe number of
hypervascularised clusters the age of the male can thus be esrimared.
In the female the only marks are
mineralised cwrlt/li
Anorher is fish with acellular such as rhe EJOX [tt(iuJ.
This which occupies a similar biorope [() the carp, shows fine
rhe deitbm. Casselman described on
mmttli whose CalP ratio is tbe same as
acrive periods. However, these
of growrh marks is easy since it is
suffIcient to observe tbe deitbra in loto in
LetbrilUt.r nebulOJUJ is a fish that lives in
donia. The skeletOn of this fish, a consists of acellular bone
and it shows fine marks: zones and lines
on the dorsal spiny rays (first dorsal fin) and on the verrebrae (Meuniet
et af., 1979). The bony tissue of the larrer shows imporranr and irreg-
ular resorbed areas so they are nor at all suitable for skelerochronology,
unlike the rays. This fish is very long-lived (at least 25-27 yeats) and
one or two testing lines may have disappeared , but comparative study
with the oroliths has confirmed the utility of the spiny rays in esti-
mating the age of these fish (Meunier et Cif., 1979).
Similar alternations of fast-growing zones and annuli are very marked
in other acellular boned fishes, such as the ilficia of monkfish, Lophills
spp., (Peronnet et af., 1992; Yoneda et al., 1997) or the dorsal spine of
the gtey trigger fish, Bahstes spp., Oohnson & Saloman, 1984), Ot cel-
lular bone fish: pectoral spiny rays of the sturgeons, Aci/Jenser spp.,
(Magnin, 1962; Brennan & Cailliet, 1989). In the larrer species the
anrJuli are also hypermineralised (Meunier, unpublished data). The
Thunnidae, which are Perciformes, are remarkable because they have
cellular bone (fig. JI.C.4b) (Stephan, 1900; Amprino & Godina,
1956). For example, the skipjack (KatsuwOrJltS pelamis) displays cyclical
growth marks on its spiny rays in the first dorsal fin (Barrs, 1972) and
on the verrebrae. On the rays, the annuli are relatively wide and they
appear ro be hypermineralised although they do not show resting
cemenring lines. However remodelling, which is related ro the high
metabolic rate of tbe fish, may destroy some growth marks in some
cases, resulting in a underestimates of age (Cayre & Diouf, 1980).
3. Regulation of the incremental deposition
Despite the extraordinary diverse biology of fishes, bony tissues pro-

vide an accurate recording of mean biological events for animals
including the annual cycles. At the presenr time no subannual growth
cycles (lunar and/or daily cycles) have been recognized in bony growth
marks in fish (but see "supernumerary growth marks") conrrary ro the
oroliths. Annuli have a very similar structures whatever the taxa which
are very differenr in terms of both phylogeny and bioropes (e.g. carp
and tuna).
3.1. Growth mark formation and seasonality

In fact, we may ask if yearly variations of climate direcdy affeCt the
hisrological structure of bone so as ro deposit the growth marks or if
their exist relays and more precisely biological relays , between these
external facrors (e.g. temperature, rain) and rhe regisrering property of
bone.
Fishes are poikilothermic species. When they live in temperate climares
they ate exposed ro seasonaJity of an external facror which induces a
yearly cyclical biological rhythms, especially for body growth, i.e.
84
of bone marks, Orher
same role as winter and in temperate

of these alternated and wet sea-
sons well marked in fish bones (Quick & Bruron, 1984; lecomte et
al) ,1 1989).
For rhe Neocaledonian Lethrimts ftCl.//tl{)Jt(J
stable rhe year; temperature is
in the course of
20.'5°C to 26.'5°C (Meu-
of temperature seems enough to
of the fish and to induce fine growth
zones and lines on the dorsal rays
Seasonal bur non-annual increments have been found in fish from

I t is
for several consec-
utive years in French Guiana have shown that some fish
(Ot/ma, A. proops (two Si
and rhomboidafiJ
rays for the
"zones" and twO apparenc "ammlt" per year (Lecomte et al.) 1985,
Boujard eta!., 1991; Meunieret
also known rropical Africa for freshwater fishes

111
Okedi, Bruron & Allanson, 1 Blake & Blake,

1984) as for fishes (Poinsard
& Another case of ctnnllft/J formed
Izifotl(t1J in Lake Awassa
which have two seasons
about six monrhs (Yosef &
85
3.2. Calcium metabolism

The hypermineralisarion of annuli is common found in carp. cat-
skipjack (Una) bur with like Letbrim/x neb/lloJ/ls for
which lacks
1979). When preSent, this
vana(Jon In calcium concentrations. in the case of
adult carp, for there is an annual Hl bur
only during (Meunier & Pascal, 198 i.e. at the time where
rhe of the skeleton is active. The difference of mineralisation
"zones" and slow "ann/lti" must rather
differences of rhe matrix.
Moreover, these differences in the of bone mineralisation last the
life of animals; there is not standard mineralisation in fish bone.
under the control in temperate couo-

tries. food is scarce or winter season.
Accordingly, food intake and consequently growth
countries feeding can also be an
between and the rainy season rivers
overflood and fishes invade rhe flooded forest where food is abundant
and rich. For in French Guiana, rhe coumarou,
boidalis, which show obvious growth marks (Lecomte et
feeds the wet season on seeds which have high nutritional value
and the dry season on leaves of podostemonacae (coumarou
with lower nutritional value al., 1 Another
Guyanese which is an omnivorous essen-
tially feeds on seeds, fruits and terrestrial insects thac find under
the overflood forest the wet season and seems in the
fiver season (Boujard et a/., 1990). Such observa-
tions have also been noticed at the whole Amazonian scale (Lowe-
McConnell, 1 1979; I and
3.4. Reproduction
another vital function that is tightly related to seasonal-
on be in synergy with low metabolic time
winter) or shifted forward to the metabolic season. In
rhe second case, can lead to the formation of a "false
mark" inside rhe "zone".
sexual an imponant component of the metabolic
that for growth
the successive cmntdi
narrower (Meunier et al., 1979).
86
Wh en the fish become older (specially in long life fishes) the "zones"
can become so tight than the counting of annuli can be very difficult
and unreliable : see for example the sturgeon.
We have seen before (see chap. II.C. 2. 2) that rep rod uction can be
responsible of the spawning marks on scales especially in Salmonid s
(Johnston, 1905, 1907, 1908, 1910; Dahl, 1907, 1911, inter alia)
Thi s is ce rtainly linked with calcium m etabolism because of the high
need of this cation during spawning (see Persson, 1997, inter alia).
Calcium is panly supplied by scales that is why their integrity is
affected (Crichton, 1935; Van Someren, 1937; Persson, 1997; Kace m
et at., 2000).
4. Conclusion
It appears that bony tissues can accurately adapt their stru cture
according to the variations of the environment throu g h physiological
relays. Many events durin g the life of a fish that will be finely regi s-
tered in the bony structure including more or less seaso nal cycles,
changes in biotopes and/or life mode beca use of mi g ration s and first
sexual maturation (tab. II.C. 3) . Consequently, th e hi stological study
of g rowth marks in primary bones can be a suitable method for indi-
vidual age estimation in fi sh especially when o tolithometry and
scalime try fail age es timation. However, the use of ske letochronology
requires as precise a knowledge as possi ble of the biology of the ani-
mals, since skeletal growth is sensitive to many internal and external
facrors. As for sca lim erry and otolithometry, validarion (vital
labelling, monthly sampling, see chapter IV) is a necessary step to test
the annual g rowth rhythm.
Tableau II.C.3 - The internal and external factors and the spatio-temporal organisation of the skeletal
growth marks (SGM) (from Castanet et aI., 1993)
Internal factors External factors

Internal growth rhythms Seasonal rhythms
(hibernation, estlvation)
Formation
and
chronology Occasional growth
variation
'"
physiological relay
(birth, metamorphosis, ~
~
Momentary variation
/
physiological relay
/ Occasional changes of:

- environment;
. individual (pathology,
breeding cycle) of osteogenesis
rate competition)
General growth
/"00+. '
Modifications of the Individual s " Variations of amplitude
of structures and of various skeletal and climatic factors
elements
and spatial
organisation Bone morphology \ biannual
Supplementary (non peflodical)
87
When skelerochronology is appropriarely applied and if rhe skeleral

srrucrure is suirable, ir can provide many pracrical applicarions ro rhe
srudy of popularion dynamics. They can also have orher applicarions in
paleobiology and archeology. Paleophysiological byporhesis (Casreels,
1974 on verrebrae) and /or paleoclimaric hyporhesis (Burdak, 1979
wirh scales) can be proposed in any Osreichrhyans owing co rhe srudy
of cyclical growrh marks on appropriare bones (or scales or even
orolirhs) and wirh comparisons wirh exranr species (Van Neer, 1993a).
The skelerochronological analysis of bones of rhe archeological sires, if
rhere are abundanr marerial, always compared wirh rhe living can allow
valuable dara on rhe food of prehisroric peoples and on rheir fishing
rechnigues: size of fishes, seasonaliry of fishing, fishing appararus
(Desse & Desse, 1983; Van Neer, 1993b; Van Neer et cd., 1999).
88
Sclerochronologlcal studies
Sclerochronological studies
89
90
SclerochronoJoglcai studies
sclerochronology, and
time-consuming. Before sllch studies it is necessary [0

know in advance the consrrainrs on avaiJable time and cOStS, and espe-
the final and resulEs. This will
ro choose from among the multitude of relevant techniques and
research methods:
between studies devoted (0 age estimation and those
of life features of individuals (age
at
[0
marks in three-dimensional space

The complex shape and
culties in initial
A. estimation
~--""'---.~««
B. Morales-Nin, Panfili
estimation involves several stages, from rhe choice of the CS to be

and the level of required, ro more technical problems
such as and the observation of marks. The incre-
menral panern also has to be selected and its
ing determined. the reader needs to in
in order to obtain a level of
criteria and [0 rransmit them to, or calibrate them
other expens. Once this process has been it should
be refreshed at imervals in order (0 be consistenr over time.
estimation is a dynamic process which can be summarised in
seven separate steps:
selection of one or several CS,
the of and tbe in the
patterns, (he periodicity of [he

and the range of the time scale covered
choice of the method of depending on the technical

of readability of the marks using the
of
by one and/or several (chap.
91
validation of the accuracy of the

the
the growth
of the results obtained V and VII).
1. Criteria for the choice
on the CS it may be necessary (Q kill che fish to

exuacr them incernal bones and The choice of CS will
elms initially on whether or nor the fish can be killed (fig.
llLA.l). Moreover, at the does nor permit the fish
for
taken when oroliths (chap. VIIl.B.]'.l
of the CS will determine the choice of tissue. For example,
m or with small scales that
are easily lost or 10 that lack owliehs Elas-
mobranchs), other structures will have co be used. ln
fishes sllch as tunas, swordfish and Jsciophorids, which have minuee
ocoliehs to rhe size of the the vertebrae are frequently
rules with many Other bones, such
as fin rays, deithrel and
However, they can be useful for
of their ocoli(hs is still very limited.
are also employed in some
such as Balisridae.
age esrimation and the life
to be studied are importam in the choice of one
structure over anocher Hl.A.I). As daily incremems are
found in they are rhe obvious srrucrure co be used when age-
ing is to be done ar rhis level. For larvae and juvenile specimens
are (he choice. Owlirhs have another characreristic
thar makes them very appropriate, in rhat they do nO( act as calcium
stores, unlike scales and other bony parrs (Simkiss, are
therefore not resorbed except under extreme stress (chap. and
rhus maintain a more seguence seructures.
Of the three types of ocoliehs, the is the mosr
prrll",mIPn except in in which the aJterisctls is
some studies oflarvae and fish, or even adults, the has
been due to its smaller size and simpler daily
increments can be observed on whole or slightly polished
However, the onset of formation is not simultaneous in the
(hree owJirhs, with and first. They can rhus nO(
be employed
92
SclerochronologlcaJ studies
III.A.l <1.1
W)
of a calcified
structure
....«I Adults
on the accuracy time '"
required, the life stage
studied and the
of inolvidual
for sclerochronological
study. Sacrifice yes yes yes no
structure
Time scale
precision
2. Selection of growth increments
The selection for increments in cs are:

- their disrinC(iveness and ease of
- rhe constancy of their
The in the and the life stages co be

addieional eypes of informarion for che
cion of an
Larvae fish lack seasonal and annual
choice. This choice
one (less than one or two
when [heir time-scale is
suitable for [he found
in all CS from bones to otOliths. Most estima(Jon studies are based
on seasonal increments,
fish from. the
but
even on a very
inherent J[1 Jl1cre-

ments make the merhocl less suirable. \'Ve cannot recommend use
increments to estimate in years, unless for reasons.
to obtain [he thin ocolirh sections that are neces-
sary for mcremem in adults also limits the of
this method for thick ocoliths.
93
Methods of cs arc generally more

marks rather than daily ones are being as
such do from the whole CS to preparing trans-
verse embedded sections in resin thin slices The time
for a single is very variable. For very small
increments can be observed
tion bur it is often necessary (0
methods ro observe these incremenrs and polish-
thin SEM, etc.). The user must therefore be careful (0
evaluate these parameters (level of preparation, duration of prepara-
tion, ete.) before srudy. He must the
previous used by
found in the literature.
The identification of seasonal or even daily ones, is never
rr.,·rl-"Nl,nri [Qnon-seasonal events

tion is based on their their continuity around the CS,
their thickness and their relative width (chap, IILe). These should
decrease in width from the central parts ro the of rhe cs, in line
with rhe fall in rates of growth with age (chap, V),
Another relevant criterion for an appropriate Struc-
ture is the number of fish (() be This also involves the COStS of
collection and CS including the time required to
them. The use of daily incre-
ments in (see above and
r",,,.,,-",,"p increment sequence from the core to the is
the time required for a skilled person (()
and polish an otolith to obtain a thin slice is one to twO
numbers of thin slices may therefore limit the
usefulness of the method. Another fact to be considered is that in some
and at certain periods in their life (e,g, migration), the
increment widths may be below the detection limits of the com-
pound Morales-Nin, 1988),
the CS and the incremental
pattern to be selected should be carefully considered, Table
ULA. L summarises all these considerations. The presence
centres or other structures that
pattern should also be taken into account in the evaluation of the
suitability of a strucrure for purposes.
94
Table IIIAl - Some considerations regarding the choice of calcified structure and the growth marks
for a sclerochronological study.
Otolith Scale Bone
Preparation +/- long none t/- long
Growth marks - primary (daily) - rirwli - opaque (seasonal)
(time scale) - opaque (seasonal) - discontinuity (seasonal) - translucent (seasonal)
- translucent (semonctl) - discontinuiry (seasonal)
- discontinuity
(irre 'ular or Jett.rnnal)
Advantage - no resorption - no sacrifice - somerimes no sacri fice
- sometimes no preparation - no p repararion
Disadvantage - sacri fi ce - regeneration - resorprion and remod elling
- somerimes long preparation - resorp rion (sometimes strong)
and interpretarion rimes and remodelling - somet imes long prepararion
time req uired
3. The process of age estimation
The first step in the age estimation process is to read the selected cal-
cified structure using an incremental growth pattern approptiate for
the objectives of the study; the next is ro interpret the age and provide
some appl ications (chap. V).
Some inevitable interpretation ctiteria for the populations and /o r the
species must first be defined; for example , the location of the hatching
check, the first increment, the transition zones on the cs, the narure
of the edge, ete. The biological information available for the species
should be used ro define these critetia empirically. They can then be
compared in order ro establish an "a lphaber" (identification of incre-
mental growth patterns) and ro determine the "g rammati cal rules "
involved (the interpretation criteria based on existing knowledge) ro
attribute ages (Sycb , 1974).
The consistency of the age estimation process tben needs ro be deter-
mined. Tbis means the ability to identify the same structures consis-
tently. The repeatability of the age estimation procedure (internal
bias) must be derermined and the ages calibrated (external bias) witb
other experts (chap. IVD). The experience of the reader is very rele-
vant to the success of limiting the bias . Even experienced readers,
however, might show low level s of accuracy when starting work on a
new species. Once a satisfacrory level of expertise in age estimation has
been reached , steps should be taken ro prevent the methodology from
deteriorating or changing over time, in order to maintain adequate
quality of the ageing procedures.
Finally, the ages must be validated , in rh e sense that the accuracy of
the ageing has to be established. The temporal meaning of the inter-
preted strucrures must be determined in order to evaluate the close-
ness of the estimated age to the accu rate age of che fish (chap. IV).
95
Manual 01 fish sclerochrono!ogy
3.1. Specific age estimation

estimation on the basis of lOcremenrs direcc and
value Ir is calculated from the number of validated
increments (number ofD-zones or i-zones) the interval between
hatching and formation of the first increment (chap. II.A.2.!).
In contrast, ages on the basis of seasonal incre-
ments some additional calculation: it can be calculated as the
number of months or years. Once all the increments on a CS have been
the information should also be taken into con-
sideration in
• date of capture;
• individual date of birth or average or standard for the popu-
• main of seasonal increment

the CS
The true date of bitth of an
and/or known.
In such cases, for convenience and ease in age data with other
statistics collected on a calendar year the convention is [() accept
1st as rhe dare of birth for the entire the Valida-
tion studies (chap. IV) allow the of seasonal incremem
formation [() be determined. III.A.2 shows of che
method age can be calculated in terms of number of months
and years for fish at different times of the year. The conversion
of an incremem count to an age esrimate involves the rela-
between the date of increment formation and the date
tore and nominal birch date.
3.2. Age group and assignation to age class

The indIvidual age group and age class are used in
and life in both of which a
years IS
reader can establish [he age of the fish
ber of seasonal incremenrs on an annual basis. As in the
age other information should also be taken into accoum:
.. dare of capwre;
.. the or average
of seasonal mcremem formarion (see validation
.. na tu te of the CS
In order [Q able [Q establish from the cs the age to
which a fish it is necessary [Q coum the number of annual
increments from the centre cowards the outer of the CS. If the
dare of capture is It 15 W calculate the year in which
Translucent Translucent
Month 0 N o n::'M A M J J 'X A S o N o *"J F M

Month rank 10 11 3 4 5 6 7 I8 9 10 11 12 \ 1 2 3
Translucent edge~
'U
Birth
Amonths = 12 x N + ((rank Cm] - [rank Bm])
Translucent edge date

Ayears =N Age group = N+ Age class = N+ 1
Amonths = 12 x N + (12 - [rank Bm] + [rank Cm])
~ A; 12x2+12-4; 32 months
Ayears =N Age group = N+ Age class = N+ 1 ~ A = 2 years Age group; 2+ Age class = III
~ A; 12x2+12--4+2; 34 months
Opaque edge
~ A; 2 years Age group; 2+ Age class; III
Amonths = 12 x N + ([rank Cm] - [rank Bm])
Ayears =N Age group = N+ Age class = N+ 1
Opaque edge
«e> A = 12x3+7--4=39 months
A = 3 year s Age group = 3+ Age class = IV
Amonth s = 12 x N - [rank Cm]
Legend
Ayears = N-1 Age group = N-1 + Age class =N A = age
N ; number of translucent increments
~ A; 12x3-2 ; 34 months
~ A; 2 years Age group = 2+ Age class; III
*"= date of capture
rank Bm = rank n° month of birth
(e.g. Bm April = 4)
en
n
ii)
rank Cm = rank n° month of capture (3
n
(e .g. Cm July = 7) ~
(3
~
o
Figure III.A.2 - Theoretical calculation of age (months, years) and assignation of age group and age class from a calcified structure. The birth date for all individuals of the population corresponds to 0'
April 1st, which is also the beginning of the formation of the opaque increment on the CS. Validation has shown that each yea r both a translucent and an opaque increment are laid down. The age ".
OQ
~
is calculated by counting the number of translucent increments, but taking into consideration the date of birth and the date of capture. As a rule, a translucent marginal increment is not counted as Vo
C
an annual increment. Some examples of individuals captured at different times of the year are shown. On any given date of capture (e.g. February), the nature of the edge of the CS may differ (opaque a.
re
""
-.J or translu cent), but the age is the same. Vo
Manual ot fish sclerochronology
rhe fish was spawned. Arabic numerals are rraditionally used co reflect
an age group and Roman numerals the age class. In the example given
in figure II1.A.2, the translucent increments are counted for ag eing.
As a rule, if the marginal increment is translucent it is not counted
because it is not fully formed. The age group or age class co which a
fish will be assigned depends on the year in which it was spawned and
on the dare of capture. The age group is the number of calendar years
afrer the birth date. The age class corresponds co the number of years
since birth. The mosr rece ntly spawned cohort (rhe "young-of-the-
year"), consisting of individuals between 0 and 12 months of age, con-
sritutes age g roup 0+ and age class 1. Individuals spawned during the
previous year make up age grou p 1+ and age class 11. The year class is
rhe calendar year in which a fish was born. In figure II1.A.2, for the
fish captured in July 2000, the year class is 1997 and it was born in
April 1997.
For the purposes of srock assessment the true birthday is generally
assumed ro be unknown as spawning time may vary slightly from year
ro year or, in the case of some species e.g. Merlangitts merlangus, the
spawning season may extend over four or five months. Thus, in order
ro ease the task of collating data from a variety of sources most fish
species in the northern hemisphere are given a nominal birth date of
1st J anuary. Normally, an annual increment is only counted when the
translucent zone is complete, usually about March or April. However,
because of the 1st January rule rhe marginal increment, even if it is an
incomplete translucent zone, is counted as the annual increment in the
period January ro March. On 1st January of each year, the fish in each
cohort thus become one age group and one age class older.
98
B.
1.
a severe energy demand on fish, and after the

onset of maturity there may be a reduction in somatic In
favour of storage for This affects the of

calcified strucmres and the influence of is often
recorded in scales and otoli(hs. So-called spawning
checks (Williams & Bedford, have been In many
srudies have established the
and the features that have
been observed.
In the process of utilises borh energy and C(11-
cium reserves that could otherwise be used for the of calcified
tissues. in parricular involves the production of vitel-
Kalish observed
and
endolymph associated with increases in the gonadosomatic index.
in female there is a decline in the amount of Ca available
for otolith Narrow translucent zones in the moliths of female
fish have been assumed to be the result of reduced
calcium availability gonad maturation (chap. VII.E.2.2).
since translucent formation is mineral-rich and protein-
poor and since in a number of is also reduced
the final stages of maturation and rhis may
influence the rate of synthesis available for otoliths as well as
for other tissues and result in the observed translucent structures, This
is clearly in line with early cod
1933) of a between age of matura-
tion and the onsec of narrow annual otolith zones with relatively
wider translucent zone.
Because the pattern of calcified tissue may change
afcer maturation, age at first can often be determined
from the
marure lace may display numerous regular and age at first
can be easily determllled by means of conventional methods.
In other those that mature at less than one year of
age, it is necessary to use otolichs, and to decermine che age at first
from primary increment counts. Diadromous may
present a further because the effects of and
sexual maturation are nO( always with spawning
99
migrarions presenr less of a problem, and rhe rransirion berween fresh,

esruarine, or ocean warers may accenruare spawning checks (or vice
versa). However, feeding migrarions may induce checks or changes in
rhe growrh parrern of calcified rissues rhar may obscure rhe parrern
induced by reproducrive acriviry.
2. Lifespan (longevity)
The abiliry ro esrimare rhe age of fish from cs may direcrly supporr
rhe dererminarion of lifespan or longeviry in individual fish popula-
rions and species. Shorr-lived species, especially rhose rhar live for a
year or less, can only be aged on rhe basis of counrs of orolirh primary
incremenrs (chap. IILA). The accurare dererminarion of lifespan may
be complicared by rhe effecrs of reproducrive acriviry in such species
(see above, chap. IlLB.I).
There is a choice of merhod for orher species, since annual growrh
zones can be observed in many d ifferenr rissues . As far as esrimares of
age are concerned, esrimares of longeviry can ofren be verified by com-
paring differenr cs (chap. IVD). However, rhe apparenr advanrage of
being able ro compare age informarion using differenr merhods and
srrucrures can lead ro conrroversy. In Sebastes spp., rhere are unresolved
conrradicrions in rhe maximum ages recorded in various CS. Similarly,
rhe analysis of macro- and microincremenrs in Hoplostethm at/anticus
orolirhs produces differenr ages. Longeviry has been esrimared ro be
up ro 70 years on rhe basis of annuli, bur as low as 40 years when
microincremenr counrs are used. When differenr srrucrures such as
orolirhs, scales or bones are being compared, ir should also be nored
rhar differenr cs have differenr aIlomerric relarionships ro fish growrh
(Casselman, 1990), which may influence rhe abiliry of rhe CS ro reflecr
annual variarions ar grear age. Ir has rhus been shown rhar in very old
fish, scales may underesrimare age relarive ro rhar esrimared by
orolirhs .
Esrimares of longeviry can somerimes be validared by radiomerric age-
ing rechnigues (chap. VII.E.3). These chemical chronomerers do nor
provide precise esrimares of age because of currenr insrrumenrarion
limirarions, and so far rhey have only been applied successfully ro
long-lived species.
3. Metamorphosis and settlement
In mosr fish species meramorphosis is a rransirion rhar marks rhe end

of che larval srage. Ar rhe complerion of meramorphosis, fish are
regarded (and ofren defined) as resembling rhe adulr form of rhe
sfecies: The difference berween newly meramorphosed fish and adulrs
·100
is sometimes considered to be only a maner of size and sexual matu-

rity. In reality, metamorphosis is a transition phase, which varies
widely from species to species. The key discriminating features of
metamorphosis, in the context of sclerochronological studies, are the
termination of cutaneous respiration and the completion of ossifica-
tion . Thus, by the end of metamorphosis, fish have complete skeletal
structures and scales, while the exchange of ions with the environment
is limited to transport across the gills and through the digestive tract.
Because complete ossification defines the endpoint of metamorphosis,
only the otoliths can be used to study events and processes that occur
before or during this stage. Metamorphosis is associated with physio-
logical, morphological, behavioural, and often habitat changes. In
some species these changes are dramatic and occur over a short period
of time, in others the changes are gradual. Any of these changes can
result in changes in otolith formation, leading to differences in otolith
shape or in the pattern of increments. Eels in particular undergo dra-
matic changes in both the appearance and composition of otoiiths,
that can be linked directly to physiological changes during metamor-
phosis(Otakeetctf., 1994, 1997; Antunes&Tesch, 1997).
The most frequently observed change in otolith shape is the result of
accessory growth centres, which usually form at the end of metamor-
phosis. These have been described in several Gadiformes and flatfish
(fig. IH.B.1). A close link between the progress of metamorphosis and
the appearance of accessory growth centres has been described in
plaice (Pleuronectes platessa) (Modin et af., 1996). In the lesser sandeel
(Ammodytes marinas Rain) the completion of metamorphosis is associ-
ated with the formation of the post-rostral secondary growth centre
(see glossary; Wright 1993). In the Pacific Dover sole (Toole et af.,
1993) Mic-rostomus pacificus the accessory growth centres seem to form
throughout a lengthy period of metamorphosis. Changes in the opti-
cal density and spacing of increments may also accompany metamor-
phosis. These changes may be gradual and encompass several incre-
ments, as observed in herring (Clupea harengus), or may be more abrupt
as in some coral reef species (Victor & Brothers, 1982, see also "settle-
ment", below).
These results permit detailed studies of the duration of the larval
phase, useful in investigations of drift, metapopulation dynamics, and
spawning behaviour. In some cases, the formation of daily increments
has been validated before, during and after metamorphosis, thus
enabling detailed studies of early life history transitions to be made.
In other cases, where validation has not included the period of meta-
morphosis, it is often possible at least to be able to determine the age
at the start of metamorphosis, and the date of its completion (fig.
IU .B.l).
tfRE";;eR
eft)lim~
C3ntre c;e B."S8t 1
BP 7'0 - 2~80 PtO:2JWE I
I- - - - - -- -- - ,.- "-- - - - --'
Figure III.B.l
Otoliths 01 juvenile
Pleuronectes platessa,
indicating the initial high
contrast increment (M) and
accessory growth centres
(AC) formed at the end
of metamorphosis.
The minimum time since
settlement is estimated from
the lirst increment
completely surrounding the
accessory growth centres.
Scale bar = 10 ~m
(photos A.J. Geffen).
a) 30 mm fish,
approximately lour weeks
after settlement.
b) 25 mm fish,
approximately two weeks
after settlement.
102
The physiological changes associated with metamorphosis may also be

studied by means of otolith microchemistry. "Ontogenetic" variations
in element composition can be observed in data collected along tran-
seers from the otolith core to the edge. Changes in the Sr/Ca ratio in
particular have been described (chap. VIl.E.2). In future, it may be
possible to compare the rate of change in elemental ratios to growth
after metamorphosis , as a means of measuring the physiological con-
dition of individuals in the past.
Most marine and many freshwater fish species produce planktonic lar-
vae. When the adult habit is demersal, there is usually a transition
ftom the planktonic larval to a demersal juvenile stage, and this tran-
sition is termed "settlement". Both the timing of settlement and the
duration of the preceding larval stage are of interest in recruitment,
life history and ecological studies. Settlement marks can often be iden-
tified on otoliths, and are the result of either physiological changes at
metamorphosis, changes in behaviour and diet caused by the moving
out of the plankton, or both.
There are numerous examples of these applications in the literature,
the most common probably being the use of metamorphosis or settle-
ment checks to define the end of the larval period in order to estimate
larval duration and drift. Studies of coral reef species often rely on
otolith checks to fix dates of settlement, identify settlement cohorts,
and determine larval drift (Heath, 1992; Kingsford & Atkinson,
1994; Schultz & Cowen, 1994). In temperate species too, otolith fea-
tures associated with metamorphosis and settlement are used to study
sub-cohort dynamics (Al-Hossaini et a!., 1989), mortality patterns
(Van der Veer et a!., 2000), larval stage duration (Suthers & Sundby,
1993) and drift (Desaunay et al., 1996).
4, Migration
Many fish move between different warer masses in the course of their
seasonal migrations. Many species also exhibit ontogenetic shifts in
habitat that consist of a long-term or permanent move into new
waters. Questions of species and population biology often require
information about the timing of these migrations. These questions
can be addressed through examination of changes in the appearance of
increments in otoliths and scales.
Changes in the pattern of scale circuli are used to identify anadromous
migrations in Salmonids, and these also provide information about
relative rares of growth in freshwater and marine environments. Simi-
larly, the pattern of otolith increments is often used to identify the
timing of catadromous migrations in eels. Similar analyses can also be
used to discriminate between anadromous and resident individuals in
waters in which tWO populations with different behaviour mix.
103
The composition of scales and owliths also changes when fish move
into new and this can be exploited to obtain derailed
data aboU( fish age at and che duration of che transition
between different environments, Oxygen racios and elemental
show in response to and are chus
diadromous migrations
VILE, L 1),
104
Sclerochronologlcal studies
C. Influence of shape and structure
B. Morales-Nin
cs growth patterns, except for those of involve three-dimen-

sional bodies whose dynamics of and size are determined a
combination of endogenolls factors and
exogenous factors (environment, the
size) II). Their function and
internal growth srfUccures with different
the observation of whole or thick
binocular or compound microscope will reduce the amount of 3D
in increment identification to a 2D view.
the problem is even more
of many
increments will have different appearances (Williams &

A failure to section close to the core will result in
errors in age and in in the aspect of the
growth increments between samples (fig. IIl.e I). The more
the preparation the more difficult are observations of the
pattern of increments. In otoiiths, for
and SEM can
1992). At this level, errors can occur

made.
Pr
of a theoretical
otolith (Onion model) 'fll\h
zones and the effect
different cpr""'"na
(a), (bl, (c),
of the same otolith.
The double arrow indicates
the of section. (a'l,
(b'l, observation
of the and location , , I I J I I 1 ! I I I I If! I I 11 I
! I I I
of the growth zones. I I i I
I I
I I
! I
11
I
I
The images offered
the three sectioning
are quite different
A, anterior; D, distal; A p
Do, dorsal; P, posterior;
Pr, V, ventral.
(from Panhli
1992, v
publisher
105
The mosr obvious consequences of shape and srrucrure for observa-

rions of rhe growrh parrern are found in rhe orolirhs, because of rhe
exrraordinary variery of shapes rhar can be found in differenr species.
Ir is worrh remembering here rhar shape can be used for species iden-
rificarion (Torres et aI., 2000) and in paleonrology (Nolf, 1995) (chap.
II). The problem would be somewhar reduced in les s complexly
shaped orolirhs, as are found in hake, Perciformes or orher highly
evolved species (fig. III.C.2), bur can be very norable in many species
Figure III.C.2 whose orolirhs are bulky or irregular in shape. Orolirhs rend ro
Example of the effect
of the 3D shape on a 2D become more complicared and ornare wirh age, wirh curvilinear
observation for a typical growrh produced by preferenrial growrh on rhe inrernal side. Ir can
Teleost otolith (Vinciguerria
nimbaria). The growth pat-
rherefore be very difficulr ro obrain a single plane of secrioning rhough
terns are presented on thin rhe core of rhe orolirh ro irs edge.
sections for a transverse
plane (b) and a sagittal
plane (e). (c) and (d) show
details on the transverse
section and (I) and
(g) details on the sagittal
section. A, anterior;
D, distal; Do, dorsal;
P, posterior; Pr, proximal;
V, ventral.
Scale bar = 100 ~m
(photos J. Panlili).
'-.. Pr
106
Because the otolith grows at different rates depending on its axis of

orientation, the increments in would display differences in
on the otolith area. This is for
with sub-daily increments and split on the rime-
scale studied IILC3). In the fastest area,
the axis, the sub-daily incremenrs are wider and
appear more clearly than in the dorso-ventral axis. The same is true for
that might obscure the seasonal increments. Three-dimen-
sional strucrures such as rhe accessory centrum laid down around the
mtdem in flatfish and Gadoids would appear on
III.C.3
microincrements
(arrows) on the transverse
section of the otolith
of Vrnl:Ii2IJerrI3
axis.
Not all microincrements are
visible on the proximal face.
0, distal; Do, dorsal;
p, proximal
Scale bar
(photo J
III.C.4 - Differences in the observation due to changes in the section in an otolith of Mer/uecius mer/uccius. Central area
core and accessory primordia (arrows). a) observation before and b) after further polishing. Scale bar = 60 ~m (photos
107
rhe plane of secrioning (fig. IILC4). Furrhermore, rhe increments laid

down afrer rhe accessory centres may nor march rhose in rhe nuclear
area (fig. III.B.l).
The observarion of rhe parrerns of increments in rhe internal srructure
of rhe orolirh is more complex because of rhe different scales of obser-
varion, which range from daily ro annual. Observarion depends on rhe
merhod of prepararion bur also on rhe magnificarion employed. The
correspondence berween daily increments observed under compound
microscopy and under SEM, for example, is known precisely (chap.
II.A), bur rhar berween daily microincrements and seasonal incre-
ments is srill a marrer of controversy.
The problem of reducing a 3D observarion ro 2D is similar, bur less
pronounced, when preparing skeleral bones such as rhe spines (fig.
III.CS). The basal (ventral) parr of rhe spine rerains rhe mosr growrh
increments bur is ofren affected by bone remodelling whereas rhe
ourer (apical) edge may miss several or all increments. The spinal sec-
rion should be stricrly transverse ro rhe longirudinal axis in order ro
avoid disrortions in rhe incremental parrerns. Vertebrae are generally
observed whole, bur frontal secrions may be necessary in order ro
reveal increments in rhe vertebral bodies (Loubens & Panfili, 2000).
In rheory, incremental parrerns should be homologous in all axes, bur
due ro different growrh rares rhar cause particular rhree-dimensional
disrortions rhey may become hererogeneous. Srandardisarion is rhus
necessary when secrions are being prepared (chap. VIII.C2.3) and
when growrh incremenrs are being read. The need for a srandard
prepararion is obvious in rhe case of complex orolirhs and spines.
Alrhough rhe continuiry of rhe parrern of incremenrs around rhe CS is
a marrer of interprerarion, a srandard reading axis should be selecred.
In rhe case of countS of daily increments, a single reading axis may nor
be feasible due ro breaks in rhe visibiliry of rhe growrh parrerns.
Counting parhs need nor be linear, and anorher reading axis close ro
rhe main one should be selecred after a single disrincr increment
(Campana, 1992) (fig. IIr.C6). Even afrer secrioning, any remaining
3D informarion on rhe cs preparation may srill obscure growrh pat-
rerns under lighr microscopy, due ro problems of parallax and focal
plane selecrion (Campana, 1992), causing subjecrive errors in rhe
interprerarion. These ambiguiries are reduced following surface
prepararion and observarion rechniques such as SEM or sraining (chap.
VIII.D.2 and VIIr.C2 .8).
Selecrion of a suirable measurement axis requires rhe same orientarion
of rhe axis and c1ariry of increment crireria as rhose associated wirh
increment countS (Campana, 1992). Because 3D growrh is exrracred
from a 2D image, rhe measurements should consisrently follow rhe
linear or curvilinear growrh parrerns along rhe axis selecred. This is
now possible wirh rhe aid of image analysis systems (chap. VI).
108
Figure III.C.5 Various levels of section for the pectoral spine of Pangassius hvc,nnilth;,/ml
different between all the levels of section: the information is lost towards outer apical parL Standardisation of the sec-
tion is thus essential in any sclerochronological A, anterior; D, dorsal; P, posterior; V, ventral. Scale bar 200 um
for the sections and scale bar = 15 mm for the whole (photos J. Panfill).
109
Figure III.C.6
Changes in reading axis
for daily increments
on transverse thin sedon
in otoliths of Oreochromis
niloticus . The reading is
from the core to the edge
along the sulcus acusticus
on the proximal side (Pl.
The arrows indicate
the reading paths, which
changes when
microincrements
are unreadable or unclear.
The reading cannot be made
directly from the core
to Ihe edge on a linear path
because growth is reduced
in the proximal side during
the first days of life.
Scale bar = 20 ~m
(photo A. Malam Massoul.
110
Validation and verification methods
Cha r IV
Validation and verification

methods
111
Manual of fish sc!erochronology
112
It is impossible to know the absolute age (true

random in its natural environment. Many are based on
accurate estimates of the age structure of the catches. The
that errors are has stimulated interest in the valida-
tion estimation methods. Validation means that a tech-
nique is accurate, and accuracy can be demonstrated or esrimared.
Estimates of accuracy are less valuable, bm in some cases only an esti-
mate is possible et cd., 1983). Beamish & McFarlane (
om the of ages and, in their review of
the literature, that few studies validated their own
Since many validation studies have been
Theoretically, a validation should be made of every popu-
lation of any since there may be differences
between them: for this is the case for
as salmon and eels. Ideally, validation would entail
viduals from
fen, Although an age estimation
entire age range of the population is very a combination of
methods offer greater certainty in the accuracy estimates.
Validation should be an obligatory step in all sclerochronological
especially in in which no age estimation studies have
been Two aspects must be determined: that the incre-
ments are laid down with a periodicity that can be related to a regu-
lar time scale (i.e. and (2) that the Structure has a con-
sistent interpretable panern of increments (i.e. The
available for the frequency of formation of
incremems can be into the following four main
which constimte the basis of this chapter:
.. direct validation, which takes into account a rder-
ence mark on a cs relative to the other
vidual-based that often uses
.. semi-direct validation, which
number of individuals; this is a
• indirect validation, which of the individual

age estimates with statistical age estimated from length
rributions as well as other age this is also a
.. corroboration, which involves multiple

after one or more of one or more CS).
113
A. Direct validation
P.). Wrighr,). Panfili, A. Folkvord, H. Mosegaard, F.). Meunier
Direcr validarion leads co knowledge of rhe age on rhe basis of a cs of

a single individual. Ir ofren refers ro a precise remporal reference
growrh mark compared ro orher marks. Direcr validarion procedures
include a series of observarions of marked individuals released in rheir
nawral environmem and /o r reared fish under comrolled or semi-con-
rrolled condirions (Brorhers et al., 1976; Neilson & Geen, 1981; Gef-
fen, 1992). The inrroducrion of darable marks on rhe CS of fish before
rearing and recaprure is a common preliminary srage. These marks
may be chemical ly or imrinsically induced.
1. Marking
In rh is secrion, we consider rhe whole process of "marking" from rhe

caprure of individuals in rheir narural environmem, rhrough marking
(fish and /or CS), release in rhe narural (or anificial) environmenr, CO
recaprure ar a given rime. As rhe imerval berween release and recap-
wre is known, rhe durarion of rhe growrh periods can be calcu lared.
As CS marking usually cannor be derecred exrernally ir is necessary co
pur an exrernal mark or rag on rhe fish.
l.l. Marking fish

The marking and ragging of individuals is an exrremely widespread
process in popularion dynamic swdies . In rhis secrion, we summarise
rhe main approaches co fish marking. Funher derails of marking and
ragging can be found in Parker et al. (1990). Clearly, individual mark-
ing and rhe rype of mark used depend on rhe size of rhe fish, being
unsuirable for larvae. Marking is essenrial for age validario n swclies in
which ir is necessary:
- co locare individuals or groups of fish which have marked calcified
srfllcrures;
- co locare individual fish from whom a calcified srrucrure has been
removed. This is only possible afrer scale exrracrion (Beamish &
McFarlane, 1983; Marlock et al., 1987; Beall & Davaine , 1988) or
somerimes afrer exrracring parr of a fin ray (Rochard &]aneau, 1991).
The incremems in such s([ucrures are rhen compared berween rhe
dares of inirial caprure (marking) and recaprure.
A wide range of exrernal rags is available for individual marking
(McFarlane et al. , 1990). Some examples are shown in figure IV.A.1a .
Fixing rhe rags is made easier by rhe use of suirable g un s (fig.
IV.A.1b). The inrroducrion of small inrernal fish rags , such as rhe
114
coded visible implants th at are implanted und er the skin in the cra-
nial reg ion, and coded (micro)wire tags, may be preferable to external
tags because the former do not affect the hydrodynamic performance
of the fish. Electronic pit-tags (passive integrated transponder)
injected under the skin allow information on the individual to be read
with minimal handling. These tags minimise the biological impacts
of marking, such as causing changes in behaviour, and provide clear
and unbiased data. For further informarion on fish tags , see interna-
tional distributors of external tags such as Hall prim Tags (Australia),
Biomark (USA), FishEagle International (UK), Northwest Marine
Technology lnc. (USA). Newly developed electronic tags are now avail-
able which mark the fish externally and record several types of daca
a
Figure IV.A.!
External marking of fish.
a) Principal types of external
tags and anatomical site s
for attachment on fish (from
McFarlane et al., 1990).
b) External marking with
anchor T-tag for an African
tilapia (Sarotherodon
melanotheron).
The numbered tag IS put
under the skin in the mu sc le
using a special tagging gun
(photo J. Panfili).
( f W> 32 ) SUb-cutaneous
~---~
( ' ....."1 \.. ) Internal anchor
......."·0
115
from the natural environmenr water temperature, pressure) solar

and sometimes aspects of fish physiology inrernal tem-
Derails of these data storage tags can be obtained from Star-
and Lotek
too-ink marking and

a under the skin or by means of a inoculator ro
apply coloured dots ro selected areas on individual fish (generally on
the if this is less As an a suitable method
of Alcian blue at a dilUtion
of 1 % in a solUtion of physiological serum. Fluorescent elastomere
may also be suitable for small fish and provide more discernible marks
(suppliers include Northwest Marine Inc., USA).
inoculators are only suitable for juvenile fish ( 10 cm). The
cold-branding allows numbered marks ro be made on the
skin. The method involves numbered tools in liquid nitro-
gen and then to the skin. These
may allow individual bur they tend to be only suitable for
short-duration as (he or brands may as the
skin is renewed. Hr"""",,,·, they can be visible for several months after
1.2. Marking calcified structures

i ( is necessary to a
mark on the cs to act as a reference. The various methods
used to mark calcified structures can be as follows:
with radioactive strontium,

- elemental with Sr or lanthanides.
methods 2-5 are
induced from either a in
events, can also be used as a reference point.
1.2.1 Fluorescent dyes

Chemical marking with fluorescent has been the main
method used for direcr validation The method relies on
the incorporation of a chemical compound into the mineralis-
ing surface. Since the 1960s, several markers have been used in Oste-
ichthyan the earliest of which were Bont,
1 Weber & 1967; 1974;
Meunier & Boivin, 1974; McFarlane &
fluorescein or cakein (Meunier,
116
son et at., 1987; Tsukamo[Q, 1988; Beckman et al., 1990), orange

xylenol (Meunier, 1974), and alizarin (Meunier & Boivin, 1978;
Tsukamo[Q, 1988) have been used. These substances have a broad
specrrum and are suitable for cs in all Verrebrate species.
These compounds all have the capacity [Q emit a specific coloured f1u-
orescence under ultraviolet light, and are thus located a posteriori.
Tetracycline emits yellow f1uorescence, f1uorescein yellow-green, and
both alizarin and orange xylenol emit red. The coloration of the marks
depends on the wavelength of the light source and the optical filters
used (rab. IVA.1).
Table IV.A.l - Various fluorochromes and their excitation and emission characteristics under compound
microscopy (reflected light).
Fluorochrome Excitation Fluorescence Excitation Excitation Filter type
wavelength wavelength rays colour (reflected
(mean in run) (mean in nm) light}
Tetracycline 90 560 blue/violet yellow 0
Alizarin 556 596 green red N2
Fluorescein 490 525 blue ~e llow-green A
Xylenol orange 470 530-650 blue orange-red 12 /3
Tetracycline (TC, C22H24N120S) and its derivatives (oxytetracycline ,

OTC, tetracycline hydrochloride, TCHC, tetracycline dihydrochloride,
TCDHC), are broad-specrrum antibiotics, and they generally produce
excellent results in all species. However, during the last decade, the
use of tetracyclines has been restricred in some countties. In many
countries including the USA, Canada and Japan, they may not be used
in the field , or be teleased into natural or semi-natural waters. This is
because these antibiotics have a very low rate of natural degradation
and some microorganisms become resisrant [Q tetracycline (Coy ne et
al., 1994 ; Kerry et ai., 1994; Smith , 1995; Vaughan & Smith, 1996).
Researchers ought therefore [Q check their national legislation on
environmental protecrion before using tetracycline.
For f1uorescein (FC, C 20 H 120 ), the results tend [Q be more variable,
from slightly poorer marking (Tsukamo[Q, 1988), to better than other
f1uorescent markers (Thomas et cd., 1995). Since the restricrions on the
use of tetracycline, twO dyes of the alizarin family that efficiently pro-
duce f1uorescent marks in fish otoliths, alizarin complexone (AC, 1,2-
dihyd roxyan thraqui none- 3-y l-methylamine-N, N -d iacetic acid,
CI 9H1 )NO s 2H 20) and alizarin Red S (AR, 1,2-dihydroxyan-
thraquinone sodium sulphonate , C 14H 7 0 7 NaS), have come into
widesptead use.
11 7
The fluorescenr are available in different forms. Tetracycline is

found in twO forms:
a In vanous
ro accive tetracycline with an as used

Ani-Terra
- various concentrations of a stable solurion Flu-
orescein (calcein) is available as a powder Of in solurion. Alizarin is
only available as a and solutions must be U1C:UdlCU
These are in various ways; by injenion,
immersion, or incorporation in food (see following). There is some
variation in the results of between the markers and their
method (Thomas et al., bur all calcified strucmres
can be labelled at the same time Further methods
ing internal markers have also been proposed.
or intramuscular is the most

technique used to mark juvenile and adult fish. Intraperitoneal
tioo is recommended IV.A.3). Some stable solutions ofTC exist for
use (e.g. Injections of
ceouations of TC can cause the death of the fish
1978; Beamish & 1987). Meunier & Boivin (1978) esti-
mate that concentrations of 50-100 mg ofTC per do
not affen later McFarlane & Beamish (1 recommend
of 25-35 mg TC per On the basis of several smdies it
appears that concentrations between 25 and 100 mg , and espe-
of 50 l live
upon growth rate, longevity, and the strucrure

1983). Multiple marking can be achieved
fish at imervals of a few weeks or months.
fish with fluorescem
thetise them. Several that are are
available for this purpose. Phenoxyethanol at a concentra-
tion around 3%0 can be recommended. The concentration must be
rested before use, since to this anaesthetic
differs between species and sizes.
1.2.1.2. Whole-body immersion

This is useful for young fish when ir is to
dye. Individual larvae or are bathed in solutions of a
concentration for a time. There are many references on this sub-
ject, which have used a wide range of dye concentrations and duracions
(cab. IV.A.2).
118
Figure IV.A.2 - Tetracycline marks ( iT ) on diHerent calcified structures. All the preparations are observed under epifluorescent
UV light microscopy (photos J. Panfili).
a) Whole otolith of eel (Anguilfa anguilfa) observed under reflected light. Scale bar = 200 ~m.
b) Whole vertebrae of pike-perch (Stizostedion /ucioperca) under reflected light. Scale bar = 1 mm.
c) Slice of the dorsal ray of pike-perch (Stizostedion lucioperca) under reflected light. Scale bar = 400 ~m.
d) Detail of the dorsal ray (c) of pike-perch (Sfizostedion /ucioperca) under transmitted light.
figure IV.A.3
Internal marking with
tetracycline inJection for an
African tilapia (Sarotherodon
me/anotheron). The fish was
anesthetised in advance.
The soJution is injected
intraperitoneally at a
concentration of 50 mg
tetracycline per kg of live
fish. This fish had been
previously tagged with
an anchor T-tag (fig. IVA 1)
(photo J. Panfili).
119
Table IV,A,2, - Concentrations of fluorescent dyes and duration of immersion marking for eggs
and/or larvae,
Subsrance Concentration Solmion Duration Quantity Reference
(mg.l- I) of fish
Te[racycline 100-S00 NaCll % 1- 2 h (H e[ rler, 1984)
Te[racycline 200-300 no 24-48 h eggs (Tsukamoro, 1985)
200-300 3-24 h larvae
TerracycJine 100-600 no 12 h eggs, larvae, (Dabrowski &
juveniles Tsukamoro, 1986)
Oxy[erracycl in e SO no I2h,d- 1 87000- (Lorson & Mudrak, 1987)
(4 d) 126000
Te[racycli ne 200-300 no 24 h (Tsukamoro, 1988)
Terracycline 400 NaCllS % 24 h 200 (Siegfried &
Weins[e in , 1989)
Oxytetracycline 400-S00 no 24 h IS.!-I (Tzeng & Yu , 1989)
Ali za rin com pl. SO-200 sea water 24 h 10 million (Tsukamoro et af., 1989a)
Te[racycline 10000 hyperosmo[i c 3 mn 30 s SOO kg (Alcobendas et "I" 1991)
Auorescein 20000 1. 25 million
Oxy[e[racycline 3S0 no 3h 1.2-7 million (Secor et aI" L991)
Alizarin compLl 100-200 34 PP[ seuwa[er 24 h 1.18 million (Blom et aI" 1994)
alizarin red S 100
Alizarin compl. 100 no 14 h lOOO (Ah[enhoIz et al" 1995)
Oxy[erracyciin<; 10000 hyperosmori c 1-10 mn 100-40000 (Rojas-Belrran et aI" 199'»
Alizarin compl. 2S0 2S PP[ seawa[ ISh 114 (Szedlmayer & Howe, J 995)
Oxy[err3cycl ine 35 0-400 no 6-8 h 600000 (Reinerr et aI" (998)
To mark a large number of larvae, some authors recommend increas-

ing the densities of fish (Secor et af., 1991), or accelerating processing
time while plunging the larvae inro a hyperosmotic solution (sodium
chloride 5% ) for a few minutes then in 1% TC (Alcobendas et ai"
1991), Other studies show that embryos can be marked in the egg
(Tsukamoro, 1985 ; Ruhle & Grieder, 1989; Muth & Bestgen, 1991).
For larvae, multiple marking (incorporation of several fluorochromes)
is possible by subjecting them to successive baths at intervals of a few
days (Tsukamoto, 1988; Hendricks et al, 1991), It is important to
beat in mind that too high a concentration of TC can kill larvae
(Nagiec et al, 1988),
The immersion technigue is guite simple: fish are pur in a tank with
an air supply, the approptiate concentration of fluorescenr dye is added
ro the water and the process is conrinued for a given duration and then
the bath is emptied and filled with freshwater (tab, IVA,2), This pro-
cedure can be adapted ro the species and the material used, The most
importanr facror affecting treatment success seems ro be the oxygena-
tion of the water, as bathed fish can be particularly sensitive ro a lack
of oxygen, The procedure given below is generally suitable for egg and
larval stages (Blom et al, 1994):
120
of needed to a final concentration

of alizarin AC or AR of 100 IS and added to a
beaker (50 of AC has also been shown to be sufficient in several
cases);
the is dissolved in IN potassium hydroxide until its colour
from brown-reddish to a darker bluish-red colour (at of
around .5-8.0 for AC and AR);
the soimion is diluted wirh distilled water (0
solution of 50 to 100 ml before further dilurion in
furcher dilution in saline is made while solu-
rion to the unit used for (0 achieve a bath solution of the
desired (see
- it is imporcant to check that the
in order to avoid If necessary acid
should be added co lower the
- since the solution will be stagnant rhe
essential co maintain the oxygen saruration above 80% aeration;
may last for 12-24 h, after which the fish eggs and larvae
should be removed from the bath or the solution with clean
salt water.
This has not found
number of individuals co be marked with

has been added at a concentration of 109
Oral administration of fluorescent markers seems
co be successful at concentrations of 25 I food
for calcein and and at 50 for alizarin

aI., 1995). These authors have demonsrrated
of alizarin compJexone is poorer than that of
calcein and teuacyciine.
1.2.1.4. Fluorescent dyes

While fluorescent are suirable for various scales of vali-
the seasonal of all types of cs and tbe daily
of otOlirhs, they necessitate the use of ultraviolet light. The
of the CS will to some extem determine the success of mark-
In cod and for prior to hatching
and this is therefore a suitable period for
Marks induced at the egg stage are detectable rhroughoU( the
larval stage and well into the (Blom id., In rhe
case of herring, tbe demersal eggs are commonly incubated on
slides and sheets of This makes them sui cable for
ar this stage since small volumes of bath solution can be llsed
121
without the problem of

alizarin are
As mentioned mulriple can done and used as

group marks on sub-popularions with different characceristics
et at, Ir is important to allow sufficient rime
between to prevent separate marks from ng (fjg.
IVA.4). the larval srage the rare of growth
to marking will also affee[ marking success. For 10
success varied berween 10 and 55% in

groups red maintenance rations for tWO weeks before white
success was 100% in groups fed m excess during the same
(Folkvord et at., 2000). In cases in which success was
low, the otolith accretion rate was also with
increment widths of less than 0.5 Jlm.
For validation studies of annual growth it is necessary m main-
tain the fish for more than 12 months, either in the wild or in experi-
mental The of the fluorescenr mark is
cially for ne and fluorescein) in internal organs such as
omliths or vertebrae bur is of shorter duration for the scales because of
the deleterious effeCt of sunlight on these Several
authors have described that continued for several years.
For elvers in the Rhine showed fluores-
cent marks five years after immersion Beamish &
McFarlane (2000) have also observed marks on the
otolith5 more than 20 years after they had been
1.2.2. Temperature-Induced marks in otohths

has been shown ro have an immediate affect on both
otolith accretion rate and microstructural (Mosegaard el
at., Berghahn & et al., Volk et
al., Munk & 1993). Optically opaque or translucent
otolith formation is believed to reflect the of the
content and structute. fluctuations will
therefore leave discernible traces on the microstructure. In a labora-
tory temperature fluctuations a
coded sequence of checks to the otOlith formation
rate at the temperatures used between a baseline level of
LOoC and of 14°C; variations have
been used m create "bar codes" in commercial salmon stock with
numbers of individuals (Volk et al., 1995). Brothers (1990) and
Yolk et al. (1999) have extensive reviews on otolith thermal
marking
122
Figure IV.AA - Example of double alizarin complexone marks on a larval herring (Cfupea harengus) sagitta visible under fluores-
cent light (a) and the same otolith viewed under transmitted light (bl. Scale bar = 20 ~m (photos A. Folkvord).
Figure IV.A.5 . Example of temperature·manipulated increment formation in brown trout (Safmo

trutta) alevin sagitta, manipulated at 135 degree-days after hatch. Basal conditions were
11 .5' C in darkness (photo H. Mosegaard).
a) Six manipulation periods with 2 h at 14.5'C and 6 h at 11.5' C.
b) Four manipulation periods with 2 h at 14.5'C and 2 h at I1.5'C . Lights were on during the
warm phases of each manipulation. m = otolith margin.
1.2.3. Light cycle·induced marks in otoliths

OtOlith calcification is entrained to ligl1C-dark rransirions (Wrighr et
at., 1992). In some species a 6L:6D photOperiod can mask rhe diel
periodiciry of increment formarion, leading to rhe formarion of an
indisrincr zone of increment formarion. An example of 6L: 6D pho-
toperiod marks in juvenile salmon is shown in figure rv.A.6. This
marking approach can be improved by hearing rhe environmental
warer to 3°C above ambient remperarure during rhe lighr phase of
sub-daily lighr cycles (fig. IV.A.7)
123
IV.A.7
of mark (M)
by combined 6L:
60 photoperiod with 3 C
Q
temperature rise the

light phase in
aculeatus.
Scale bar = la
(photo PJ
Sr and lanthanides)
Otolith
regulations uu,/v.,,,u
dispersal of larval
hatched larvae with
radioactive label in the fish larvae could be detected in whole larvae
for up to 60 after The extent to which otolith compo-
sition contributed to the retention of the mark is and further
studies would have to be carried Out to evaluate
which would leave detectable radioactive Sr in the otoliths.
124
larval and juvenile orolitl1s have been marked by altering the ele-
mental composition of the rearing water. Two strategies that differ in
principle can be employed. First, the addition of a relatively common
element such as strontium (Sr) has been used ro alter the Ca/Sr ratio in
the oroliths. Strontium chloride has been used ro mark salmon fry
with good success (Schroder et al., 1995). A 24 h exposure to SrC! bath
solutions in concentrations of 120 ppm and higher yielded at least a
five-fold higher Sr concentration in the experimental oroliths than in
controls. The Sr marks could be detected after 21 months with Wave-
length Dispersive Spectrometty or Backscattered Scanning Electron
Microscopy (chap. VII). In a second approach, marking has also been
achieved by immersing fish in solutions containing elevated levels of
tare earth metals (lanthanides). These elements are normaJJy very rare
in fish oroliths, and marking with different proportions of these ele-
ments can produce several unique group marks in different sub-
populations. Ennevor & Beames (1993) used different concentrations
and procedures for marking coho salmon (Onwrhynchus kislttch) fry and
smolts with lanthanum and cerium, and the elements could be
detected up ro 10.5 months after treatment. However, the mode of
incorporarion of these rare elements into the otoliths is uncertain, and
sensitive analytical techniques need to be employed ro detect their
presence (chap. VII).
2. Breeding and rearing
Generally speaking, methods rhat release fish to the wild, or in which

fish are maintained under semi-artificial conditions which provide for
good growth and natural behaviour (i.e. mesocosms), are preferable for
validation studies (Geffen, 1992). The best approach for age valida-
tion is ro release marked fish into the wild, as has done with Japanese
red sea bream (Tsukamoto et cd., 1989a), eel from the Rhine ri ver
(Meunier, 1994) and Pacific salmon (Volk et al. , 1990, 1999). How-
ever, because of the large numbers of fry that need ro be marked ro col-
lect sufficient returns, releasing fish is generally not feasible (Geffen,
1992). For this reason, validation generally requires fish to be reared
from eggs or wild-caught fish. Caution should be raken with the rear-
ing conditions as these should be as close as possible ro the natural
environment of the species (fig. IV.A.8). Field enclosures or ponds (fig.
IV.A.8c,d), have been employed in several studies (liew, 1974; Gef-
fen, 1982; Simoneaux & Waden , 1987).
The conditions under which validation studies are conducted are also
very important, since facrors such as feeding level, feeding periodicity,
container size and temperature all affect the rate of growth of fish and
may also influence increment periodicity (Geffen, 1982; Neilson &
Geen, 1982; Radtke & Dean, 1982; McGurk, 1984; Al-Hossaini &
125
Pircher, 1988). In some laborarory studies, an incremenr periodiciry

lower rhan one per day has been relared to rhe inabiliry to mainrain
rhe high larval growrh races observed in che wild (Al-Hossaini &
Pircher, 1988). Rearing condirions in che laboratory ofren produce
unnarural stresses, leading ro che formacion of sub-daily incremenrs
and inrerruprions or checks in growch (Pannella, 1980; Morales-Nin,
1987a). There also rends to be a less clear disrinction berween L- and
D-zones in fish reared under conscanr cemperarure and lighc levels
chan in fish reared in field enclosures (Geffen, 1982; Campana, 1984).
Figure IV.A.S . Various types of aquacul!ure systems usable for validation experiments: from artificial (a) to semi·natural environ·
ments (f). The experiment should be as similar as possible to the natural environment of the fish. a) artificial ponds for freshwater
aquaculture (Thammasat University, Thailand); b) concrete tanks with freshwater in ambient weather conditions (Centre de
recherches oceanologiques, Layo, Ivory Coast); c) cages submerged in a brackish water lagoon (Centre de re cherches
oceanologiques, Layo, Ivory Coast); d) cages in the open sea (Research Institute for Marine Fisheries, Sulawesl, Indonesia); e) Wide
concrete marine tanks open to the lagoon (Albion Fisheries Research Center, Mauritius); f) semi·natural ponds for freshwater aqua·
culture (Centre national de recherche agronomique, Ivory Coast) (photos J. Panfili).
126
Validation and verrtication methods
For rhese reasons, validarion srudies should ideally be carried under

borh laborarory and field condirions or in rhe field alone (Geffen,
1982; Gjosaerer et al. , 1984; Campana & Neilson, 1985).
3. Data treatment
Srarisrical rrearmem of dara from a validarion experimem is relarively

simple. In general, rhe relarionship berween rhe number of days
elapsed afrer ragging and/or breeding and rhe number of incremenrs
on rhe CS analysed is caiculared from a linear regression, as follows:
(lVA.l) number of incremenrs = a· number of days of growrh + b
where a is rhe slope and b is rhe imercepr of rhe regression.
The nexr srep ro analysis involves resring wherher rhe slope differs sig-
nificandy from a slope equal ro 1 (a = 1) and an imercepr equal ro °
(b = 0). Significance is usually derermined using a Srudem r-resr.
Several resuirs can be obrained:
• If a = 1 and b = 0, rhe coumed incremems are relared ro rhe num-
ber of days of rhe experimem. The validarion is accomplished;
*
• If a = 1 and b 0, rhe rare of deposi rion corresponds ro rhe days bur
ir does nor srarr from [he firsr day of rhe experim em;
* *
• If a 1 and b 0, rhere is nor direcr relarion berween rhe number
of incremenrs and rhe number of days and rhe deposirion is nor daily.
However, in rhe case where a change in rhe incremenr deposirion rare
is apparenr, separare resrs can be made in each age inrerval ro check if
rhe deposirion rare is daily during one of rhe rime inrervals. This is
especially imporranr in cases where incremenr deposirion rare may be
Ini[ially lower rhan 1 per day, bur subsequenrly increases. Including
all rhe early incremenrs-ar-age in such a case will lead ro underesri-
mar ion of rhe incremenr deposirion fare ar subsequenr ages/srages
where rhe deposirion rare could be daily.
The above approach is ofren rhe only srarisrical jusrificarion given for
daily incremenr deposirion. However, rhis approach of raking non-
rejecrion of rhe null hyporhesis as posirive evidence rhar rhe null
model is cO(recr, ignores rhe porenrial of Type II error, rhar is con-
cluding incremenrs are formed daily when in facr rhey are nor. The
probabiliry of avoiding Type II error can be measured by caicularing
rhe srarisrical power. By calcularing power for rhe regression of incre-
menr counr on rime, one can derermine rhe likelihood rhar a signifi-
canr difference in slope would be derecred if one really exisred and rhus
evaluare rhe conclusion rhar incremenrs are formed daily. Merhods for
de[ermining sra[isrical power of regressions are now widely available
in srarisrical packages. Rice et al. (1987) also provide a useful review
of rhis issue.
127
128
B. Semi-direct validation
Paofili. B. Morales-Nio
Ooe of the most used validation semi-direct validation, coo-

the evolution of cs marginal zones over time. This
is a method which
the time of
ments (daily, seasonal or otber) under and the observarion of a
oumber of individuals. The method consists of the
of the CS, and its formation
through time in a The result is an average and/or
percenmge of obsetvations at population leveL The selected mark
must be to the detection of its formation at
of the CS, which may be difficult. Semi-direct vali-
dation is usually used to validate seasonal and twO types of
srudies are one of which uses gualitative data and the other,
data.
1.
1.1. Qualitative data

This process consists the presence or absence of the mark
at the of the CS, and the results in percentages for (he
population under The evolution of this percentage through
time is then studied: IVB.l shows the result for one growth
per year IVB.la) and two per year (fig. IVB.l The
second case is less common in a temperate environment but It has been
observed for fishes (Yosef & Casselman, 1995). In Nonh
a system of codes, first in Casselman
and elaborated in Casselman ( is now widely used.
1.2. Quantitative data

This method consists of the dismnces the latest
marks at the of the CS. The axis of measurement and the
tion of tbe marks being utilised must be standardised. The
absolute distance is the distance that separates the
last mark from the (fig. IVB.2). The relative distance
(RMD) is the ratio of the absolute distance to the distance
rhe twO last marks l)
(VIB.I) RMD
129
100 350
300
80
250
60
200
150
40
100
20
50 E
3-
Q)
Q) u
OD 0 0 c::
"0
Q)
~
'"
'of' 100 350
ro
c::
OD
300 ~
:::;;
80
250
60
200
150
40
100
20
50
_ %opaQue
Time
% transtucent
Marginal distance
Figure IV.B.I - Theoretical graph of the evolution over time (two consecutive years) of the per-
centage of opaque edges, the percentage of translucent edges and the margin width on a CS.
These examples are totally artificial and they are not taken from observations in the field. The
nature of the edge, opaque or translucent, is determined macroscopically. The margin width
corresponds to the distance between the beginning of the last opaque zone and the edge.
a) Theoretical graph of the appearance of one cycte per year. The opaque zone is laid down at
the end of each year (maximum percentage) while the translucent zone is completely deposit-
ed in August. For some CS, the evaluation of the nature of the edge is not possible and the sum
of percentages differs from 100% (e.g. July Yl). The margin width follows the evolution of per-
centages over time.
b) Theoretical graph of the appearance of two cycles per year. Th€ Gpaque zones are laid down
around May and at the end of each year (maximum percentage) while the translucent zones are
deposited in April and August every year. An inter-annual variation is observed with a slight shift
of the zone deposition every year: the first opaque zone is totally deposited in March in year I
and in April in year 2. For some CS, the evaluation of the nature of the edge is not possible and
the sum of percentages differs from 100% (e.g. March YI). The margin width follows the evo-
lution of percentages over time.
130
The use of AMD is recommended when working separately on differ-

enr age (or incremenr) classes. le takes direcdy inro accoum the dif-
ferences in growth rates between individuals. The RMD , which is
sometimes expressed as a percemage, compensates for the effects of the
reduction in growth with age: it is less sensitive to the variations in
growth rate since measuremems are relative (fig. IV.B. 3). le is often
more difficult to follow a given cycle with the AMD than the RMD. In
general , quamitative data regarding the margin cannot be measured
for O-year-old individuals (without incremenrs) , unless there is a
Figure IV.B .2
Edge aspect of transverse
stained sections of otoliths
of Colossoma macropomum
(Serrasalmidae). AMD, abso-
lute marginal distance;
D, j.\, distance between the
last zone i and the previou s
one i-I. The relative
marginal distance is then
RMD = AMD / Dj,i. \ .
Scale bar = 500 ~m
(photos J. Panfili).
a) Stained zone on the edge
of a 455 mm (SLl individual
caught in September
in Bolivian waters .
b) Un stained zone on
the edge of a 457 mm (sL)
individual caught in March
in Bolivian waters .
131
demonstration of between the period of birth and that of

the formation of the considered increment, the
tive method is used with fish more rhan one year old,
1.3. of semi·direct validation studies

We here an of the observation
stained section of the o(()lirh of Colossoma nla'I'1'f.W()"iWm
from the Yfamore basin (Bolivian Amazonia), For
otolith is eirher srained IVB.2a) or left unstained
It can be difficult to the because of the accu-
mulation of srain at the resin-orolirh interface, For
the measurement axis and where to measure the increment (start, mid-
need to be deft ned, In rhe the of
rhe stained zone is taken as the point, The dimensions of the
stained zones may vary because a srained zone consists of several d1ro-
mophilic bands (fig, IVB.2a), The semi-direct validation applied to
(he C macropOmtl111 otolith has shown that stained zones are laid down
annually and to the season in Bolivian Amazonian
freshwaters & Panfili, 1997),
graph of
240
the evolution over time
(two consecutive of
the margin width the last 220
growth zone on a calcified
structure, The relative 200 ....E:;
width in percentage Q)
u
c:
is division 180 ro
11)
of the distance between the is
of the last zone 160 -;;;
c
the (AMD) 6iJ
the distance of the 16
complete zone (D",;L
140 E
2'
:;,
The lone is fully formed 0if>
at the end of each 120 .0
(maximum width). signal <r:
is clearer for the relative 100
distance than the absolute
one because in the last case 0
all classes are mixed
and reduction of
with age is not taken
account
Time
......0-- Relallve marginal distance
- _ Absolute marginal distance
The marginal increment has been used mainly to determine

the of annual increment formation for all types of cs
otoliths, in temperate Teleost (Beall &
D;lvaine, 1 Crawford et at, 1
132
Schramm, 1989; Vianer et aI., 1989; Mann & Beaumom, 1

1991; Hales& 1991; etai.,1992; &
1995; al., 1
Gordon &
aI., 1999).
The semi-direct validation of increments has been much less
but [his method has also been (Tanaka et [11.,
1981; Re et ai, 1987;
been used in [his way because it is
orolith
SE M). Because of the low power of resolution of the
the SEM must often be used.
2. Problems of
All semi-direct validation studies have mentioned the difficulty of

evaluating the nature of the orolith opaque v. translucent,
ere. There may remain a cenai n number of
otolirhs when we examine the Three
can be put forward for the observation of whole
to The otolith is thicket in [he central area than at its where the
marks are less clear. Under transmitted rhe can be highly
If this is the case, the whole otoilrh should be
refers to the observation

opaque and translucent) and the of
to the observed orol irh area.
fare differs in the various otolith faces
dorsal, 1'0 thicker incremenrs on the nr,'rp"pr,_
IV.B.4b). The observation is thus differenr

the otolith area, ic necessary co define
cion crireria (incremenr connnuity acound the otolith, presence or
absence on a area), In older is often lunited to
the internal and dorso-venrral sides of the otolith and incre-
menrs are only visible in these areas.
These remarks also concern observarions of otolith
as those near the tend to be less visible
133
Figure IV.BA -Difficulties in interpreting the edge aspect of otoliths. A, anterior; D, dorsal; I, internal; P, posterior; V, ventral (pho-
tos J. Panfili).
a) Whole otolith of plaice (P/euronectes platessa) under transmitted light. The edge of the otolith is particularly refringent and it
is difficult to interpret it. Scale bar = 2 mm.
b) Whole otolith of the European eel (Anguilia anguilia) under reflected light. The aspect of the edge is opaque on the ventral and
anterior face and more translucent on the posterior and dorsal face. This is due to two factors: (1) the reduced thickness of the
otolith on the edge and (2) the differences of growth rates in each face. Scale bar = 500 ~m.
c) Thin transverse section of the otolith of the oxeye scad (Selar boops) under transmitted light. Edge interpretation is also dif-
ficult when looking at the microincrements which lose contrast near the edge. Scale bar = 20 ~m.
134
c. Indirect validation
B. Morales-Nin, J. Panfili
Indireer validation methods are based on corroborative information

that supports age imerpreration but does not validate the periodicity
of the CS incrememal growth patterns. The methods most frequently
employed are based on length frequency information, which is routinely
gathered in fishety studies. These methods were originally used in the
19th cemury and had a second peak in populari ty when cheap com-
putet hardware became available and fast software was developed.
Other methods are based on age information obtained at seasonal or
daily levels.
1. Comparison with length distributions
Length-based methods analyse the length composition of catch land-

ings in otdet to idemify the various age-groups presem, based on the
assumption that each age group has a normally distributed length
composition and a differem modal length. Petersen (1891) was the first
to idemify the modes in the distribution of lengths as corresponding to
°
age groups. Age class cotresponds to the smallest mode presem in
the sample obtained after the spawning period, while subsequent
modes correspond ro age 1, ete. (fig. rv.c.l).
Since then, many methods have been developed to identify the age
classes in a given length composition. These include: a) graphical
methods, which identify the groups of poims obtained by mathemati-
cal transformation of rhe length frequencies that correspond ro an age
class (Cassie, 1954; Bhanacharya, 1967); b) computational methods,
which employ the maximum likelihood (Hasselblad, 1966), com-
bined with previous information regarding the age classes presem and
their mean length (McDonald & Pitcher, 1979), or incorporating bio-
logical information (Schnute & Fournier, 1980, inter alia).
If age-length keys from cs readings, corresponding ro the same sam-
plings as the original length frequencies, are available, predicted
length-at-age disttibutions from the cs can be compated with
observed distributions by means of the chi-square test.
The usefulness of statistical methods for rhe desegregation of age
groups by length frequencies is limited when:
- the species has a relatively long spawning period;
- there is variability in growth rates, which generally increases with
age. Both of these conditions cause high overlapping of lengths
between age groups and impede the idemification of rhe age groups.
135
Figure IV.C.I
Basic principles involved
in methods for
length-frequency analysis.
Hypothetical set 01 length-
frequency samples captured
N time = t
at three different times
(t. t + I, t +2). At time t, there
are three age groups
(AI> All, A2 1) with normal
length-frequency
distributions. The analysis
hypothesises that at time
t + I, the ages become At+ I,
AII+l and A2l+l and that at
time t +2. the ages become
AI+2, AII+2 and A21+2·
Note that at time t+2, a new
age group appears in N time = t+l
the sampled population.
The interconnecting lines
follow the hypothetical
modal progression of the
lengths.
Length
Thus, lengrh-based merhods may only be reliable for rhe firsr age
groups or for species wirh a shore lifespan.
Anorher approach is ro calculare rhe von Berealanffy growrh parame-
rers from rhe lengrh frequencies (Pauly & David, 1981; Casselman,
1987) and compare rhem wirh rhe growrh paramerers obrained from
age-Iengrh keys elaborared from cs readings.
2_ Methods using age information
There are srrong indicarions rhar rhe age-reading merhod is accurare if

rhe age composirion of exceprionally good or weak year classes can be
followed over a long period of rime. The srrengrh or weakness of a year
class would be losr rapidly if rhe age reading merhod was nor accurare,
because of rhe assignmenr of ages co wrong year classes (Elrink &
Kuirer, 1989). However, reader bias, which is defined as rhe subjecrive
assignmenr of ages caused by knowledge of rhe exisrence of srrong or
weak year classes, is likely co have an effecr on rhe age reading resulrs_
136
This reader bias can be excluded a set of cs of an excep-

tionally strong year class collected in different years
number in each of these age
mine wherher a CS sec from an extremely strong year class can be used
in CS workshops ro estimate the accuracy and rela-
tive/absolute bias in age (an example for horse mackerel is pre-
sented in ICES (999), and to estimate rhe effect of age errors
on the assessmenL
There now exists considerable evidence that daily increment observa-
of otolith macrostructure (see
review in Ameri et et!., CountS of daily increments
have been used ro verify that one opaque and one translucent
zone represenc an annulus (Victor & 1982). rhis
is limited
fine rrr>'r,,,nlr increments in fish more
chan one year old;

the need w validate the of incremenr structures;
- the difficulty of idenrifying the macrostrucrure when (he
microstructure is observed at high Due ro these
problems II1crements are used co validate the first
annulus.
An alternative method is ro follow a cohort of juve-
niles recruited the same year, with a number of samples col-
lected during rhat period, count rhe increments in numbers of oeoliths
and compare the results with the number of days elapsed between sam-
The linear between dates of captures and the mean age
of the should have a equal to 1 (0 indicate
incremem (Hoedt, This method that there
should not have been any migration to or from the area.
When fish are the increments in their
owliths, their dates of birth can be determined from their date of cap-
ture and their age in days. If rhere is a march between the spawn-
and the back-calculated date of birth the nature of the
increments has been supported (Morales-Nin & Aldebert, 1997).
137
D. Verification
B. MoraJes-Nin, Panflli
-------------------------~ ,,~- ..
Verification confirms the of the of age, i.e.

the repeatability andlor of a numerical that
may be independent of age. For if tWO readers agree on the
number of increments present in CS, or if twO different CS from the
same animal are as the same number of incre-
ments, verification has been achieved (Wilson et al, Indices of
and these can
cion
means that increment formation

to major life events, not that it has any tem-
L Different .... ,.r1i.,a" or several readers: bias
When interpreting a CS, it is always necessary to read it more than

once in order to reduce parr of the subjectivity. In order to maintain
the of physical data date of
Several statistical indexes and
the of agreement between
methods is to compare the results of
~~'.'H"hJ from one or several readers for the same CS. The per-
centage agreement can then be calculated: this is to the
ratio between the number of coincident and the total num-
ber of PA depends on the of
one a 95% PA to within one year between two age teaders of
Pacific cod would indicate poor the few year classes in
the On the other a 95% PA to within five years would
be good for a spiny 1(S J
138
(Beam ish & Fournier, 1981). These authors have therefore

an Percem Error (APE) index:
OVD.1) .APE 100%--

1 R
I,----
I
R 1;1
where XI; is the age estimation of the jth

(he fish, and R is the number of times each fish
the standard deviation in che

rather than the absolute deviation from the mean age. The
resulring an estimate of the Coefficient of vatiation
and does not assume that the standard deviation is proportional
(Q the mean:
CV 100%
CV can be across a number of fish (Q produce a mean. CV is

more robusc than APE and is thus more flexible (Kimura
1991). There is no CV chreshold value for or
on [he and the range of
ages. Laine et al. a maximum CV value of 5% as the
limi t for accepGlble
Considerable efforts are made by imernarional committees to stan-
dardise che
can be
1997).
For daily owli rh incremencs we also recommend that rhe same
reader should read the orolirh twice, first from rhe (Q [he
and then from the

aXIs 1992). If no
t-rest, the mean
w estimate 1992). These
amhors also some form of weighting in order to rake
of individual
139
2. Different from calcified structures
If the formation of the increments is due to major life

events, should be present in all the CS of one or all members of
the sample, Different from several CS should the
same results, In order to compare these the same indexes as
those described above (chap, Iv.n APE and/Of CV, are used, Another
test for of rwo cs oroliths v, is to
prepare a two-way table and carry our a symmetry test
(I-:!oenig et et!. , 1995),
Although left and oroliths are similar in flat-
ItA), in order to avoid bias oroliths from the same side
should be If any problems of should
the orher otolith of the can be used,
3. Trends in of
Another verificarioo criterioo is the

marion, Increment widths should show a rate of cs growth
with age, This linearly interval between increments forms
the basis of age estimation (May, Furthermore, if increment
formation is a response to a major environmental event, it should be
throughout the population, The regularity of the
patterns may thus be demonstrared by
menr-to-cenrre distances for all cs IVD.I) and
of rhe distributions means of a test.
Figure IVD,l IOl

First three distances.
measured as ring radius 9
from the centre, in otoliths 8
of SaJpa saJpa, showmg
7
a normal distribution
and decrease in growth
with age,
""'"
'"
C
(l)
6
u 5
0;
Q. 4
3
2
I
0
Otolith radius
Another method of trends in patterns of

the obtained from direct CS
VA,2), The observed from
140
the cs interpretation must be calcu lated from fish sampled during the
period of the measured (for back-calculation) increments (rranslucent,
opaque, check, etc.). The observed length-m-age will otherwise corre-
spond to a period of a complete year for a given age group , while the
back-calculated length correspo nds to a particular time of the year.
This will give rise ro differences between both mean lengths and stan-
dard deviations.
4. Accuracy and precision
Accuracy is the closeness of the estimate of a quantity (measured or

computed value) to its rrue value (fig . IV.D.2) . Precis ion is the close-
ness of repeated measurements of the same quantity (fig. IV.D.2). For
a measurement techn ique that is free of bias, precision implies accu-
racy, but the two parameters are not identical. In any study of ages, it
is necessaty to determine both of these parameters and to take steps to
Figure IV.D.2
Accuracy versus precision •
in sclerochronological
studies. The age estimation
results (black squares) are
• • •
plotted against the target
true age value (cross point • • • •
of the XY axis). Accuracy
is the closeness to the true
value whereas precision • • • • • ••
is the closeness of repeated
measurements. •
•
•
no accuracy accuracy
no precision no precision
.r:;
••
. •
no accuracy accuracy
precision precision
141
improve them. accurate estimates need not be and

vice versa & Moksness, 1991), Thus a mean age can be accu-
rate while the individual observations that led to its estimation were
not
within or among
accurate (Campana &
142
Some uses of individual age data
Ch rV
Some uses
of individual age data
l43
144
Growth and rates are of fundamental in fish

and normally age information is needed (0 esti-
mate these rates
some
In the first part of the
of age dara
are may grow at differ-
em rates summarized in the
context of and relative condition. A major of the
incremental structure of the CS is that it also enables the estimation of
fish size at different earlier its thus pro-
viding a sequence or on an individual basis by
means of back-calculation The and assump-
tions underlying back-calculation of fish size based on cs are
The second parr of the deals with
cations where the use of individual age data is
age-t)as:ed data is considered to be essemial for the
processes the recwirmenr of marine fish popu-
[at ions. Potentially mortality rates can be associated with indi-
viduals of small- or low rank size, sub-oprimal and birth dare
andlor and the mechanism may be docu-
of cs from individuals sampled over the period of
Meekan &
Many world fisheries are
assessed by age st[uccured population models, Worldwide fisheries
laboratories utilise CS to determine fish age by
formed structures. and IJ"''-C''''
cs like scales and bone structures amollnts to a substantial
for fish stock assessment. In the last parr
are of how data are
in fish stock assessments. '"' fTH..,''''<
the benefits and of lLsing models versus other
types of models, and on how the added information the
age estimation can the increased effore,
145
A. Growth and analysis
1. Models of
1.1. Measures of growth

Absolute growrh refers ro rhe rotal increase in body material or
and absolure growth rate is defined as the absolute
growth over a time period (see below). An is the daily
incremem width in larval fish oro11(hs, which represents (he radial
orol1(h growth over a 24-h period. If the absolute growth rate is
constam over time or the increase is constam on an absolute scale, i.e.
the increment widths are constant or the orolith size is
in absolute terms, we have linear growth.
In the following we use S for the size of the calcified struccure rep-
and T for time. The size may refer to
such measures as area, or We can define abso-
lute growth rate as (AG R):
(VI) A G R = -;;;c;--;;;;;-
Measures of absolute are sometimes of limited value since they

are dependent on the size of the strucrure we are
One way of with the problem of different sizes is ro calculate
relative growth, which is absolute divided by initial size. Rela-
tive thus expresses the increase in size. In a similar
manner as for absolute
rate as relative growth over a given time
l(G[~
When the relative growth rate is constant over a short period of time,
we can express it as:
dS
(V3)
g = dt·S
where g is also called the i nstanraneous growth rate. over

time we obtain the it to be con-
stant):
(V4)
\46
In cases where the rate 15 the above

we have a case of Le, the propor-
cional increase in size is constant per unit time, If a day is the time
unit, the daily growth rate (DGR) can [hen be calculated as:
5) DGR (eLl)
where g is calculated as above on a daily basis 1975). g

and DGR are as daily We can multiply each of the
measures by 100 to obtain the rare in percent per day.
is often used for or for
When che growth rate is very unit time basis, the SGR and
DGR will values, This is due to a "com-
pound interest" situation where growth is added to rhe within
a day, This effect may be illustrated by DGR witb g
in the below:
(V6)
The use of SGR instead of DGR In the equation will result in an

underestimate
Most growth curves can be treated as linear over short of time.
When the time duration increases,
become non-linear. An
for shorter e,g, in the larval stage in (Fiksen &
Folkvord, are unable to maintain
COnstant growth rates in the long term, due to variations
in food supply andlor physiological constraints (size-related surface-
volume constraints),
An distinction has to be made when with individ-
ual growth data between and cross-sectional dam
& Miller, data are of the type where
individual are available at var-
ious times, whereas with cross-sectional the sizes at sam-
(or harvest) are available (fig, VA, 1), In fisheries it is very com-
mon co use data based on individual size at catch as the
basis for growth curves (cross-sectional This CO[-
the size of individual fish at the intersection of the
vertical I ine of sampling) with the individual growth
curves at the lower left In VA.l b, Obviously, the col-
lection of these data poims will not represent the actual
of the individual fish and any selective loss of or
small individuals over time will bias our estimates, On the
other hand, if several measures of of all the individuals
from the lower right projection in
individual estimates can be obtained. The CS enables the
147
exrraction of such long itudinal data from individual fish , and depend-
ing on rhe questions asked , rhe extra efforr of ag eing and construcring
individual growth rra jecrori es may be worrh whil e (chap . VB and
VC). The use the cross-sectional data will still require the fi sh ro be
aged , but in this case only average popularion growth-at-age measures
instead of individual growth measures will be obtained. In these cases
comparisons of si ze-ar-age berween g roups can be carried om using
analysis of covatiance (ANCOVA) based on the size-at-age daw from
respective g roups. On rhe other hand, the staristical rrea tm ent of lon-
gitudinal da ta may solve for rhe seri al correlation of increment wi d ths
of the CS (Chambers & Miller, 1995; Ralsron & Howard , 1995; Jones,
2000) . Both repeated m easures ANOVA and time series mod els (e.g .
ARIMA) are designed fo r this purpose, and a furthet di scuss ion of these
techniques is presented in the references above.
In many cases, when the selective morrality in the pop ularion is negli -
gible, or can be corrected for, cross-sectional data may suffice. A pos-
sible way ro account for si ze-selective mortality between samplings is
to rank the fish of the initial sample according to size, and ro exclude
the proporrion of the initial sample that corresponds to the selecrive
morta lity berween the samples, before calcula ting the population
g rowth rate (Folkvord, 1997). There is also the possibiliry of obtain -
ing averag e indi vidual g rowth meas ures using cross-sectional data.
The si ze of a five-year-old harvesred fish will provide a suffici enrl y
accurate measure of average g rowth from birth, bur no time-resolved
individual g rowth informarion will be available in this case.
b
,-
...... . ~ ... ....
···~··~··~Tf.[ : ..
.,........ . ........! ········L. ........ ...........

.. ! . ........ i.··· ... . / ... >1-..-' »~.-.+ ':... .-.
~_-'---_ ;..J
-. .
......
; :.:
... ..... ~
•.
.........
,"'
..'. ' ... ~
Cl)
N
'
... .... )J
Ui ·······.1 .....
······· · N
Figure V.A.! - Three-dimensional representanon of growth and size-at-age (modified from Chambers & Miller, 1995). Individuals
from different cohorts are presented in different lines and colours. a) The growth of individuals over time and age. b) The pro-
jections of individual growth trajectories to age and size plane, nme and size plane, and time and age plane respectively. The
intersection between the dashed vertical line on size and nme plane and the respective growth curves represent the typical cross-
sectional size-at-age data obtained from a given sample.
148
1.2. Allometry and age-independent growth models

Different body parts grow at different relative rates. In th e case of
weight and length, th e relationships between these m easures are fre-
quently used in various ways to assess the overall growth and condi-
tion of the fish (e.g. Ferron & Leggerr, 1994) . The rel ative size of cs
(e.g. otol iths) in comparison with the size of the fi sh may also be influ-
enced by the previous growth and environmental history of the fish
(Casselman, 1990).
In the foll owing, we use Was the measure of fi sh size (weight) and S
as oto lith (or scale) size. An allometric relati onship berween fi sh size
and otolith size can be expressed as:
(V 7) W = a ' So
or in log-tran sformed form:
(VS) log W = loga +b logS
In fish-length - fi sh-weig ht relationships, b tends to li e between 2 and
4 and is often close ro 3. In the case of orolirh-size - somatic-size rela-
tionships , b will depend on which measures are employed. When a
one-dimensional measure of otolith size (e.g. radius along the anterior-
posterior axis) and a three-dimensional measure of fi sh si ze (e.g. dry
weight) are used, a valu e of b cl ose to 3 wo uld indicate isometric
growth. This implies thar the re lative growth rate is simil ar in all
dimensions (axes) (chap. II.A). A b lower than 3 would indicate that
otolith gtowth in th e anterior-posterior axis is relatively higher than
in the other partS (dimensions) of the fish (fig. VA.2). When fi sh
length is used as a one-dimensional measure of size, b should be closer
to 1 when otolith size is also measured usi ng a one-dimensional mea-
sure as above (e.g. H are & Cowen, 199 5).
Figure V.A.2
Allometric relation of otolith
radiu s (sagi tta) and larval In DW = 1.232+1.604 In Radius .0
dry weight (DW). Data from

herring larvae (Clupea 2000
harengus) with otolith radius
> 18 ~m and reared at 8°C
00
(data from Folkvord et aI.,
OD
2000). Note that the slope
of the regression line is tess i 1000 c)
than 3, indicating that o 800 , .. ... o"60 - .
the growth is not isometric. o

600
400
40
Sagitta radius (~ m)
149
It has been found that of different cs only rransitorily scales

isometricatl y to somatic growth 1990). Instead, the
growth of bone and to even greater extem, of scales is positively allo-
faster bone and scale than somatic
rates), whereas otOlith growth shows
slower otolith
gtowth rates). The for northern
fate of cs and rate of body size B can be as a
polynomial:
dCSldt a·
where b is less than but greater than 1 for the

of scales and deitbrum This model
some general observations on nutritional conditions as indicated by
prey availability, which influence the relative sizes of calcified struc-
i.e, when mote prey was northern pi ke
c!eithra. The relative size of CS is thus an indicator
In rate and nurritional status,
This simplified model, however, does not accoum for certain extreme
cases such as the continued of otoliths concurrent with
checks or even the rp,:",!>",,, of scales when fish Stop
et at.,
rates in variolls dimensions will not
remain constant over long and the plot
between the variables will thus appear to be non-I ineaL A marked
transition In (or orolith-size fish-size
relations), may occur imporrant
as meramorphosis in flatfish or smolrificarion in
in different in the different stages or stanzas, In these
cases it is normal ro determine separate relations for each stage
nal & 1978),
In (he same manner as one can llse residuals from the
weight directly as measures of relative condition, the
residuals from the somaric-size - otolith-size can be taken
as measures of relative orolith size (Hare & Cowen,
condition is a cumulative result of previous
growth and stage of individuals
condition are characterised
As far as oroliths are rf\f1na rrlPr!
(Q be associated with
at (Reznick et a!., 1
man, 1990; Hare & 1995, If CS size and somatic
150
Some llses of individual age data
sIze are the pattern of residuals of and

otolith should be positively correlated. This is indeed
the case in shown in and the higher the
correlation between the the lower is the
tion, y= 1, there is no
somatic size would imply that the exacc CS size was
una, the correlation of the age-on residuals from
VA.3d with the residuals of the orolith
residuals from VA.3e will be if
viduals rend to have otOliths at a
If relarive otOlith size is independent of then tbe correlation
between the lam;>r residuals will be 0, and (his correlation has therefore
been termed the rate effeet" (GRE) (Hare &
If fish
(ate a function
can be solved fot a group of individuals following a similar pattern,
with the allometric of
rate of growth i)
von Bertalanffy, ii)
types of relationships between somatic rate and otolith
variability in the parameters of each growth
of this when individual fish do
not in the same paper
the author numerically the common observation of so-
called rate effeer on the otolith-size fish-size relationship
Reznick a!., made the
observation that in haddock the ratio of
otolith to total decreased with fish size bur for fish
size increased with age, Xiao used these data on haddock in a
simulation exercise of otolith size v, fish rate the three
earlier mentioned models ending up with some
quite unrealistic panems in the between otolith
and somatic growth rate for any of (he models
demonsrrates that even otolith
may scale to fish [() some age nPI,Pf"\P
individual otolith size - fish size growth may not
derived from averages. The of new
models comrolled owiith growth may in
rime yield more realistic pauerns of somatic rate - otolith size
151
a d
.9
ID
co
«
Age length
b e
Age length
c Relatively large fish

Slow growing fish With
relatively large otoliths
~
Relatively small fish

.4
Fas t growing fi sh wllh
relatively small otolilhs
Residual of otolith radius-on-age Residual of otolith radius-on-Iength
Figure V.A.3 - Schematic examples of growth of diHerent body parts of a fish, a) length, b) otolith, and c) the corresponding cor-
relation of length-on-age and otolith radius-on-age re siduals from a) and b). A high correlation between the residuals indicates a
low age independent variability of the otolith - fish length relationship, suggesting that cumulative otolith growth to a large extent
reffects somatic growth and vice versa. The correlation between the residuals from the age-on-Iength relationship d) and otolith
radius-on-Iength relationship e) represents a measure to what extent relativety fast-growing fi sh have relatively small otoliths (in
the case of a pOSitive correlation) at a given fish length (or in the case of a negative correlation to what extent the relatively slow-
growing fish have relatively small otoliths). Positive correlations are indicative of a growth rate effect (GRE) where slow growing
fi sh have relatively large otoliths and fa st-growing fi sh have relatively small otoliths f) (modified from Hare & Cowen, 19951.
152
1.3. A2.'e-DaSE~a growth models

The most common model used in fisheries research is the von
function (VBGF), In ifs form it is
wnrten as:
(V 10)
where L, is mean at rime t, and L"", K and to, are (he parame-
rers co be determined, An implicit of rhe VBGF is (hat the
Insranraneous rate:
(V 11) dL = a+bL
where b is
rate decreases as size increases
o as size Leo, the mean asympwcic
1995a), The paramerer K is rdared w the curvamre of the
curve, where higher values of K indicate a more rapid
in the rate of length with The last param-
erer, to, represen ts che age ar which rhe mean
would be 0, The curves of individual fish in
based on the VBGF, The differences in
certain assumptions, it is
for A way of
involves the use of a Ford- Walford J is
To obtain rhe paramerers rh is merhod we rearrange rhe
VBGF and ser e -K = k, and solve for t+ 1 co obtain:
(V12)
ThIS can be w:
(V 13) J Loo
which is of rhe form y = er where the paramerers can be esrimated
The lS to k (or which
us the K parameter, The Loo parameter can be found
dererm the inrerseccion between [he regression line and the line
of Y X. These lines wil! intersect ac che size where Lt J , which
will to rhe Alcernatively, can be
k in che for the L oo '(
(V 14)
where K and to are as above and \11 is the
00 (Cam-
pana & 1992). This in contrast to the
based VBGF, has an lOfle([ion race is at its
153
Another used function in fishes is the

function. This also has an inflection in its growth curve,
and like the VBGF it is around the inflec-
The
lVI _
(V.IS) w1 -
g is a constant related to the

ID in growth rare.
The parameters of VBGF and other models can now be accu-
rately estimated non-linear methods
The inclusion of models will
with the interdependence of
and thus avoid the common error of running the estimarion with
inflated of freedom.
2. Back-calculation
One of the main of the incremental information

stored in calcified structures in fish is the possibility of
information about size at earlier ages at the individual level (longiru-
dinal
2.1. Background and assumptions

Back-calculations of fish size have been made since 1910, when Lea
and used patterns in scales to predict her-
flng rates. Later applications have involved the use of
oroliths, bones and rays, and the use of annual
srrucrures i ncremems
or microstrucrure) 11). Back-calculation is an
method of estimates of individual and
In field-based studies it is the main alternative ro mark-
recapture buc it has the that it can potentially be
employed on virtually any harvested members of the population
and not to the limited number that have been
marked and then . The methodology has strong resem-
blance to (the study of which
has been used to infer climate-induced variations in growth rates
Cook & Kairiuksris,
The back-calculation can be defined as fish size
at an earlier rime (or on the basis of a set of measuremems of cs
size and poim in time (usually at
we use L for fish size length) and
scales or otoliths). The size measures are
indexed with i, an age
154
at capture. In this conrext a back-calculation formula (BCF) is one that

enables Li (Q be back-calculated from L(, S( and Si.
In order ro carry our a back-calculation correcdy, three mai n assump-
tions have ro be fulfilled:
- the size of the CS mark is the same as the size of the CS at the time
the mark was formed (no resorption or degeneration);
- the assumed time of formation is correcr;
- the BCF formula accurately relates body size ro CS size for each fish.
Regarding the first assumption, the back-calculated fish lengths
would be biased if the size of the mark in the CS had changed with
time since deposition. In the case of oroliths jt is generally believed
that the strucrures do not change after deposition on ro the orolith,
parrly because the orolith is regarded as an inerr non-cellular structure
(chap. II). On the other hand, scales may be liable ro some resorption,
especially in fish approaching their asymprotic length (Casselman,
1990), and for this reason the validity of scale-based back-calculations
of growth in these fish may be uncerrain. For the second assumption
it is essenrial ro know the age at the formation of the incremenral
marks in order ro correcdy assign the correct age ro the back-calcu-
lated size. The proper validation of the incremenral strucrures (chap.
IV) is mandarory regardless of which cs is being used. Finally, it is
essenrial ro esrablish a relationship between cs size and somatic size
that enables measured cs size ro be rranslated inro estimated somatic
size. There is a large body of literarure on how ro proceed with back-
calculation (see Francis, 1990 for review), and in the following sec-
tions we presenr a summary of some of the techniques employed.
2.2. Regression methods versus proportional methods

Earlier fish size can be calculated on the basis of a relationship between
cs size and somatic size (some transformations could be involved in
either variable, bur that is not the main poinr here). The regression
approach is based on a common relationship between somatic size and
CS size, which can be described as:
(V16) Regression BCF: Li = h(S;) where h is a funcrion.
In its simplest form this would ignore the poinr at capture (So Le>
which uniquely characterises the individual fi sh (fig. VA. 4a). In such
a case all the back-calculated sizes would be derived from the same
regression line. Alternatively, one might use the parallel lines (com-
mon slope) from each end-poinr (5" L), and determine earlier sizes
from a series of parallel lines (fig. VA.4b). The problem of such an
oversimplified method is that it implies that the same slope between
the CS-size - somatic-size relationship can be applied ro all fish. A sim-
plified regression approach is usually not ro be recommended for
back-calculations (Francis, 1990).
]55
a b
o
o
o
00
~ .-
·············· 0··
.~- .......
.- . " ;
c d
0
0
\-) 0
0 0
'! •.•
0 0
'! -
...
~._ .. _•.••..•..••••.•.•.••..•.•••••.. .o .•• ~ ••
o () .. .
···-····· ······--· · ····· ·o~· :~~· ..

o ~ .""" l.i
."
.- .-
e
o
0 0 0 0
_ ••• H •••• ••• •••••••••
CS size
CS size
Figure VA4 - Back-calculation of fish size based on different ba ck-calculation procedures . a) Regression approach assuming
equal growth trajectories of all individuals, b) Regression approach assuming similar slope of CS size - fish size relation for all
fish, c) Dahl-Lea procedure using direct proportionality (intercept at origin), d) Fraser-Le e procedure using set intercept on ordi-
nate, e) Biological intercept method using a biologically determined starting point (marked +), fJ linear SPH method, and g) lin·
ear BPH method. The same data set is used in all cases, and the growth is only calculated for one of the fish (solid points). The
three arrows on the abscissa represent the CS mark sizes of the fish in study. The dashed line from the size at capture repre-
sents the back-calculated growth trajectory, whereas the vertical and horizontal da shed line s illu strated how cs sizes are trans·
lated to corresponding fish size s. Difference s in back-calculated sizes can be seen on the ordinate. The solid regre ssio n line rep-
resents the relationship between CS size (5) and somatic size (L) used to estimate the parameters in the BCFs. L on 5 regres-
sion is shown in a·b, d and g, while f is based on 5 on L regression.
156
In comrast, methods define a series of lines

from a common one f()f each individual fish, These lines will
have different and the would determine which line
should be used for each in the back-calculation, The under-
ng rationale for (his is that fish with
moliths at a given somatic size at capture are most
ocolith5 at somatic sizes as well, while fish
with relatively small ocoliths relative (() size can be to
have had some ti me before capture,
The principal difference between a and a propor-
tional visualised in figure
the improper use of the
an unrealistic reduction in fish size since the last increment
due to (he small ocolith size at capture, The alternative of
from the
1[1 unrealistic fish sizes would be inferred at

small cs sizes,
2.3. Starting points in proportional methods:

statistical determination versus biological reference points
There are that methods are
preferable (() methods in back-calculation,
First of all thete are no indications that one CS-size somatic-size rela-
tionship will suffice for all individuals in a On rhe con-
trary, we observe fish with differem CS sizes at a given
very much in (he same way as we find fish with different rel-
ative conditions, we have abundant documemarion to the
effect that fish scales (andior nar-
row age and size windows, On a wider age and time '''''r'''',pr'r this
will manifest itselfas a well-defined
size - somatic-size relationship (i,e, the individual lines will
from a common point rather than run in
ways of the common
methods in back-calculation, The point is either determined
estimation or is manually set on the basis of ([]-
tefla fish at the onset of scale
The most hasic based determination is
the Dahl-Lea method, which assumes a conscanr
between CS size and somatic size, This forces all lines to go through
the Le, if fish size is 0, then CS-size is 0
(VI7) Dahl-Lea BCF:
157
A further extension of the concept has intuitive

appeal since it can be that the size at which a fish
for example, is relatively constant, In herring this takes
mm, This would ('()f'rp';"£1,nn
VAAd (i,e, the fish size when the cs size is 0), This
correction in the Fraser-Lee
whether this is on a biological basis or determined in
the from In such cases, the back-calculation formula
(BCF) can be written as:
(VI8) Fraser-Lee BCF: Li =
(or determined by the regres-
sion L c + The rationale for the use of the determined intercept
from the L on 5 in the BCF is that L will be esti-
mared from 5, The mathematical validity of this has been
Francis (1 formulation is
provided below,
One of having the point set manually becomes apparent
when there is non-linearity in the CS-size somatic-size
in the early stages, that is not resolved by suitable transformations, In
sllch cases the back-calculation by methods
will be on the relative number of small and individ-
The inclusion of many small individuals may cre-
line that does not reflect the overall
the CS-size somatic-size in older, fish
1990)
An extension of the Fraser-Lee correction method is pro-
vided who the biological
correction that is not set at the but
rather at some point off the axes where both CS -size and somatic size
are than 0, This is more in the case of otOlirhs, since
these are present in fish at the time of hatching, in contraSt to
scales which appear later in In the biological
may also be determined by mean CS-size and somatic size ar
the starr of proportionalIty, The BCF for the
method can be formulated as (Campana, 1990):
(V. L!
where So> represents the V.AAe), The for-
mula can also be wrirren as:
When 0 and So 0, the IKF identical to Dahl-Lea pro-

porcionality through the when only So = 0 it is identical to the
Fraset-Lee BCF,
158
the method often credible

back-calculated it has been criticised for its lack of mathemati-
cal rigour and questionable s(acistical The
method does nor rake imo account the
CS-size - somatic-size
absolute terms. Since the biological is set more or less
manually, it is difficult to compare the of various back-cal-
culation since it is not clear to what extent the result is
pn,'n(iPr1t on the choice of a given
Although the utilisation of a realistic [or accu-

rate back-calculated sizes at intermediate ages, the acrual correlation
of fish size versus otolith size at this initial age and the
variation in proportionality between somatic and otolith growth are
the two [actors that determine the accuracy of back-calculated size at
hatching. le has been found that hatch stage variation in
the otolith-size - fish-size relationship is with the result that
otolith size may explain less than of the variation in fish size. For
this reason, strict adherence to (allomerric) is essential
for the back-calculation method.
An of the variation in proporrionality between otolith size
and fish size in small fish may be obtained
ofa salmon
values from their between 0.5 and
1 g were marked individually and weighed initially and
28 period. The otoliths were measured
and back 28 increments. The assump-
in the form of a non-linear power function
and otOlith radil-is may be in the fol-
form:
where kj is the individual of the otolith-rctdius -

A plot of fish v. otolith radius for the
two evems combined showed a linear trend and at first
offered no comradicrion to the assumption.
If kj remains constam for an individual fish j, the correlation between
individual v. at 1st and 2nd
evem (It and unity. A re-examination of the values pre-
seored by Wilson & Larkin ( showed an of
about O. a high degree of ctOssover between otolith
growth This exercise shows one of the
tifiable proportionality in back-calculation
159
2.4_ Proportional methods: SPH versus BPH

When we consider methods of back-calculation, we have
basically twO means The individual CS size
of a fish may be (0 the average CS size at any fish
size or the individual fish size can be ro the average fish
CS size. In the former case we have what Francis
hypothesis (SPH) whereas the
laner case is (BPH).
the same terminology as before (from
and somatic size wc can express the twO relationships as S f (L),
where average CS size is funnion of observed somatic size, or alter-
L g where average somatic size is cl function of observed
cs size. In the linear case this is as:
(V.22) a+bL
ndS
In the fIrst case we use observed fish length to estimate average scale
size at any fish Since the benveen average
scale size and observed scale size should be constant over the entire size
range of the Individual this proportionality can be
terms as:
= cortsla rt t
The constant between scale sile and observed (mea-

sured) scale size may for i.e. the average scale size is
90% of the measured scale size of the individual flsh at any gIven
length (i.e. the scale size of the fish in is about 10%
than the average at any given fish The BCF for tbe scale prop or-
tional can be wtitten in a form as:
25)
In the common linear case rhis translates to:

a + . (et +
With some minor rearrangement, the BCF for rhe SPH propor-
tional h"'h"'-hP« is rben:
SPl-J: Lj - (a I b) ct / b)
where a and bare and slope) from the

Son L (fig.
if we employ a body (BPH), rhis
(V28) g .....g
160
If the constant in (his case is 1,01, (his implies that the average length
size) is 1% greater than the observed size at any scale
size (i,e, the size of the fish in is abour 1 % shortet than
the average at any given scale The BeF for
the BPH is then:
Ig
which in the linear case can be wrirren as:

BPH: L=
I + I (c +
where c and d arc coefficients (intercept and slope) from rhe

L on S (fig,
The Fraser-Lee procedure is not
1990), It lacks a clear
is correct to esrimate average L for a given S in the way this

done, The estimated parameter in the Fraser-Lee procedure (when
estimated by will be than (a / b) in SPH (see
and the back-calculated will thus be
smaller the SPH, The Fraser-Lee
similar results to the BPH since they are based on the same
(S on L)
There has been some controversy which method
should be employed for the estimation of the parameters to be used in
the BeF, francis (1990) has advocated the use of
due ro rhe model description and the statistical
that are desitable in connection with
Ricker (l992) has recommended using the GM
difficult to assess which variable should be
The central line obtained from the
GM would thus represent a better fit to the data, The
of the GM line can be obtained by:
(v'31 ) v
where v estimated GM b = estimared slope of the of

Yon r correlation coefficient between X and Y, and d esti-
mated of the of X on Y
The type of to be used, nr.'[Vp'lfPr
rhe choice of proportionality (see
there are no firm rules for which of the
methods should be used, A comparison of back-calculated size from
both SPH and BPH will an indication of (he inherent
sion of the back-calculation procedure itself. A high correlation
between cs size and somatic size for the target population will reduce
the difference between SPH and BPH results, In all cases of back-cal-
culation it is useful to compare the SD of back-calculated
with observed sizes at the same age, Inflated SDs of back-calculated
sizes to the observed sizes may be an indication of problems
wirh the back-calculation
2.5. Non-linear BCFs

As indicated by the
rhe between CS size and somatic size,
can be and we presen t
two alternatives below:
(V32) Air. 1, L g (5) uS"
In this case fish size is a non-linear (power) function of CS size, The

equation can be to a linear form taking the natural
arithm on both sides of the equality
(V33) L ~ log u + v log 5
We determine v by and the BCF:

(V34) BCF:
When v is determined from the of L on 5, the BCF is con-

sistenr with a BPH. The back-calculation lines are linear and parallel
on a plot (common and will converge towards Li 0
and Sj = 0 on the linear plOL Ifv = 1 the BCF is to the
Dahl-lea BCF,
In the second of a non-linear BCF, fish size is as
polynomial function of CS size:
(V35) Ale. 2: c + dS +
The BCF can be

BCF: + + +
When c, d and e are fit by non-linear of L on 5, the BCF is

consistent with a BPH, If e = 0 then the BCF is identical to the com-
mon linear BPH, In the same manner as in tbe linear case, a BCF can be
derived on tbe basis of tbe SPH tbat average scale size is
a polynomial function of observed fish length, in mind tbe
underlying proporcionality, a wide range ofBCFs
can be constructed on tbe basis of machematical
Tbe advantage of tbis is that the statistical of tbe estimaced
parameters in the BCF will be known, and measures of the precision of
the back-calculated sizes can be obtained.
162
2.6. Problems of back-calculation

The validity of the back-calculations is highly on the ful-
filment of the listed above. Bm even if the back-calcula-
tions have been catried Out in a correct manner, the results themselves
need to be with camion. As in other ecological the
inferences that can be made from back-calculations are related to the
population (or part of the population) under Although accurate
inferences can be made about individual we are
pattern of the population as whole. In
these cases are essential in order to
ensure that we have obtained a random sub-set of the population or a
random stratified in mind that there may be
selective removal of members of the through natural mor-
tality and , the population characteristics at any age will
on the age of the fish used (0 assess these characteristics
average size at age 2).
It is common to observe that the average back-calculated size for
a age-group is smaller when calculated from older fish than from
younger fish 1975). This effect is termed Lee's
and it may be due to selective mortality, biased
with the or a combination of these explanations.
One essential element in proportionality methods is the of
constant proportionality in an individual fish. Several authors have
this and have concluded that there is varia-
bility within an individual over time in the proportionality between CS
size and somatic size 1 studies have also
shown (hat the of the CS can be "uncoupled" from somatic
for some time, there still exists a strong overall corre-
lation between CS size and somatic size et at., Secor &
1989). In the case of otoliths, these have been shown to continue
to grow even somatic growth has completely ceased during food
restriceion and starvation (Folkvord et at., 2000). One could argue that
even starved individuals would be less to survive in the
field, and that the problems under narural conditions is
less to occur than in the because only
healthy fish live enough in the field to be
Also seasonal deviations from a allometric
between cs and somatic growth may rum back-calculation into a non-
trivial matter. In Northern pike che instantaneous
rate ratios of both scale and cleithrtlm bone showed values above one
during summer and below one during winter low growth
(Casselman, 1990).
163
The relationship between fish size and the CS size is also under envi-
ronmental influence (e.g. temperature, Mosegaard et af., 1988), and
this adds uncertainty ro the results of the back-calculation procedure.
A temperature-dependent fish-size - CS-size relationship can generate
a bias in the back-calculated sizes if a common relationship is used,
and several examples of temperature-dependent relative orolith size
are available (fig. VA.5). In such cases the back-calculated fish sizes
will ro some extent reflect the ambient temperature hisrory of the fish
in addition to their previous si ze development. The problem of such a
temperature relationship may be less if the fish in the population have
been growing under similar ambienr temperature conditions.
3. Recommendations
Growth analysis may appear ro be a trivial problem, bur the proper

treatment of data is not always straightforward (Ricker, 1975; Kauf-
mann, 1981 ; Ftancis, 1995a). In the following, we offer a series of
recommendations that are relevant ro growth studies that involve the
lIse of calcified sttuctures (CS).
Figure VA 5 350r-----------------~------------------~----~
Stock- and temperature- __ CC la· C _____ ..... __•__ .. ,. _____ .._. ___ ..
dependent allometric 300 __ ccw c : ·· ·--·f· ···----
relations between fish length - 0- NA la· C :
and otolith (lapillus) radius 250 -0- NA 14· C .
in juvenile cod (Gadus
morhua). Offspring of cc 200
(Norwegian coastal cod)
and NA (Northeast Arctic
cod) have been reared
at constant temperatures
(Otterlei et al., submJ
100
50u-__________________ __________L -_ __ __ _J __ __ _
~
~
10 20 30 40 50
SL(mm)
The data structure of the incremenr widths in CS should be analysed

using multivariate statistical techniques (Chambers & Miller, 1995;
Ralsron & Howard, 1995; Jones, 2000). Both repeated measures
ANOVA and MANOVA (multivariate ANOVA) can take inro account the
inherent aurocorrelations, bur MANOVA is generally preferred due ro
164
the underlying data structure

Serial correlations of Increment widths
dealt with time series models
1995). The effect of unlvaflate
without for data dependence includes intlated
of freedom in the statistical tests, high
and thus an increased likelihood of a type I error.
attention should be to that combine
from the same otolith. This is
with microstructure where many increments are
counted and measured. In many instances this involves the combina-
tion of incremenc and thus
some of increments (0 achieve a common increment
1981). In cases where the senors read are of different
sizes, the use of relative increment widths to the total may
provide the basis for a common parcern of otolith
From a statistical point of of individual
otoliths would yield a
rical methods when the underlying

rical methods cannot be mer.
imervals of increment widths and check sizes
A proper strategy is also essential. A dear definition and
documemacion of the population units under i
required if we are to make valid between
rions or temporal within Different panerns
obtained from two samples coliened at different times and
may be due ei ther lO differences between
differences within a sub-population or
as better than all others in

no can therefore
be recommended as such. the user should be aware of any
underlying and limitations of the different back-calcula-
tion and should check the pattern of back-calcu-
lated sizes observed patterns of sizes at ages. Fran-
cis (1990) recommended the use of more tban one
and this would be useful for several reasons. First of
ity which model
best with the actual data at hand. the variability in the back-
calculated sizes using different methods would an indication of
the variability in back-calculated sizes from the choice of
165
An alternative means of working with longitudinal data obtained

from cs is co use the incremental data from che cs instead of
them into fish size (Anderson, 1 . Hare &
1995). Provided that (here is a high correlation between fish size and
size of [he CS, the incremental CS dam could be used as or
indices of fish size. The incremental dam can then still be used to
study (see
166
B. Ecological applications
H. Mosegaard, A. Folkvord, P.]. Wright
1. Recruitment studies
The development of age estimation techniques co determine age at the

annual level enabled Hjorr (1914) co put forward his ground-breaking
hypothesis of year-class fluctuations in fish scocks. The distribution of
age-classes in elt/pea harengtts revealed that one year-class, the 1904
year-class, was clearly more abundant at an early stage, and remained
the dominant year-class for nearly twO decades. The underlying reasons
for the abundance of this year-class were nor evident, although its size
was evident at an early age. The discovery of daily increments by Pan-
nella (1971), opened up new possibilities of studying the dynamics of
growth and survival within the first year of life in fish, thus enabling
researchers co answer the fundamental question in fisheries biology of
why there is variation in pre-recruit survival.
1.1. Documentation of selective mortality

One of the most powerful applications of individual age-structured
data from hard parts (for example, and particularly, otoliths) is the
combination of studies of growth and size-dependent mortality. Pre-
viously formed increment widths-at-age in an otolith (and also oroiith
size-at-age) will remain unchanged as the fish continues co grow over
time. If the temporal significance of these increments has been
established (see chap. IV), comparisons of increment widths at given
ages offish sampled at later stages will have the potential to reveal any
changes in the increment width pattern (or orolith size distribution)
among fish from the sampled population.
While many srudies have investigated growth and survival rate, few
have attempted to test a specific hypothesis. Anderson (1988) pro-
vided a general framework for considering the importance of feeding
success and predation co mortality; the growth mortality hypothesis.
Three mutually non-exclusive mechanisms have been proposed for the
functional basis of the growth mortality hypothesis.
The "bigger is better" mechanism: if mortality is a decreasing func-
tion of size, then the larger individuals at any given age will have a
lower probability of mortality than smaller individuals of the same
age (i.e. Leggett & Deblois, 1994)
167
rate" mechanism: if the probability of mortality is size-

growth rates will minimise the time over which
individuals high mortality rates and
larvae 980).
The mechanism: if mortal rates are size- and
srage, small percemage
in instamaneous rate can have n"",o"_At
quences for the number of survivors
stage if the later stage is to a lower rate. incli-
viduals that faster and make the transition at earlier ages
would have a lower of monality than individuals that
make the transition at greater ages & 1987;
1987)
It can be seen that mechanisms 1) and 2) are
a larger size at any age is
rate; mechanism 3) is also potentially related to the first two by the
extent to which development rate is correlated with growth rate and
size on an individual basis. These mechanisms may have a distinct
definition in terms of the tOtal or basis of selective mortality if
rhey are in terms of each other.
One approach would be to show the effect of one mechanism
on survival when the others are either held constant or
accoumed for. Verification of the is better" mechanism would
of individuals with equal
at stage transition. This is of course not
mechanism" since growth results in
the mechanism would demand indirect evidence
vival of initially smaller than of
growth, until a certain size was reached.
To the by Hare & Cowen (1997) has
these authors examined whether the distribution of

as a measure of with agc.
An increase in the of individuals with large mdii-at-incre-
ment with age was seen as evidence for
dent selection. Tbey and others
as a measure of growth,
increments with age was seen as evidence for positive direc-

rional selection. correlations
168
between otolith size and otolith growth rate prevent a resolution of

the of the is better" mechanism and the
have been made of
increment widths instead of several increments (Gallego et al.,
although such an to the technical limita-
tions of daily increments.
The presence of otolith structures associated with and larval
ILA.2) enables comparisons of the stage dura-
tion co be made. for selection on the basis of stage
duration is difficult from field sampling because there is no
cross-sectional sample of fish before to indicate size and age
for for with a cross-sectional sample of frsh that
survived co this
compare the distriburion of size and age at transition of
tally reared fish with field of survivors (Hare & 1997).
this assumes that there is no selection and no difference in
the studied trait in the reared group.
The of frsh with relatively small increment widths at a
age may decrease with time, an effect of
dent (i.e. higher loss of relatively smaller individuals in a
In tbe shown (fig. V.B.I), the mortality of the
smaller slower ind ividuals is shown to occur between
20 and 40. After day 40 none of the individuals with
narrower increment widths at day la was left in the The
timing of this mortality would be difficult if not
impossible to detect without of the recorded
available in the cs. An IS
with in which the initial conditions were poor
V.B.2). The ultimate survivors became on
average than the initial population at stage, and this
suggests that the high mortality initially observed in this mesocosm
was indeed the of fish with rel-
atively slow otolith
with time, the existence of
The interpretation of such will
of the same
the same
169
Sample day lO
Sample day 20
Sample day 30
Sample day 40
Sample day 50
Increment width day 10
Figure V.B.I - Schematic view of increment widths of a cohort of fish larvae (born on the same day) sampled jusl prior to midnight
on different days. The upper panel represents the distribution of outer increment widths on the day of sampling. whereas the
panels below represent the widths of the 10th. 20th. 30th and 40th increment from the edge respectively. thus corresponding
to increments formed on day 10 (assuming daily increment formation).
6,-------- -- - - -- -- - - - - - - -- -- - - - - - - - - - - - - - - - - - - ,
Mesa a Mesa b
Age of capture
5 - - - 20-29 -- - -
- - 40-49
- 6%7 - -
cQ)
4
3
-
E
~
<.>
E 2 ..... ......
......
O~~~_r~~~~~~~~~~~~~~~~~~~~
o 10 20 30 40 50 60 70
Age (days)
Figure V.B.2 - Increment width pattern (least-squares fit) of larval herring (Clupea harengus)
reared in two mesocosms (thick lines mesocosm a. thin lines mesocosm b. modified from
Folkvord. 1997). Larvae were sampled with tw(}Chambered nets during the experiment (dotted
lines. day 20-29. dashed lines day 40-49). and the survivors at the end of the experiment (solid
lines. day 65-67) were sampled by draining the mesocosm_
170
1.2. Bigger is better

of an analysis that employed the method described above
by Meekan & Fottier Larval, and setded
cod were sampled over a grid parrs of the
Scotian shelf in the Northwest Atlantic. On the basis of an annually
invariant linear correlation between radius and cod stan-
dard length (R-square:O.97) they used the
method & to back-calculate standard
at age. intervals of back-calculated
statistical
approach, they were able to demonstrate different survival patterns of
cod in two consecutive years. In one of the years the ptopor-
cod decreased during the winter, and
recruited to the stOck con-
sisted of fish that were at the stage. In
"rf>~PlntF'rl a correlation between ulti-
mate recruitment and of juvenile cod rhe
nile stage. These studies evidence that the ultimate survivors
were not a random of rhe initial of cod
and that in both cases there was a survival
relatively faster
1.2.1. Correlations of sizes at different ages and hierarchies

The maximisation of survival until recruitment has led ro the
ment of quite different early life hiStOry in differem phylo-
or groups. It is believed that lOvestmem in
the survival of eggs and has led to traits that
the size of individual The increment pat-
of further fitness-related life
transitions during
In a swdy on Frazer River ""~l<f'''P
orolith check formation to back-calculate distributions of individual
size at emergence in a of size-selective survival at later
stages. were able to show thar individuals with a owlith
size at emergence had a much probability of survival. A
of the same material Mosegaard (1990) showed that due
to a low correlation between fish size and otolith size at the end of the
yolk-sac stage selection for size could nor the
in the observed distribution of owlith sizes. With a low
size-selective has to very high shift in the disui-
bution of fry sizes to make a modest shift in otolith size
as demonstrated in VB.3.
171
Manual of fish scierochronology
Selective survival causing strong shifts in otolith size distribution may

be explained by a coupling of latge otolith size at emergence with
individual competitive performance during the establishment of
dominance hierarchies at first feeding as demonstrated experimentally
in both brown trout, Saf1llo trut/a (Titus & Mosegaard, 1991) and
Atlantic salmon, Safmo safar (Metcalfe et af., 1992). A physiological
explanation of the weak relationship between otolith size and fish size
a
600
y: 5.05x + 147
500 R2 : 0.20
o Original
cS 400
g!' Survivors
.!!! Survival probability
300 _ Linear (onginal)
~
E
0 200
----------------~~--------__
7
100%
100
0% survival probability
0
20 25 30 35 40 45 50 55 60 65 0
Fish length
b 12 %
10 %
r- Original mean 48.0
>-
u
c
Q)
=>
0-
~
u...
8%
6%
4%
r Survivors' mean 52.4
11 Survivors
o Onginal population
2%
0%
34 38 42 46 50 54 58
Fish length
c 12 %
>-
u
10 %
8%
rr Original mean 390
Survivors' mean 411
c
Q) Iil Survivors
=> 6%
0- o Original populahon
~ 4%
u...
2%
0%
240 280 320 360 400 440 480 520
Otolith length
Figure V.B.3 · Simulated data demonstrating the shift in otolith size distribution by selection for
large first·feeding salmon size (size scale is arbitrary).
a) Individual otolith length versus fish length. A random logistic survivorship Junction based on
fish length was employed in the simulation (red curve separately in the plot); survival: I if
1/(1 +EXP(l00-2' Lalev;n))> random number [0; I), where Lalev;n indicated normally distributed
alevin lengths, and otolith size a linear Junction of fish size with random normal error.
b) Fish length frequency distribution before (blue) and after selection (red).
c) Otolith length frequency distribution before (blue) and after selection (red).
With a low correlation between fish length and otolith size (R-square < 0.25) at first feeding, a
strong selection for fish size will produce only a minor shift in the otolith size distribution.
172
afrer yolk-sac resorprion would rely on rhe merabolic conrrol of orolirh

accretion rate (Mosegaard et al., 1988; Wrighr, 1991a) and the
increased energy cosrs reducing somaric growth in embryos with a
high turnover rate and a posr-emergent increase in growth porential
(Titus & Mosegaard, 1991). The coupling of dominance sratus and
otolirh accretion rate with resring metabolism has further been sup-
ported by work on masu salmon, Oncorhynchus mdSU (Yamamoto et aI.,
1998). Implicarions of individual characreristics at life history rransi-
tions for later survival have also been studied by orolith microsrruc-
rure analysis in orher sysrematic groups. Large harch check size has
been correlared with increased survival in cod (Gadus morhua) larvae in
rhe Balric (Gronkjaer & Schyrte, 1999).
The CS size-ar-age data ftom individual fish can be used to srudy size-
rank correlations wirhin a cohort. In an example from field-caughr
larval menhaden, rhe rank correlarion of otolirh size was shown to
decline from 100% to abour 80% after six days, and further to abour
60% afrer another 12 days of growth (fig. VBA). This shows that
rhere is considerable "crossing over" in size rank wirhin a short period
of time. This may reflecr medium-scale variations in environmental
condirions and food availabiliry experienced by cohort members or ir
may be due to generic differences in growrh capacity or ro social inter-
acrions relared to size-dependent hierarchies berween individuals in
the population (Imsland et aI., 1998).
2_ life history timing
Age-relared growth sequences in rhe calcified srrucrures of fish have

rhe porential co dare the occurrence of imporrant life history events,
when fish experience sudden changes in rhe environment and/or
undergo major developmental changes.
Figure V.B.4
Rank correlations based
on otolith radii sizes of
Atlantic menhaden larvae
(Brevoortia tyrannus).
1.0
0.9
+.
-0 '-40
,,>-
-- -t..._ -- ..
e-_
Correlations are calculated

for otolith sizes at ages 6 c
o
to 18 for subsequent ages .~
.... , .
6 to 21 at three-day inter- ~ 0.8
. '+
,
vals (data from Chambers
& Miller. 1995). "
u
oX
c
& 0.7 --0-- Age 6
--0 -- Age 9
. .+.. Age 12
--t.- - Age t5
0.6 - .. -- Age 18
0.5 L-~-------L-------L------~------~--------~
6 9 12 15 18 21
Age (days)
173
2.1. Birth date distributions

A key argument proposed to variability in early survival rares
concerns the between and hatching times and
favourable environmental conditions. Asynchrony between the tim-
of larval occurrence and favourable environmental conditions
would be to lead ro between the "hatch date"
composition of recruits and the actual temporal pattern of
hatching. Such a of hatch date is possible
through the analysis of otolith increments (Campana & Jones,
1992), Birth date can be derived the estimated duration
of embryonic development from hatch date, This
field measurements of temperature and an
between and temperarure.
a detailed account of hatch date
distribution of
hatch dates from at least twO successive of fish collected at
random from a The timing of the of harched
larvae would be determined means of sampling
throughout the The distribution of true
hatch dares would then be with the back-calculated hatch
dares of survivors of a age, as estimated from sam-
of the population, Statistical comparisons of hatch-date fre-
quency distributions involve similar to the analysis of
mortality, The main problem with hatch-date
analysis is rhat early-season larvae will have greater cumu-
lative morrality than those hatched lare in the season and therefore
will be in the back-calculated hatch date distribu-
tion, Thus, in order ro derive a true survivor hatch-date frequency dis-
rriburion, survivors should be coJJecred over a to
the duration of the period. In most studies have
to overcome the of differential. cumularive mor-
tality by the numbers at a given age by the inverse of the
survival rate berween the age at capture and the age of the youngest
fish in the This survival rate should be based on an indepen-
dent field estimare, As mortality tends to be related to age,
the need for differentiation of cumulative declines with age,
For this reason hatch-date is more suitable for later life his-
tOry stages such as which tend to have a lower rate
than larvae,
the birrh date technique it is possible to explore the firness con-
sequences of Most srudies of temperate
have found selection for late-hatched individuals between the time of
harching and larval (Crecco & Rice et at.,
174
Moksness & 1991; Wright & Bailey, However,

this form of selection may reflecr the stage of development under con-
sideration rather than life-time fitness in mOSt temperate
in
conditions for early tend co improve for

thus selection for late However, as early hatching
this trait may be
for an date of
events such as &
first maturity
tal stage in the of hatch-time effects on
of hatch date should ideally include samplings of a
development, as in the study by Schultz
2.1.1. Approaches to testing for size-dependent mortality and hatch date

Differences among actual and back-calculated hatch date and size dis-
tributions have usually been tested for by means of the
Smirnov test, which compares cumulative distributions.
However, several approaches have been developed to characterise how
the pre- and differ. Two are
below.
The of Schluter (1988) as modified
relative survival functions. This method uses cubic to estimate

the form of selection aning on quanritative trait. This
method makes no of the underlying fitness function,
assumes that the function is changing and allows calculation
of the confidence intervals. The non-linear form of selection is calcu-
lated using a "before-selection" group and an "after-selection" group
of otolith size measurements. A spline-based regres-
sion is fitted to the before and after selection dara,
log-likelihood methods and confidence limits
rhus allowing for between
This allows estimation of relative whereas the abso-
lure fitness function can only be found if population sizes or che rela-
tive sampling are known 1995). It should also
be noted chat otolith size is not che trair under selec-
tion bur is correlated with other traits of interest, e.g.
scope for growth or metabolic activity (West &
1991; Wright, 1991a).
175
Miller (1997) proposed an approach based on a resid ual analysis

In rhis approach an orolirh-radiw age
>tUBUle" is used to derive the "before-selec-
tion" sample using a best-fit function. This function is then fitted to

the otoli th-radius - age data of the "after-selection" group and the
residuals are deviation represents directional
and disruptional phenotypic selec-
pattern in the residuals.
trair. any assessment of selection to

characterise the prior of traits in the individuals that are
selected. This task is simple for traits such as hatch date, since
in the between collection rimes can only reflect selection.
However, traits such as which change independently
are far less easy ro because both size and rate can co-
vary. body size may be back-calculated from orolith size
there is no framework available ro account for the combined
effeCt In and although longitudinal approaches
based on the reconstruction of have been (see
Miller, 1997)
2.2. Ontogenetic transitions

Hatch and checks are often used for
of birth date disrribmions in species with small embryos and
short periods. with a pro-
stage, such as Salmonids, the formation of owlirh
mary increments this development separate estima-
tion of dates for and first to be made.
from the larval to the stage involves
a marked and transition. Most larval
sagitta and fapillus oroliths grow from a more or less spherical
appearance at rhe yolk-sac stage to a gradually more disc-formed and
somewhat at che late larval stage. The transition
to the juvenile stage is in some taxa by the appearance of
accessory growth centres in the sandeels (Wright,
plaice (Modin et at., 1996), while the accretion rate of these accessory
growth centre exceeds the concentric growth and thus
influences the shape of the otolith (chap. II.A and
III.B.l). Counting daily increments outwards from the cencre of the
otolith or in from the to the first accessory centre per-
mits the estimation of age or date, for (his life history
event. In a number of demersal also coincides
with (he transition from a to a bottom-dwelling habit. Where
176
the pelagic and bemhic habitats differ the

induced otolith growth transitions may be accompanied by environ-
mentally influenced in patterns of otolith
in their environment
are found among Salmonids,
many of which migrate from the freshwater environment
where they were born to the saltwater environment where they feed
umil they eventually return to their native rivers to breed. The tim-
of the downstream can be traced in the o(Olith, from the
marked that are reflected in its pattern or chemical
composition after the smolts have entered the sea. In chinook salmon
(Orluffhynchus juveniles display various migration strate-
and otolith microstructure characteristics to particular
freshwater and ocean habitats enables the differem life history
types to be identified and the of their migration to the ocean
studied In Baltic brown trout
from small freshwater streams to the
brackish estuarine environment was indicated by the early accumula-
tion of Sr/Ca ratios in the otoliths of some individuals et
earlier direct observational studies of O-group
from this area & 1991).
One of the first examples of is from American eel
otoliths where the chronology of upstream migration from the sea
could be traced Ca and Sr concentrations
using WDA \~''''''><:Ull''U
Seasonal differences in food and temperature may also be reflected in
the temporal record of otolith increments.
2.4. Spawning
Immature fish allocate their excess energy to somatic while as
reach maturity need to of their
net energy on gonadal
is usually noted in CS as a change in pattern.
LU.'''"","U otolith pattern are found in fishes from a wide range
of climatic zones: Areto-Norwegian Gadus mlJrhua (Rollefsen,
Mal/lJtus vil/oSUJ et al., 1986), plaice, Pieu-
ray/ectes L (Rijnsdorp & 1991), orange roughy,
Hopfostethus atfarlticus (Francis & Another CS charaeter
connected with is the zone in scales often found
in which is caused by hormonally induced in cal-
ci urn metabolism maturation (Persson et aL., 1998).
177
3. 1;!""m",lina
The differences in patterns of owlith can also be used w

study the effects of sampling avoidance and the time at which this
becomes a problem. It is often found in studies of larval and
fish that the fish eventually become ro avoid the nets
used ro catch younger larvae, If a non-biased of che
population can be obtained using a different gear, a of the
increment panern of fish
indicate the point in time at which avoidance
In this example, the larvae from mesocosm B were
up co 20 with che two-chambered net, but thereafter the
fish this net the smaller fish in che
popuiacion, with che survivors at the end of the
the benefits of the pattern of individual fish
are evidem in studies of this type, adherence to che made
the of the increments and the proper
sampling of the population remains essential.
Differences in stage- or behaviour may also bias estimates
of vital rates when using standard sampling from the fisheries, Juve-
nile and adult sandeels display behaviour chat influences
their ro the nocturnal and seasonal
of of this is the estimated
growth of lesser sandeel in the North
sen et at., 1 where a decline in
cowards the end of the growing season, In a
of sandeels were obtained out
viduals from the sedimenr in February/March by means of
Back-calculation analysis of the ocolich size distribution at the initia-
tion of the first translucent zone showed that different size fractions of
che wefe in differem
periods of [he fishing season. individual size to the width
of this first annual Stfucture the seasonal estimates
could be correcred for sampling bias & unpubL).
178
179
c. Demographic structures
in stock assessment models
B. Mesnil
Scientists dealing with the dynamics of many terrestrial or marine

ate often forced to take intO account that these popula-
rions are Charlesworth, 1994; Tuljapurkar &
1996). The main reason for this is that several parame-
ters and survival that determine how the size
or biomass of such populations change through time differ noticeably
among age groups. In the case of fish (and shellfish), much scientific
work is concerned with the dynamics of populations, i.e.
populations that are subject to borh and anthropogenic pro-
cesses, with a view to providing advice about the sustainable use of
resources. "Srock assessment" is the term used for such
As pointed out by Hilborn & Waiters the aim
of slIch swdies is not only to assess the state of stocks and fisheries
relative to historical states, reference points or management
targets, but also to evaluate the consequences, for both fish stocks and
fishermen, of alternative management scenarios.
This is not intended to a review of methods and

models used in s[Qck assessments. it attempts to poim our the
mmives that underlie the of some classes of age-struc-
tured models. In other words, the focus is on the "added value" of
for age structure in comparison with that
it. This is put in the context of other chapters of the manual by
calling attention to two aspects. which is treated in the discus-
sion of each is the extent to which to a more elaborate
class of model the in terms of age determina-
tions. The other aspect (section 2) refers to the processes that take
the ages of a of fish and the stage at
age of catches or of some associ-
ated are used to estimate the parameters of
models. Jr will be shown thar the improvements afforded more
detailed models have a COSt in terms of the quantity and quality of data
required, thus the recourse to the more efficient methods of
age structures in this book.
180
1. Age-structured models for stock assessments

and management advice
Most methods used for stock assessments involve a combination of a

population dynamics model with models that relate observations to
some attributes of the fish population (NRC, 1998). Fisheries scien-
tists are also concerned with evaluating the effects of different fishing
regimes on the future states of stocks and fisheries. For this reason,
they also need models that translate changes in the control variables
(catches and/or fishing effort), consistent wi th proposed management
options, into appropriate changes in the population dynamics. The
introduction of age structures into fisheries models follows from the
recognition that the distinct age groups that make up a population
(fig. V.C1) provide different contributions to biomass production,
reproduction or harvest through time or are not equally sensitive to
managem e nt measures. This is in contrast to the simpler biomass
dynamic or (surplus) production models (Schaefer, 1954) which
describe stock dynamics only in terms of overall biomass.
Years
Figure V.C.] Ages 1992 1993 ]994 1995 1996 1997 1998
Typical structure
of a population on an annual 0 NO.92
basis. Here, numbers at age I NI.93
are con sidered and one
cohort (year-{;Iass ]992) 2 N2 .94
is outlined diagonally. 3 N3.95
A similar structure applies
to other variables such 4 N4.96
as catches, biomasses, 5 N5,97
mean weights at age,
fecundities, natural
6+ N6+,98
and fishing mortalities.
1.1. Single species, single fishery

The core age-structured models from which the extensions presented
hereafter have evolved are the so-called analytic (or dynamic pool)
models associated with the pioneering works of Beverron & Holt
(1957) and Ricker (1958). These models treat the population as con-
sisting of individual cohorts (broods born in a given year or season)
and consider four basic processes at work in the evolution of popula-
tions through time: recruitment and growth, that increase the
biomass of the population, and natural morcality and mortality due to
fi shing, that decrease it. The parameters associated with these pro-
cesses may depend on age. This is obvious in the case of recruitment,
which only concerns the youngest age group entering the fishery each
year. Many stocks show considerable annual variations in the number
of recruits, and it is important to accounr for this in assessments.
IS]
Manual of fish 5clerochronology
Growth is obviously a function of age, whether we are with

the iscrete set of weights at age or with increments from one
co the next. The pattern of most fish is such
chat the growth rate is at early ages and slows down as fish grow
until some asymprotic value is reached. Growth is the
first process ID any of a fish sroc k and is the most
immediate application of age data VA), The reproductive
of a is also matter of great interest for biolo-
as it is an essential of criteria. In most
(a combination of the number and quality of
IS on the size and age of the fish. Natural mortality is
inherently difficult to and even more so its variations with
age. it is often treated as constant in many applications bur,
as we will see later with models, it is that the
model structure should allow for parameters.
values for natural mortality is often the most
critical issue in assessmems and Finally, because the
various age groups are not equally vulnerable [0 different types of fish-
is bound [0 vary with age for any
This is sometimes formulated as the
fishing monality at age a in year y is the
factor Sa
recruitment" in North
f."lctor fy which is related to the overall
and are also processes rh at may affeCt the
biomass of a popularion, wirh parameters usually, bur
all models considered hereafter assume closed populations.
In recent decades models have been used to advise

on TACs Allowable Catches) and catch quotas, which are the
prevai management wol in Norrh ArIantic fisheries, as in many
others. In this case, the models are used in simulation
due account of the structure at of the inirial popula-
tion and, if of the abundance of recruitments. In
some cases, changes in or fecundity at age are also
taken into account. Each cohort is traced individually time
and the catch and biomass in each year are as the sums of
contributions of each age group outcome of
these simulations is a rable of
patterns).
predicted carches to set a TAC that is consistent with conservation
needs and/or social and economic considerations. In
182
stocks, recruits ofcen conrribure significandy to the

pv"p,~rp'rt
catch, However, because recruitment is difficult
to even with surveys, for more than a
few years ahead, TAC been resuicced [0
The concern that this pro~
vides managers with insufficient to their decisions has
been addressed by broadening the scope of simularions in such a way
that the medium-term consequences of managemem can be
shown explicidy, This involves stochastic (in the broad
variants of the models, The purpose is also to
explicitly how uncertainties in the data (including age composition
estimates) and in the parameters of the models translate into uncer-
tainties and risks in the outcome of management decisions
Hilborn et at., Smith et at., Francis & 1
YEAR 1 YEAR 2
- ---- -
1 N R R
11 2 3
2 N N N
21 22 23
3 N N N
31 32 33 etc,
4 N N N
41 42 43
5+ N N N
51 52 53
+ +
+ +
V,C,2 Outline of an age-structured simulation,

~V~)UIO"'UII
number at start 01 years; recruitment R is the number at the age,
As simulation proceeds, Incoming recruitmenls must be input, either or as the product of internal computations
(stock-recruitment relationship),
B: stock biomass, sum of population numbers multiplied
C: catch in number calculated from supplied fishing at age consistent with Ihe simulated management scenario,
Y yield in weight, i.e, sum of catches in number multiplied by mean weights at age,
183
If we wish to pur these models ioro use, we need ro estimate their

parameters. Favoured methods for the parameters of ana-
lytic models belong ro the class of srock analysis (ASA)
techniques, in the sense proposed (1989), These
methods are also used ro reconsrrucr the hisrorical abundance and age
structure of fish srocks and to assess their current stare.
nr,(W"nF" an excellenr review of the numerous tools
over the years, which include various forms of Virtual or

Population Analysis (VPA or SPA) and of statistical
analysis. In essence, all these arrempt to estimate <JVIJU'''-
tion numbers at age and fishing morraJities, also by age, over rhe
years, the number of fish from each cohort that have been
in successive years. Of imeresr is the estimation of numbers
at age in the most recenr year, since this is the initial structUre from
which simulations are starred ro forecast rhe of the fish-
ery in the short term, which is the main concern of managers and
industry in TAC-based this is pre-
where most reliable esri-
mates for paSt states data are accurate
our the time bur estimates of stOck numbers and fishll1g
morralities at age are uncertain for the recenr past, unless additional
information can be used to redress this deficiency. In most of che
research deployed during the past decade around this methodology
has focused on implementing appropriate methods to
incorporate additional data in order to "tune" or "calibtate" the esti-
mat ion procedure, so thar the in the system (i.e. more
unknowns than there are for) can be reduced, In most
information consists of rime-series of abun-
dance age, obtained
derived from catches per unit effort fleets.
indices are all the mote useful in this conrext, as they pro-
vide information for individual cells in the
bur this implies yet more and age
back of most ASA rechniques, wirh implications in the comexr of age
dererminarion, is thar in the provision of
any disruption in the rime series creares almosr
insurmounrable unless very strong (but
are made.
1.2. Single species, multiple fleets

Ir is seldom the case that a given stock is fished by a single,
neous type of vessel as is assumed in the basic model above. Most fish-
eries ate by several types of vessel a of mobile or
set gears with different seleCtion (mesh or hook sizes)
184
Some uses individual age data
rhe year or seasons, and dif-

ferem componems of the s[()ck v. adults). These fleets
may also be to differem sets of Many fisheries con-
fliers arise because some fleets rarget young fish ar the expense of
orhers rhar on the availability of older due [Q their
gears or fishing fisheries).
In which fleets ar
balanced managemem may have
of rhe stock and for the sustainability of the whole
~"~""A with distinct exploitation patterns in differem segmems of

is that biomass dynamic models are
unable to do, for which reason analytic models are needed. To be
models are more suitable than models for
managers and fishermen are mainly concerned

simulations are nO[ complex to set up if the initial popu-
and/or factors
in nominal effon in the different fleets. It should be
cedure is used ro treat
To the extem that time-series of catches at age are available for all
the
model. This
the total fishing mo(talities at age. If it is assumed that
any age group is equally available to all then the
mortalities are derived on tbe basis of the ratio of the catch of
each fleet to the total catch at each age in each year. In there
is no extra demand on for ages.
1.3. Multiple species, technical interactions

An obvious weakness of the still
to assessment and management, and one reason they have been
is that fail to account for the fact that no in any
part of the sea can be as bei ng or isolated from
Others. any management decision that is guided one
is bound to have an impact on associated to some
extent, and may nO{ be for these or for tbe aggregate. We
will the usual distinction between alternative
185
which put the emphasis either on biological interaCtions

or on technical technological) imeractions, in mind that
both kinds occur simultaneously in many fisheries. Because the latter
are based on rather extensions of the above model,
will be dealt with first.
Technical interactions arise whenever the distributions of different

so that any aimed at some desired
imentionally or nOt, in catches of sympattic
each amount of effort deployed in an
area generates fishing A, but
also on the incidental (or B, ere. the dis-
rribution of fishing morralities across ages may be quite different for
the various concerned: the may be adults of A
together with les of 8, or vice versa. Thus, a fishing strategy or a
managemem measure appropriate for A can have unwanted
side effects on the dynamics of the associated
when rhe latter are less productive. If we wish to evaluate whether
some strategy or regulation is consistent with management
for the whole that is for all it is important that the
should enable us to model the of all rele-
vanr
reasons as in the case, Most
technical inreractions use so-called mixed-fisheries models (Mahon,
in which the fishing fleets are subdivided into gtoUpS or
meners" eta/., 1991) that have consistenr towards
easy to
involve simlllat-
the connections among
handled through the mortality parameters: a
in effort of each fleet is assumed ro result in the same propor-
on all and ages caught by that
the initial population at age and the reference
patterns fleet are estimated separate
VPAs, i.e. the same as for the models. There
may be an additional demand for and age data for some
that are not parr of the schemes because their
low scale does not j llstify the
cosr, bur that contribute a proportion of the income of some
local fleets that would be affected the proposed regulation. If the age
data are not available or are toO to obtain, that
treat some with models and others with biomass
models are a ai, 1991).
186
The apparenr of technical interaction models hides at least

two difficult issues. One is related to discarding. In many instances,
the most critical interactions among (or their due to fish-
take at life stages that are eventually discarded some
fleets, If the fishing parameters are estimated on the basis of
landing data alone, the of the system and the assessmem of
the need for or effects of can be seriously biased,
including discards in such assessments is and since
fishermen are usually reluctant (0 report this often entails
increases in the costs of data colLeCtion, to
there should be a marginal rise in tbe esti-
of all the sizes that are never landed), The other issue
the response of fishermen to in abun-
Most assume that each vessel will
maintain its current strategy, i.e. that it has a fixed matrix of catcha-
bility coefficients across and ages. In we can expecc
fishermen (0 adjust their fishing when the relative abun-
dance or market values of or when new
alter costS, the behavioural, eco-
nomic or social determinants of fishermen's choices in order (0 model
and them in assessments is a challenging endeavour (Alien &
McGlade,l
1.4. Multiple species, biological interactions

There are several ways in which interact within
ecosystems, the most obvious,
being a major element in the natural of the prey
Also, the abundance and of prey influence predator growth. In
addition, there may be for food or habitats.
It is clear that considerations of size and age structures do matter in
interactions, Whether tbey are or prey, not all
individuals in a are Predation
occurs mostly on young stages, and the threat decreases as size and age
increase. Fish of the same cohort can be prey when young and turn
when older in cases of cannibalism). Predators often
switch from one set of prey to another as grow, The
effeCts of depend on the abundance and vary as
weak Of s([ong of predator pass through the sys-
rem, This is the models mentioned below have been constructed
from tbe onset in used
refers (0 a age of a given
, To the extent that most fishery
only consider and prey
for which age data are collected for assess-
mems in any case, there is nor an extra cost associated with
187
the form. The pnce

imeractions models (Hilborn & WaIters, 1992) has to do with
the collection and of stomach data.
As memioned fOf natural mOf-

of historical states more
rhe implications of management can be dif-
fecent with to their medium- and
the of
context, and within the International Council for the

of the Sea (ICES) community (Daan & 1991).
In some DU'""....." Ilm~rp'''pr the effects of variations in the abundance of
prey on the and reproduction of are also considered
Sretimsson & 1998)
The technicalities of biological imeraction models are wo ro

be explained here. Interested readers may refer w the overviews
(1995) or (1990. estimating the frac-
tion of natural rate M at each age due ro those
included in the involves coefficients thac are "mea-
sures of what the likes to ear as well as whar it is able to eat"
1995). In the ICES area, the method most often
the size
a process that must rake into account not
bur also what has been eaten, as reflected
in any year, the pre-
darion mortality on on the abundance of all
must be solved simulra-
here is rhar the
relative weighted
the suirabi must match the com rion observed in the
sromachs of predators Since these suitabilities include a
component of of the distributions of and
prey, which depends on seasons, computarions are actually made on a
basis. This stomachs for all and
ages size classes) of all by quarrer, over the entire area,
and them to derive the and age (or
of their prey" In the 1981 ICES stomachs were col-
lected 1987) even if these were pooled to
formidable. This is why only two
such schemes have been feasible for the North
Sea ar an i merval of (en years. numbers of otoliths (by
188
Some uses of IndiwJual age data
area and also had to be processed that many

of these were also required to derive the survey indices of abundance
by age, which are a routine product),
1.5. Spatial models

All tbe models discussed so far assume that fisb and/or effort
are distributed over the area, but there is a
awareness that spacial structures, for both fish and fishi ng
sbould be built into fisheries models, This is an
issue as there is a strong (although uncritical at times) movement to
revive management tool
Guenene et at., 1998 for a "single-bucket"
models are not rhe effects of such
measures,
Although a variety models has been proposed over rbe years,

many of them depend on rbe availability of particular data tag-
surveys, logbooks) and has still not been
There is little doubt, nOWt:veT.
models for management purposes is enhanced when they take age
structures into account. In many tbe various age gtoupS have
distiner spatial distributions nursery
and their patterns of movements also differ
or spawning The cOlrreSD()n(jlO parameters
in models are thus likely to be of a pro-
teered area also needs to consider such
ages are concerned (location, season, size of the
stay in the area (its size is relevant), how fast
ere. structures are therefore eo
take ineo consideration.
2. From estimation to models
2. L Estimating the age composition of catches

Whatever kind of model is its to real
that its parameters be est i mated from data. As
out above, the parameters of most
The in pur is a matrix of

the total catches taken at each age in each year tbe fishery. Most
variants in current use also try ro take of auxiliary informa-
tion such as abundance indices at age obtained during research cruises
or derived from catch races at age from seiened fleets. These data are
just cases of catches at age.
189
Two are utilised to estimate the age of

catches. One is co sample fish randomly at landing estimate the
age of all and apply the of the various ages
observed in the to the cotal number (simple
random sampling). The alternative is double sampling: in the first
stage, fish are randomly to record their while in the
second, a of the fish measured is taken and the age of those
fish is determined. Sub-samples for age are usually stratified by size
classes (a fixed or proportional number is taken at each size) and this
leads to the (ALK; but in a different sense from Hilborn
& Waiters (1992) who call it the The ALK is a
usually with ages in columns and lengths in rows, which the
probability of a fish of of age i that it is of length j (fig.
they have been turned into the ceUs in the
ALK must sum ro for each length class (across each row). When
the vector of numbers at length is multiplied by the ALK
matrix, a vector of numbers at age is obtained. Either the
(prior to the or the age composi-
tion (after ALK) can be raised to the overall catch. Variance formulae
for the alternative with considerations
the effects of and about the treatment of
errors in stock assessments, are VIII of
& Deriso (1999). On statistical
may be equivocal
when costs are taken into account, the
turn in favour of double as a
for age estimation is far more
1993). This is why
used.
There is a recurrent issue about the ALK that needs to be well under-
stood. Whereas the distribution of lengths within ages is
determined by the pattern, which may not
from year to year, the distribution
ALK) is also influenced
ALK established for one year should not be applied to the com-
for another year. As illustrated in the case study below, it can
shown that this leads to serious misallocation of ages, and
thus to errors in assessments. More precisely, as has been
stated Kimura (1977) and other experts, the used to build
an ALK must be drawn from the same population as the one to which
it is applied. This is why some research institutes rightly
separate schemes for age (and length) for different areas and
190
seasons for any given stock in a given year, on the grounds that the
structure of the underlying populations do differ. At the very least,
ALKs should be built anew each year. Note, however, that this condi-
tion would also apply to the alternarive simple random sampling
strategy.
2.2. Case study: age-length keys in practice

The main purpose of this example is to demonstrate, on the basis of
real data (Celtic Sea whiting, Merlangitts merlanglls, data kindly pro-
vided by R. Bellail , Ifremer, Lorient, France) , the consequences of
applying an inappropriate age-Iengrh key to length compositions.
Table V.C1 shows the age-length key (ALK in number of fish aged at
each length) and, in the second column, the length composition of
the catches of a particular fleet segment for 1992. The age composi-
tion of these catches obtained by applying the ALK, using the proce-
dure explained in figure V.C.3, is shown in the bottom row. This
example shows data pooled over the year whereas , in reaJity, the treat-
ment is made on quarterly ALKs and length data, bur this is immate-
rial for the purpose of this illustration. Using annual data may result
in misallocation to ages for those age groups (typically the younger
ones) that grow fast within rhe year, and some of this effect is appar-
ent here: if caught early in the year, fish in the range 30-36 cm would
all have been of ages 2 or 3, but rapidly growing or early-born age 1
fish also reach those sizes late in the year, and their presence is
reflected in the age samples collected in the last quarter. When sam-
ples are simply pooled (without being weighted by the numbers
caught in each season) to set up an annual ALK, the proportions of
ages within length classes observed by the end of the year influence
Age Age
Length 1 2 3 ete. Length 2 3 ere.
10 6 10 100
11 8 2 11 80 20
12 7 7 12 50 50
13 3 9 13 25 75
14 6 2 14 75 25
15 9 15 10 90
ere. ere.
Figure V.C.3· Format of an age·length key (abbreviated).

Left: tally of numbers of otoliths or scales assigned to ages in processed samples;
Right: data transformed into proportions (percentages) within length classes.
For example, the catch in number at age 1 is computed as the sum of all fish caught at length 10, plus 80% of those at length
11, 50% of those at length 12 and 25% of those at length 13.
191
the allocation for the whole year, resulting in a degree of bias in the
estimated age composition. The bias can be large when, as is the case
here, the incoming year class is strong. The spread of lengths at each
age and the overlap between adjacenr ages are also amplified when
annual data are used.
These problems can largely be avoided by employing the whole pro-
cedure on a seasonal basis (e.g. using quarrerly length compositions
and ALKs), which is a reason why this is usually done despite the addi-
tional cose. Another drawback of using seasonal ALKs is that it may be
more difficult co obtain sufficienr age samples for all sizes. Table V.Cl
indicates that large and old fish have been rather difficult ro
encounrer, even for the whole year, al though these fish are those for
which the variance is largest, a facror that calls for larger samples in
order co improve precision. However, it should be noted that in such
a heavily fished scock, old fish make a very small conrriburion co the
catch and co the srock, so that knowing their size precisely is not the
main concern. Finally, it shoul.d be noted that about 1,700 fish were
aged. The proporrional sub-sampling scheme used may have resulted
in some sizes and ages being much more sampled than others, bur this
scheme has bener overall statistical properties than a fixed allocation
scheme. This number is quite considerable, bearing in mind that it
was for only one of several srocks that have to be monitored routinely
by the scienrific team involved.
Table V.C2 shows the same items based on sampling in 199 3 , to
which the considerations discussed above also apply. Comparing this
with the previous table, it is inreresting ro see how the pattern
between ages 1 and 2 in the ALK has dramatically changed as the
strong 1991 year class proceeded from age 1 ro age 2. The probability
of drawing an age 1 fish in the " normal" length range for this age was
much lower than in 1992, and this also applies co individual lengths.
Within length class 30, for example, the proportions of ages 1 and 2
were abollt 6% and 80% respeCtively in 1993, comparecl to 18% and
53 % in 1992. This illustrates the point that the structure of the ALK
is nor only influenced by the growth pattern, but also by the relative
abundance of the various ages in rhe population from which it is sam-
pled. Therefore, it is bound co change from year to year as strong or
poor year classes pass through the population, or from one area to
another if the age groups have c1ifferent spatial distributions.
192
Table V.C.l - Length composition, age-length key and resulting age composition for 1992.
Length composition Age-leng th key
1 (c m) Number Age I 2 3 4 5 6 7+ Total
caught '000 aged
24 16.4 5 0 0 0 0 0 0 5
25 75 7 7 I 0 0 0 0 0 8
26 296 0 \3 6 0 0 () 0 0 19
27 6 22 .5 22 \5 \ 0 0 0 0 38
28 91}.4 19 29 5 () 0 0 0 53
29 \ 227. 5 14 45 7 0 0 0 0 66
30 14195 14 42 22 I 0 0 0 79
31 135 6 8 \1 47 12 4 0 0 0 74
32 1298.1 8 56 13 0 I 0 0 78
3.) 1241.2 6 52 20 2 3 0 0 R:;
34 90 5 1 2 45 27 3 5 2 0 84
35 7 17.6 2 43 41 5 R 2 0 101
36 5256 I 47 28 5 7 2 1 91
37 42 3. \ 0 31 41 6 5 0 0 83
38 3220 0 30 30 8 5 I 0 74
39 2 \5 9 0 20 26 3 8 2 0 59
40 164.6 0 18 25 9 5 6 0 63
41 134 3 0 \2 33 7 13 2 0 67
42 122 .7 0 14 38 14 10 3 I 80
43 94 .5 0 12 37 8 7 3 I 71
44 69 1 0 R 27 9 13 R 1 66
45 56.8 0 7 24 I1 11 2 0 55
46 40.2 0 2 23 9 \4 3 0 51
47 3 2.8 0 0 14 8 23 4 1 50
48 24.5 0 0 II 7 9 8 2 37
49 199 0 I 1\ 12 9 I 1 35
50 14.6 0 0 J 4 12 4 0 27
51 9.0 0 0 4 2 5 7 0 18
52 97 0 0 5 0 13 4 0 22
53 9. 7 0 0 2 3 10 2 0 17
54 1\ .4 0 0 2 3 11 3 I 20
55 6. 1 0 0 1 I 7 4 I 14
56 4.1 0 0 0 0 4 6 0 10
57 5.4 0 0 I 1 3 3 1 9
58 2.6 0 0 0 2 \ 2 2 7
59 3.2 0 0 () 0 4 2 1 7
60 2.2 0 0 0 0 3 2 1 6
61 1.3 0 0 0 0 0 4 0 4
62 1.8 0 0 0 0 2 1 1 4
63 0.0 0 0 0 0 0 0 \ 1
64 + 0 .4 0 0 0 0 1 1 0 2
Toral 12477 .4 1738
Age composition
1972.0 65980 2892.0 4 169 45 2.1l 129.3 15 .9
193
Manual of fi sh sclerochronology
Table V.C.2 - Length composition , age-length key and resulting age composition for 1993.
Length composition Age-length key
L (cm) Number Ag e 1 2 ;) 4 5 6 7+ Toral
caug hr '000 aged
24 40 0 I 0 0 0 0 0 I
25 17.0 I 2 U U 0 () 0 ;)
26 75 1 0 7 U 0 U 0 () 7
27 178.6 2 12 I 0 U 0 () 15
28 379.6 4 22 0 0 0 0 0 26
29 751.7 8 42 4 0 0 U 0 54
;)() 84 7.3 3 43 13 0 0 0 0 54
31 995.5 4 42 15 0 0 0 0 61
32 97 0.6 I 33 31 I U 0 0 66
.B 1089.4 2 47 28 2 0 0 0 79
.34 886. 2 0 40 33 2 U 0 0 75
35 788. 0 0 41 50 4 0 I 0 96
36 622.9 0 44 49 6 0 0 0 99
2, 7 498.8 0 39 'is 17 2 0 I 114
38 3483 0 32 58 14 I I 0 106
39 27 1.1 0 24 5~ 9 3 0 0 94
40 2236 0 15 60 12 5 0 () 92
41 163.5 0 I1 51 14 I 2 I 80
42 LO , .1 0 6 43 12 1 () 1 6.)
43 94.4 () .3 44 10 4 1 I 63
,,4 86.9 Cl 2 37 10 3 I 1 54
15 78.7 0 I 43 L.? I 2 0 60
46 56.8 0 () 19 14 5 4 2 44
47 39.6 0 0 18 15 1 I 2 37
48 36.1 0 0 9 12 1 7 0 29
49 28.5 0 0 9 I) 0 3 2 27
50 32.9 0 0 7 9 I 8 5 30
51 19.0 0 0 2 8 2 4 5 21
52 23.0 0 0 3 10 3 5 2 23
53 15.8 0 0 3 4 2 4 1 14
54 20 .6 0 0 2 4 :) 7 }- 19
55 16.4 0 0 I 3 2 5 2 13
56 14.4 0 0 () I 5 5 2 13
57 95 0 () 0 2 2 5 I 10
:58 8.0 0 0 0 2 I 2 3 8
59 9.1 0 0 0 0 2 6 2 10
60 6.6 0 0 0 0 2 2 5 9
61 4 .8 0 0 0 0 1 0 5 6
62 4.8 0 0 0 0 0 0 6 6
63 0.6 0 0 0 0 0 0 I I
64 + 0.6 0 0 0 0 0 0 I I
Total 9825.2 1683
Age composition
3539 5 142.0 35 7 58 5166 839 94.4 585
194
Table V.C. 3 firsr summarizes the age co mpositions obtained when the
length compositions are processed correc rly, with th e ALK sampled in
the same year. Although the examina ti o n of ca rc hes alone is nor suffi-
cient to judge rh e rrue abundance (i r would need to be esrimared by
VPA based on tOral internarional carches rh rough rime), and bearing
in mind rbar age 1 had nor fully recruired ro rhe fi sher y, th e re are indi-
ca rions rh a r rh e 1991 year class was relarively srrong in borh years.
Th e posirion is s imilar for rh e 1987 year class (aged 5 in 1992 and 6
in 1993) which makes a large r conuiburion rhan rbe ad jacent ones.
Table V.C .3 - Age compositions for 1992 and 1993 derived using ALKs
sampled in these years.
Age Length 92 Length 93 Length 93
ALK 92 ALK 93 ALK 92
1 1972.6 353.9 989.2
2 6598.0 5142.0 498 55
3 2R92 .0 3575.8 2()()5 .0
41()9 51().6 4395
5 45 2.8 839 5437
6 1293 944 178.2
7· 15.9 58 .5 24.1
Final co lumn: age composition estimated for 1993 based on the ALK set·up with 1992 samples.
The parrern is consisrent and rec ent assessments confirm rbar rhese
were among rh e good yea r classes in rhe decade. But, if we make rhe
mi srake of applying rhe 1992 ALK to rhe 1993 lengrh com posirion
(Iasr column ), rhis consisrency breaks down. Firsr, rh e contribution of
rh e 1992 year class to rhe carch ar age 1 is inflared by a fa c ror of about
rhree, a r rhe expense of an underes rimari o n of rhe ca rches m ade ar ages
2 and 3. There also seems to be a larg e contriburion of ag e 5, i.e. rh e
1988 year class rarher rhan rhe srrong 1987 year class, which is ar odds
wirh available evidence. In facr , rhi s year class was rh e poores r in rh e
rim e series. If such erroneous ca rch composirions had bee n in p ur into
any kind of carch-ar-age a nalysis , rh ey would have serious ly di stOrted
rhe es rimarions o f s to c k a bundance and fishing mortaliry fo r a ll
cohorts . The particular misrake shown in rhis example (l arg e ca rc hes
sp read over adjacent ages) would like ly resulr in underes rimarion of
fi shing mortaliry for rhe su o ng cohorr, but no general rule is applica-
ble : rhe effects of ag ing errors upon assessment res ulrs would d epe nd
on rhe proximiry and size of rhe age groups rh a r are affecred by rhe
m isallocarion. Aging errors are one source of uncerrainty and should be
handled tOgerher wirh or her sources, using appropriare merhodolog y,
to evaluare rhe overall quality of assessments.
195
3. Summary and critique
If all individuals in a scock were equivalent in terms of viral rates

(growth, fecundity, natural and fishin g morraJjty) or even abundance,
there would be no need co suffet the pain of lIsing relatively complex
age-based model s with the associated costs of data acqui sition . Unfor-
tunately, thi s is nOt the case, and fisheries biologists are often com-
pe lled to consid er age struCtures in assessments. Oth er vi ews ha ve
been expressed, however. In contexts where manage ment advice is the
only perspeccive of the assessm ents, models that depict reality accu-
rately may eventually be less useful th an models that support sound
fisheries manag ement over the years. In particular, the latter should be
tobust to uncerrainti es of all sorts and it is a well -known rule thar the
more derailed a model is, the more parameters it has , and the more it
is sensitive to uncertainties. In some instances, simpler surplus-pro-
duccion mod els have proven co perform better than age-strucrured
models, when age data are imprec ise . However, this supetiority has
usually bee n established in contexts in which management is by TACs
that are defined simply in te rms of wholesale weight of fi sh. Th e con-
clusi o n may have to be revised when other management measures
(mesh regulations, closed areas, ete.) are considered, or when the fi sh-
eries are hig hly mixed . In the light of current public concern about
ecosys tems and biodiversity, we may expect that advi ce based on
assessments in which species are lumped cogether will increasingly be
regard ed as unacceptable if thi s impli es ignoring the fate of some frag-
ile species or stock components. A drawback of simple model s is al so
that they cannot make lIse of extra information or kn owledge which
may be available.
Rather tban opposing models that take full accollnt of age structures to
those that ignore them alcoge thet, it may be more construccive to con-
sider the potential of models of intermediate complexity (e .g . Conser,
1994; Jacobson et at. , 1994). Basica lly, these consider age structure
only for those ages that exhibit hig h dynami cs in their parameters , and
lump all others into one compartment. Genetally spea king , the former
are the younger ages and the operational advantage is that they are also
those for whi ch age es tim ation is eas ier and cheaper. An extreme
option, which may be justified when biomass production chang es pre-
dominantly due co vatiations in recruitment, is co expli c itly model
only tWO stages in the population, the recruits and the recruited ages.
These are fairly easy ro identify, even fro m length compositi ons, par-
ticularly when the latter are record ed on a seasonal basis. M ore age
groups in the juvenile phase can be differentiated if thi s is d eemed ro
196
be to the problem at hand. In any case, having a

model does not help to management when enf()fCement
is deficient.
Finally, the major models used for stock

assessments is not their '-''''H~'''-.". se, which is modest
to that of models used in other The main obstacle is the
cost of and the data needed to estimate their
the need to operate on routine as
lS managers. The treatment of material for age estimation
often makes up a part of that cost, given the amOllnt of data that
have (Q be handled, the skill and and the
standards to be mer. In as emphasised in senion 2, the whole
task has (0 be redone from scratch every year. Given that relatively
need to be taken for age estimation in order to
U«;U,,'V,;l. and that many and stocks have to
the amount of material orolirhs, ete.)
each year may be This calls for
very efficiem handling that should be in the form
of a line (Morison et al., Of course, quality should
also be a major concern, rigorously validated procedures for
age estimation IV). Furthermore, in the comext of assessments
which the maintenance of time time
is a critical element of quality and should be
Such mean that there is to be paid if

we wish ro reap the benefits of even in their
forms. The mere to conrinue using such models, even
in their basic form for TAC depends critically on the
ment of more efficient and automated
COSts while the precision and
tions. This is all the more necessary if clients of advice conrinue to ask
for marc details, more (whether commercial or nm), more
more areas, etc., to be included in assessments, while staff and
allocated to scientific institutes dwindle.
197
198
Computer·3ssisted age estimation
Ch VI
Computer-assisted
a e estimation
A. Benzinou
199
Manual of fish sclerochrono!ogy
200
Computer-assisted age estimation
pro-
cess of zones that associates visual
mechanisms and biological Faced with
sources of bias and the of
have sought to introduce a certain
basic data acquisirion process, which is
of their The appearance in the sixties
coupled co micco-densitometric systems inaugurated a relatively
sparse series of smdies that various methods
The fitst aim of this
this process of a computer.
It is essential to be aware from the very that the human
"machine" excels in pattern that the results of
anempts to automate the pattern process very often fell
well below the hopes raised by pattern Their
application to the calcified structure process did not deviate from this
tendency and the first attempts at automation soon tan up the
complexity the acmally find it difficult to do
whar a young child is
and growth esrimation
based on
mathematical concepts of
is to offer rhe reader a bener
and to the effeCts of their
materiaL
Tbe wide range of CA AGE
laboratories is tbe refleCtion at the same time:
of the of calcified strucmres of
modulation of the signal, etc.);
-the of tbe situations involved (macrosttLlCtures, microstruc-
(visual comfort, automation or

measurement capture or
the technical solutions available
gence, neural networks, 2D or 3D
Our tbird objective is tbus to help tbe reader to make the choice of a
tool suitable for his needs_
A. What is a computer-assisted age

and growth estimation (CAAGE) system?
An ideal CAAGE sys rem would co nsisr of a corn purer providing sofr-
wa re ancl hardware ca pable of ensuring (1) assi srance in rh e gua nrifi-
carion of cs images, (2) assisran ce in rh e d am inrerprerarion process
and (3) efficienr manage menr of clam s rorag e a nd exchange.
Among rhe CAAGE sys rems currenrly available, howeve r, we pri marily
find sysrems based on a n image analysis sofrwa re co re, which rhus
mainly offers help ro rhe dara guanrificarion srage. Some of rh ese sys-
rem s provide speciali sed funcri o ns (back-calcularion, curved profiles ,
mem orising o f rhe growrh zone locarions , in[[odu crion of biolog ical
consrrainrs , erc.) bur pracrically none of rhem offer any real assisrance ro
inrerprerarion, alrhough occasi o nal arremprs ro do so have been made.
For rhi s reason , we will concenrrare here on vi sual dara processing. A
CAAG E sys rem consisrs essenrially of rhree unirs (fig. VI. 1):
- a digiral image source (ca m e ra, scanner, digirising rabler, SEM,
microprobe, e re.);
- a cenrral processing lInir for image di splay and srorage ;
- a sofrware uni r rbar characreri ses rhe level of ass isrance offered by rb e
CAAGE sysrem by providing spec iali sed funcrionaliry for rh e id enrifi-
car ion of basic fea rures, morpho m erry and possi bly, i nrerprerarion .
1. Visual data processing
The rapid evolurion of numeri cal rec hnol og ies makes ir exrremely dif-
ficulr ro d escribe rh e compos irion of an imag e an al ysi s s ysre m in
derail. Only a few years ago we could have praised rh e performanc e
offered by speciali sed im age-process ing boards rhar offered fram e-
memory and real-rime fun c rions (array processor, morphologi ca l pro-
cessors, e rc.) bur rhe increase in clock frequ e ncies a nd srorage capaci-
ries of mi c rocompurers has made rhese almosr obsolere roday. For rhi s
reason , we focu s our presenrarion on whar we regard as rhe funda-
menral componenrs of an image analysis sysrem.
1.1. The digital image source

1.1.1. Digital image formation: 2D sampling
Srarring from a simple represe nrarion of visible oprica l phenomena,
rhe image gradually rurned inro rhe visllalisarion of radiarion from rhe
invisible parr of rhe spec rrum (infra-red, UV) and rhen of all kinds of
measured physical guaoriries (radar, scanning elecrron mi c roscop y,
romography, microchemical analysis, e re.). The rerm "digiral"' refers
202
I Frame grabber ~--------,
Scanning electron
microscope
Digital image
sources
Computer unit Control monitor
Software unit
Knowledge based
system
Scientific image ~
processing package ---'
Oigi-Pad
e
Graphic and photo 'S\a"C
retouching package ~ de{ aSS'
~ . ,,{ea
s
\"c._ea '"''
Figure VI.! - Composition of a typical CAAGE (Computer-assisted age and growth estimation) system. The digital image sources
range from a classical image camera to a chemical image produced by microprobe s (e.g . in Casselman, 1983). The steady
improvements in computer power and storage capacity enables us to handle larger images and utilise more sophisticated algo-
rithms. The type of software unit will determine the degree of assistance provided to the reader by systems ranging from a sim-
ple interactive ring location to a fully automated interpretation process.
to the discrete nature of the information that constitutes the image as

opposed to the continuous nature of analog signals. The real world
being three-dimensional and dynamic, the image formed will obviously
be no more than an instantaneous view of a projection of the scene as
registered on the surface of the sensor.
The sensor determines the namre of the information represented by
the image _ For light information, the sensor is a device that is sensi-
tive to light energy and that transforms the optical image into an elec-
tronic signal. The image sensors may be an analog video camera tube,
but most modern cameras are based on solid-state semiconductors
arrays known as "Charge-coupled devices" (CCDs). CCD sensor ele-
ments are photodiodes of a few microns in diameter that react to pho-
tonic excitation by producing electron charges that are collected
throughout the array and converted to an output voltage_ This collec-
tion process produces, an analog video signal for each row of CCD ele-
ments, implying that (1) the signal still needs to be sampled even
when using a digital sensor, and (2) tbe horizontal resolution of the
203
digital image does nor depend direcrly upon the number of CCD
columns.
The video signal sampling is performed in three srages by an analog-
ro-digital convener (ADC) (fig. VI.2):
• amplificarion, which modulares the srrength of rhe analog signal in
order ro obtain suitable levels at the inpur of the analog-ro-digital con-
verrer device. Ar this stage, mosr digiti sers allow rhe gain and offset
of rhe signal ro be modulated;
• sampling, which rransforms the continuous analog video signal,
coming from the camera, inro a succession of discrete values. In this
srage, the sampling frequency determines the spatial resolurion of [he
Image;
• quanrificarion, which rounds off the analog samples ro digital va l-
ues so rhar they can be coded inro a final number of bits. This deter-
mines rhe brighrness resolurion. Coding these measuremenrs at a res-
olution of 8 bytes provides a range of 256 grey levels. The fact thar the
Brightness
Gain & offset
resolution
~~1 Computer
RAM or Frame
Memory
Columns
9 !:6 1/68 171 95 '"

1~ 4 l t2
2 B!) ii 3~ 70 7,) ~, 36
13 B7 27 3 ,,!os:..72 57
1216 15 fIllO 71
<
h '"
J67 1 18 84 ... ........
DAC
Digital-ID-analog converter ~
~ (VGA, SVGA .. .,
Display
Digital Image
Dstorage
Video SCreen
Image File
(TIFF, JPEG __ .)
Figure VI. 2 - Schematic presentation of an image digitalisation process. The electric signal generated by the sensor is amplified,
sampled and Quantified by the digitiser. These digitalisation steps will determine the dynamic, spatial and brightness resolution
properties of the resultant digital image. The image is temporarily stored in the computer or the imaging board RAM memory,
where it can be displayed on a video screen or written to hard disk or CD-ROM for long-term storage.
204
Computer·assisted age estimation
human eye canno[ dis[inguish more [han abour six[y shades of grey
underlines [he impo[[ance of [he image dynamic manipula[ion and of
[he conuas[ enhancemem s[ep. Wi[h srandard sensors, [he resolurion
may range from 512 2 pixels [Q 2048 2 pixels, representing a file size of
be[ween 262 Kby[es and 4.2 Mby[es.
The effecrs of different spa[ial and brighmess resoJurion are shown in
figure VI. 3.
Grey
levels 8 (3 bits) 16 (4 bits) 32 (5 bits) 256 (8 bits)
Image
size
64 x 64
pixels
128 x 128
pixels
256 x 256
pixels
512x512
pixels
Figure VU . Effect of varying brightness and spatial resolution on digital image quality. Image resolution varies from 64 2 to 512 2
pixels and grey level depth from 3 to 8 bits.
205
Manual of fi sh sc lerochronology
A colour im age is g ene ra ted by covering a singl e se ns o r wirh red ,

g reen and blue filrers in rhe case of a colour mono-CCD camera, or the
rhree individual sensors for tri-CCD cameras.
Most current sc ientific digitisers provide squ ate pixels, i.e. im ages with
a 1 ro 1 aspect ra t io, but the electroni cs regulating rh e sampling fre-
quen cy is liable ro drift, and standard televi sion images have an aspect
ratio of 4:3 . In either case, this m eans rh ar rh e sampling wirhin a video
line will differ from rhe sa m pling berween lines, resulring in a di g ira l
image wirh recrang ular pi xels. Sin ce eac h is g eo merri cally distorred
ignoring rhis factor will bias rh e measurements . Mosr sci entific imag e
processing sofrware enabl e correcri on factors to be introduced.
Eac h individual pi xe l of an imag e I can be locared by its coordin ares
(x, y) whil e irs grey level value is g iven b y I(x,y ). The digirised imag e
is srored in RAM and reconverted ro an ana log vid eo signal by rhe DAC
(Digiral-to-ana log converter) in order to be disp layed on rh e scteen
(fig. VI.2)
Nore rh ar a 4 :3 rar io sampling can be particularly pernici ous as when
di spl ayed on yo ur video scree n , rhe DAC can g eom et rically modify
your image . Thi s can be eas ily checked by digitising a circular object
and measuring it horizontally and ve rti ca lly jusr by ca lculating the
Euclidean di stance from the (x,y ) coordin ates. If you no ri ce a sig nifi-
cant di sc repancy, then you must rake ir into acco unt in yo ur m easure-
ments parti cul arly when using it for sha pe features.
1.1.2. CS image acquisition

In accordance with rhe conce pt rh ar "a good orig inal imag e is berter
and avoids extensive further processing" , we should try to urilise rhe
besr hard ware avail able and to stand ardi se and optim ise the illumina-
ri o n conditions. CS consist of concentric layers rh at are more or less
crys ra lline , o rie nt ed in vari o us d irecrion s, and rhi s fe ature a wid e
rang e of contrasts. Combined microscopy a nd video techniques such
as po laris ati o n microscopy, image inr eg ra tion and imag e m osaic
building tools he lp to produce the imag es required .
1.1.2 .1. Polarisation microscopy

The illuminario n sysrem of a bin oc ul ar mi croscope, and ro a lesser
extent th ar of a compound microscope , is subjecr ro ambi ent distur-
ban ces fto m refl eCti ons or g enerated by rh e observed m a rerial irself
(birefrin ge nce of ce rrain crysr a ls ) or ir m ay g e nerare, by irs very
narure, a mulripliciry of incid enta l rays (e .g . d ark-field illuminati on).
In a ll rhese cases, th e use of a polari sing filrer enables cerrai n lighr
sources to be selecred and , particularly in rhe case o f rransmirred d ark-
fi e ld i1lumin ari on , al so e nables background noi se to be drasrica lly
redu ced (Macy, 1.995; Welle m an & Srorbec k, 1995; T ro ad ec et al.,
2000) (fig. VI A ).
206
Computer·asslsted age estimation
d
?0r---~--------------------------------
1 5~--~--------------------------------
IO~--~------------------++---- __~__
~
~ ~ ~--~~~~r-~-+~-r~~~'-nrr-T~~
~ 0 l--~"~*""I/w4:..~!fi.MIlIP.\"'~1M
i .S ~+-~~j-ll~~~~~~U+H4~+-~~~
~IO 1--------------------------------------
·15 1---_________________________________
50Q 700
Figure VIA· CS digital image acquisition: plaice (Pleuronectes platessa) otolith image with (a) normal light and (b) polarised light.
Effect of image averaging on nOise distribution: (c) false colour representation of the difference between two consecutive images
acquired with a standard CClR camera. An intensity profile (d) shows that the difference in pixel value has a significant magnitude
(blue line) on a single image aCQUisition compared with averaged images (red line, N= 10). Construction of a CS image mosaic
that allow the processing of the entire CS at high resolution on le) whiting (Merlangius merlangus) otolith 14 images), (f) SEM image
of an orange roughy (Hop/ostethus at/anticus) otolith (3 images).
207
1.1.2.2. Image integration

The noi se generared by rhe whole imag e acquisirion process (vari a-
rions in lighr incensiry, e!ecuoni c noise, ere.) can be observed as rhe
variabiliry of rhe response of a sing le pixel under consranc illumina-
rion. Fi g ure VIAc presenrs a false colour imag e of rhe d ifferen ce
betwee n twO consecurive single images acquired on a srandard CClR
camera. The incensiry profil e acquired in rhis image (fig. VI. 4 d) shows
rhar a pixel value may vary by as much as ± 50 grey levels for rhe sam e
scene . In order ro reduce rhe effecrs of rhis scarrering, several succes-
sive images can be added m eliminare rhis random noise. When work-
ing wirh srill microscopi c images, as in our case, frame averaging can
be very efficienc. Ir can be performed by sofrware or even by hardware
o n some imaging boards (ex: M auox Meteor, Scion AG -5 , erc. ). Hard-
ware qualiry (camera, frame-grabber, ere.) is consrancly impmving rhe
signal-m-noise rario, bur rhe incensiry pmfile of a difference of aver-
aged images (fig. VI. 4 d) shows rhat pixe! value variabiliry is signifi-
cantly red uced by image averag ing . Tuning rhe exposure rim e param -
erer rhar is available on some digiral cameras is anorher possible way
ro inceg rar e lig hr informari on.
1.1.2.3. Image mosaics

The relari o nship berween rhe growrh zo ne dimension and rhar of rhe
CS rarel y allows rhe CS ro be visualised as a whole thmug h rhe micro-
scope eyepiece. Moreove r, even when rhe resolmion allows ir, ce ncring
each sample by m ea ns of rh e zoom is in practice very roug h because of
rhe need for a calibrarion value for each image. Ir should be remem-
bered rhar cerrain rypes of sclerochronolog ica l processing, such as pro-
porrional back -ca lcularion methods, require rhe whole cs. In such
cases, fi eld-by -field digiri sarion is ofren unavoi dable in pracrice , and
rhis larer poses rhe problem of how m m ap rhe imag e se c.
A sarisfacrory solurion mighr consisr of building up a panorami c view
of rhe cs by means of an image mosa ie. This can be done usi ng a ser
of overlapping field imag es, ei rher manually (Macy, 1995) or auro-
mat ically, as proposed in some currenc sofrware. These rool s are now
relarively rei iable as long as rhe operaror is careful nor ro make abrupr
changes in focus or illuminari on. It is nor always easy m cope wirh rhi s
resuiction, parri cularly on cs rhar presenc growrh zones whi ch may
nor be visible in rhe sam e focal plan and wirh brighrer marginal zones.
Since such mosaic images can represenc several rens of meg abyres, pro-
cessing rhem requires an adequare amounc of RAM and rheir srorage
demands image form ars rhar offer significant compression rarios (e.g.
JPEG) (fig. VIA).
208
1.2. Digital image processing

on the screen is a or
induced an
and
series of optical, electronic and
processes which themselves generate various types of noise.
The sources of of the relevam information are numerous
and the material does not
solmions that have been on
a given process of \\/elleman & Storbeck (1995)
consider [hat image enhancement is not necessary, whereas Macy
and double iteration of a direc-
contrasted macrostructLlres and the latter on

with
can of a CS, but any misuse

such as a bad parametensarion, the of its imrinsic proper-
ties or even an abusive iteration process may have effects on
(he from a growth-mark
the imroduction of additional marks. Much current
Images bur have a poor ability (0

resolmion. It is Important to realise that the
the can affect
we will stared and
conrrollable.
The
i nformat ion
209
More derailed information can be obcained

(Gonzalez & Wi[1(z, 1987; Coster &
& 1995;
Processing
Periodic information
Data
Scene
1.2.1. Contrast enhancement with point operations

presented One of the errors most commonly made by new users of pro-
structure In relation
to the most important see". As memioned above
results produced by each of grey shades is far poorer
c!ass
than rh,l[ of arrificial sensors.
[Jons, an will be LdlUdlJ1C
tion in a reduced bm
eyes. A operaror (see
VIAl
since ir is 1st-
never nmice that it contains all the available information

The concentration of information on a few grey
levels will rhus generate:
less easy for the as result of poor concrasr;
- calculation inaccuracy cl ue to errors.
210
Computer·Qssisted age estimation
is an whose disnibution levels,

is stretched to fill rhe whole of the available
obtained by means of a linear or non-linear

sis function, V\.6 illuscrares various hIstOgram modifications
and their effects on the of
The ulrimare
but may be to highlight certain
interest ftOm tbem, We can rhus modification
funnions that operate on dues holds or more rraos-
formarions such as normalisation or However, ir is
to remember thar rhe choice of transformation
simultaneously on rhe type of and the aim in wherher aes-
thetic or funccional and thar cermin tbar enhance
detail contrast may have rhe imporcam faulr of also
can also be combined pixel,
-, x, "i-, min, or (MD, OR, XOR, NOT)
tWO
patrern or even detect objen motion

used in fiI-
filters so as to make reses,
1.2.2, Noise reduction with neighbourhood operations

Because operare on a by pixel basis, point
allow rhe modification of derai
value with its offers information on
rrends, Neighbourhood are based on transformations char
take the environmenr of each inro account, The
most usually used in such cases are:
based on modifica-
the use of convolu-
tion operatOrs;
• non-linear filtering based on transformations or local
sracistics (median
Such
so
of them appears ro be essentlaL The
a few but should
duction to (he range avaiJable.
211
15000
~ I 000
~
.0.
.0
Z 5000
0~~-9~~~~~~~~
o In 255
o 32 64 96 128 160 192 224 256
Grey levels
Inverse 15000
255
00 32 64 96 128160192 224 256

o In 255 Grey levels
Normalise
15 000
255
0-1--.......-
o 32 &4 96 128 160 192 224 256
Equalise
15000
255
~ 10000
.0.
.0
Z 5000
32 64 96 128 160 192 224 256

Threshold
200000
255
~
150000
~
.0
100000
Z
50000
O ~~~~--~~~--~
o 32 &4 96 128 160 192 224 256
Figure Vl.6 - Examples of Image contrast enhancemenl by hiSlogram transformation, i.e. on a pixel by pixel basis. Each column
will present the anamorphosed image, the anamorphosis function and the resulting anamorphosed image histogram respectively.
The computation of the anamorphosed value of a pixel is made by projecting It from the "In" axis to the "Out" axis through the
anamorphOSIS function that acts as a mapping function .
212
When We must be aware of how

ital images are Dr<)Ce'SSE~d either in the spatial or
and that both are of the same
from to done by means of the
Fourier transform, This converts the sum of sine and
component in rhe overall

known since its by
111
of a fast the FFT (Fast Fourier rrans-
form)_ Nore that )PEG image format uses a relative of the
Fourier transform, the Discrete cosine transform (DeT) using
cosine waves basis functions, The )PEG will consist essen-
to eliminare the
the human eye, On the other
may affect your
a
can be evaluated in the orher.
and dark zones on a CS
or to noise ",,--",,-,,,",,-u
the acquisition process, In the profile shown in
VI. 7, it can be seen that the presents schemarically:
component corresponding (0
componenc corresponding to electronic noise and

to the fine texture of the CS;
to the lCJ.I1IJ'Jld of the
a component to the modulation of the

zone width with time,
213
Basi cally, we re tri eve each of these signal components, more or less
clearly, in any cs signal. Neglecting any of these components, when It
is strongly represented , lowers detection efficiency. Depending on our
objectives and on th e biologi ca l material available, we try to reduce
im age noi se by suppress i ng th e hi g h-frequ e ncy com ponen tS , or to
homogenise image lumina nce by attenuating the low frequencies, or
to improve the cs co ntras t by enhancing the "ring" component.
Signal Components
Periodic biological
cycle
.
. .!I
..
..
.
...... Low frequency =
..- ... .", Trend
... ....
.. ...
..- ....
...
-.:,:....... . t r
: .: :.: .:~
High frequency =
Noise
••••••••• V
....... Frequency
modulation = Growth
------.~ X
Figure VI.7
CS signal modelling
decomposi tion of a plaice
!P/euronectes platessai
Image profile into it s main
basic signal components. l.2.2.2. Spatial convolution: from moving average to Gaussian
The symbol I refers to Spatial filterin g is mainly based on the operation of convolution of an
a light-intenSity value (grey
level) and v to a frequency. image by a window of coefficients known as the co nvo lution kernel.
The ptocess consists of moving this kernel ac ross the image and to te-
compute each pixel value as the weighted sum of the adjoining pixels
in a ne ighbourhood d efin ed by the kernel size (fi g . VJ.8) . Thus,
depending on the distribution of these coefficients, we can select slow
grey level tran sitions, corresponding to low frequencies, by using a
low -pass set of coefficients, Ot convetsely, se lect sharp grey level tran-
sitions, corresponding to hig h frequencies, by using a high-pass filter.
1 1 1 1 1
1 1 1 1 1
1 1 1 i 1
1 I I 1 1
I 1 I 1 :
1 I I 11
..........
-------------
t
----.-+
/Hiu, 0/11
~,.
006 0.18 025 018 0.1)6

'"(ilt:J l\ _j
01 8 OS!) 0.71 0.;0 o.le
0.25 07 1 1.00 0 .71 025
t hlx.yJ
0.18 0.50 0.11 050 o !8 ,I
~~ 0.18 0 25 ~\ 8 0.06
-
5X5 Gaussian kern el
...... --"
t h/x,yi
I
li
Spatial convolution
...... . ------------
t -----.-+
lHiu,vH
,," 1
c
~'~' ...I\
....~'J? .:.;.~ ~ __.....
"I
I
("")
o
3
"0
co
~
''""
V>
(n.
iD
D-
C»
OQ
Cl>
Cl>
~
Figure VI.8 . Linear filters : smoothing filters designed in the spatial (convolution kernels) and in the frequency (impulse response) domain a) The average filter is a good smoothing filter but in the 3
N frequency domain it displays lobe s that makes it po orly selective regarding frequencies, resu lting in a blurring of small ring contours. The Gaussian filter produc es better results b) a Gaus sian ker- ~
6
nel is difficult to parameter ise and is a tru ncated Gaussian function. c) A version of thi s Gaussian filter designed for use in the frequency domain wil l produce more accurate and selec tive results.
'" :0
For a berrer undersranding of rh is principle, let us observe how a mov-

ing average operares. This well known smoorhing operarion consisrs
in replacing rhe value of a pixel by rhe average inrensiry of a given
neighbourhood. The efficiency of such filrering, i.e. rhe increase in rhe
signal-ro-noise rario, is highly proporrional ro rhe size of rhe convolu-
rion kernel. However, rhe more the kernel size increases, rhe more rhe
rransirions relared ro edges widen. This tends ro delocare image con-
rours which rend ro become rarher blurred (fig. VJ.8a). Berrer preser-
varion of rhe conrour locarion necessarily implies a smaller kernel, bur
ar rhe same rime, rhe image will be smoorhed very lirde. This obser-
varion faces us wirh rwo anragonisric filrer properries, i.e. irs efficiency
in rhe sparial domain and in rhe frequency domain. The frequency
response of rhe average filrer presenred in figure VI.8a shows clearly
rhar rhis filrer cannor separare one band of frequencies from anorher.
AccuaJly, rhe moving average is an excellenr smoothing filrer bur a
poor low-pass filter, which means rhar arrenuaring rhe noise simulra-
neously implies a loss of precision in rhe objecr locarion . We need ro
find a compromise: rhis is rhe Gaussian filrer.
Alrering rhe kernel coefficienrs enables us ro balance rhe influence of
certain pixels on rhe overall average and rhus ro be more selecrive in
our choice of frequency componenrs. Instead of having a gare-shaped
impulse response like rhe average filrer, rhe Gaussian filrer has a bell
shaped one. This filrer offers rhe besr compromise between signal-ro-
noise rario and local isarion accuracy. The theoretical basis of this rool,
which is frequently used, have been widely discussed in the lirerature
since rhe work of Marr, which was based on rhe scudy of rhe human
visual sysrem (Marr & HiIdrerh, 1980; Marr, 1982).
1.2.2.3. Spectral filrering: rhe Fourier transform

Spectral filrering is based on rhe modificarion of a signal in rhe fre-
quency domain. A frequency represenrarion of a sparial signal is
obrained by using rhe Fourier transform. As we have already seen, spa-
rial convolution operares in borh rhe sparial and specrral domai ns.
Among several reasons for employing specrral filrering are rhar:
• ir is more efficienr when rhe original problem can be easily expressed
in rerms of frequencies;
• rhe convolurion of rwo signals is simply rhe producr of rheir Fourier
rransform. This single property enables us ro drasrically speed up rhe
convolurion processing process;
• ir allows rhe design of more efficienr filrers, wirh a wider suppOrt,
rhat lower rhe rruncarion effects imposed in rhe sparial domain by rhe
approximarion of a filrer shape on a small kernel.
How can we design a filrer in rhe frequency domain? The naive
approach consisrs in applying a gare funcrion ro rhe Fourier rransform
216
of the (fig. This brutal way of the number of

coefficients of the spectrum generates ringing and over-
shoor at and corners in (he
known as the Gibbs effect. Again in (he we are
faced with the of between
tiallocalisations. This remains a possibility the
the filter as described in V1.8 or by utilising recursive
mentation of analog which will avoid the Gibbs effect Slm-
infinite support.
L2.2.4. Linear
The low-frequency (or trend) component in a CS (see
VLA.l.2,2.l) drift of the average luminance, may
also be due to:
•
material
zones with age, closer the rranslu-
This phenomenon often appears as a brightening of the cs towards its

that the luminance features of a zone, whether
translucent or opaque, are not spatially constant, We can
the of a sim pie to these
of separate the different
zones adequately, linear presupposes a
stationarity, i,e" that the average value and standard deviation of a
constant, In the same way, the of
i,e, their robustness ro are increased by the elimi-
nation of trends or at least by their attenuation, For that purpose, we
filters to detect the components and
to remove them from the rough This could be done either
means of a Gaussian the best compromise between
spatial and frequency localisation VL9), or using a morphologi-
cal filter slIch as an VI. l2),
1,2,3. detection derivative operations

We may now consider the image nO( as a sum of compo-
nents, but a relief whose grey levels will represent altitudes, The
thus consists of a succession of and valleys whose
are more or less steep, A highly will present a steep
slope of grey levels on its whereas a weak slope characterises a
r.n,po,>""C area of luminance. The detection of local disconrinuities
means of by derivative operators enables

to be localised (fig. VU 0),
250
230
210
190
~ 170
>-
~ 150
130
lIO
90
70
50~-------------- __----__- -- -__----~
o 50 100 150 200 250 300
a b Distance (pixels)
250
230
210
190
~ 170
'"~ 150
'" 130
110
90
70
50~-- ____- -__----~----~----__--~
o 50 100 150 200 250 300
c d Distance (plxels)
250
230
210
190
~ 170
~ 150
<0 130
110
90
70
50 ~----------------------~----------
o 50 100 150 200 250 300
e Di stance (pixels)
Figure VI.9
Elimination of the light trend
on a CS Image and related
profile: a-b) rough plaice
otolith (Pfeuronectes In rhe unidimensional case, rhe poinr of maximum slope of a grey level
platessa). cod) image
01 the estimated light trend, rransirion corresponds co rhe extreme values of irs firsr-order deriva-
e-f) detrended image. rive, i_e. irs gradienr. The human visual perceprion sysrem applies co
Such processing facilitates
growth mark detections. all whar we see an enhancemenr of rhe second order derivarive or
Laplacian type.
218
m
Gradient ·· ·" ,~
Edge o • ,? =B
~~~
0 0
profile = V y=I(x,y)*
South Gradient 0 -I 0]=
o~@j
q
[o I
1st order
derivative
2nd order
derivative
=
East Gradient = Vx=I(x,y)* 0 -I
rf,:~
0 0 0] = j~,~.
[0 0 0 "'.~ -.-:,;~~1
I
'
~
:".~~
.. '.~
\ ,' ....:'..
= I(x,y)*
[
Laplacian
1 1 1]
~ - 8 ~
D
Prewitt
Gradient magnitude
2 ] -2 =
Gm(x,y)= [V .;::)(x,y) + Vix,y)
1
n
Laplacian of o
3
a Gaussian (LoG) Gradient orientation
'0
c:
m
= ~G(x,y)*I(x,y) e( x,y) = arctan(V y (x,y)!'V x( x,y)) =
~
V>
u;'
m
0.
Q.>
, is the convolution operator 00
ro
ro
~
3
N Figure VI. 10 . Edge detection by derivative ope rators: processing of a section of a pollock (Pollachius pollachius) otolith by different derivative 1st and 2nd order ope rators ~
0'
<D (Visilog softwa re, Noe sis). =>
The 2D gradient can be approximared by means of rhe Roberr, Pre-

win or Sobel convolurion kernels shown in figure VI.10. However, rhe
use of small kernels makes ir difficulr ro disringuish berween local
variarions corresponding ro edges and rhose corresponding ro noise.
The increase in rhe size of rhe compurarion kernel enables us ro obrain
an operaror rhar is more robusr ro noise, rhis robusrness being
obrained ar rhe expense of precision. In facr, all rhe derivarive opera-
rors operare a smoorhing funcrion and once again impose on us rhe
consrraint of making a compromise berween a good localisarion of
noisy edges and a poor noiseless localisarion. The besr compromise
will be obrained by rhe operaror of Marr & Hi/drerh (1980), rhe
"Laplacian of a Gaussian" rhar allows rhe advantages of rhe Gaussian
filter ro be associated with those of the Laplacian. In rhe same way, rhe
Canny-Deriche edge detecror is a recursive algorithm for the derermi-
narion of the gradient. In order ro minimise the effects of noise, it
smoothes the image before computing the gradient. A smoorhing
scale parameter allows LIS ro accurately determine rhe smoorhing
intensity. If the value is large, the noise will be reduced bur the edges
will be less sharp and only the most significant edges will remain. It
is important ro select the right coefficient ro lower the noise just suf-
ficiently without defocusing the edges.
One way in which ro sharpen a CS image is ro anamorphose the image
hisrogram. As mentioned in 1.2.1 there are various ways of doing so,
but linear scaling and equalisation are the most frequently used
methods. The latter is likely to be more effective, as it aims ro balance
the distriburion of grey levels, bur it will have a tendency to enhance
noise. A more effective way to do this is to use deblurring algorithms.
Blurred images are characrerised by low contrast and parricularly by
smoothed edge transitions. Deblurring images enhances their contrast
by reinforcing edge transitions. As blurring is an averaging, or inte-
gration operation, deblurring will be approximated by means of com-
puting derivative operators. The Laplacian operaror (2nd-order
derivative) is a good approximation of a deblurring operator and is
more efficient in sharpening edge transitions. An image I(x,y) will be
deblurred by (fig. VL11):
(VI.l) D(x,y) = I(x,y)-k.l1I(x,y)
where l1 is the Laplacian operator and k a constant value.
1.2.4. Structural information detection by morphological operations

Contrary to the linear methods which consider an image as a sum of
frequency components, mathematical morphology (Matheron, 1975;
Serra, 1982, 1988) treats the image as a set of opaque objects in which
superposition produces masking rather than summation. Saying that
220
Figure VI.II
Image sharpening by
subtraction of a Laplacian
(second-order) derivative.
objecc A hides objecc B is equivalent to say ing that the contour of B
This is a good is included in A. Thus, mathematical morphology wiJi examine, by
approximation
using set operators (1'1, U, c:;;;), the geometrical structure of an image by
01 a deblurring lilter
(Visilog software. Noesis). means of a reference object of known size and shape: the struccuring
element.
Erosion and dilation are the basic operations of mathematical mor-
phology. Instead of re-computing a pixel value by means of a coeffi-
cient set as for the convolution, we test in each point of the image the
inclusion or the intersection of the structuring element. In a binary
image, coded into a series of 0 and 1, an erosion will consist of pre-
serving tbe zones in which the structuring element is entirely
included. A dilation, on the contrary, will consist of adding to an
object all the points at which the structuring element intersects with
it. An erosion will remove isolated points and small objeccs and will
disconnect objects, whereas dilation fills the holes and connects the
objeccs (fig. VI.12). These operations are also applicable to grey-scale
images, in which erosion will be computed by the min function of
pixels in the neighbouring region, defined by the structuring ele-
ment, whereas a dilation will be the max function.
From these basic operators we can build operations that present simi-
lar but less destruccive properties that better preserve the original
shape. The opening of an image is a combination of an erosion fol-
lowed by a dilation, while closing is a dilation followed by an erosion.
All these transformations have the increasing property, i.e . that , if a
Ot object is included in another object O 2, then the transformation of
0 1 by a transformation T is included in T(02). On the other hand the
topologi cal properties (holes, number of related components, ete.) are
not preserved.
These basic processing operations will provide a very efficient cs
growth ring detector: the Top-Hat transform. This consists of the sub-
traction of the original image to an opening, when looking for translu-
cent rings, or of subrraccing a closing from the original image in tbe
case of opaque rings (fig. VI.12f).
221
Figure VI.12 - Extraction 01 a plaice (Pieuronectes piatessa) otolith contour (a) by morphological operators: a closing operator
01 a thresholded image (b) will close the contour and consists 01 dilating the image and then eroding it (c). Holes inside the object
are lilled by a hole-lill algorithm (d) based on geodesic dilation 01 the image complement. The outline 01 the biggest object will
correspond to the otolith contour (e). The Top-Hat lilter is an adaptive operator allowing the thresholding 01 objects 01 a delinite
size (I).
Other type of rransformations will allow us to preserve the ropologi-

cal properties of the images_ They consist, for binary images, of look-
ing for particular neighbourhood configurations and removing the
pixel corresponding ro such configurations in the case of a thinning
operation, or adding one in the case of a thickening operation_
1.2.5. The growth pattern: a limit to classical operators

One of the principal properties of the classical linear ands non-linear
digital filters is that they are shift invariant. This enables us to detect
the same object wherever it is in the image. When the image repre-
sents a scene with a perspective, for instance a highway seen from a
bridge, we cannot detect the objects in the foreground using the same
222
filrer paramerers as rhose for rhe background. Wirh classical filrers we

need ro enlarge rhe size range crireria and finally derecr rhe foreground
objecrs corresponding co noise. Wirh CS, rhe growrh parrern acrs like
a perspecrive, varying ring widrh and allowing us ro relax our size cri-
reria, and co garher noise.
When we have a rough idea of rhe growrh parrern, a possible solurion
is ro adapr rhe filrer paramerer ro rhe perspecrive or grow rh parrern
funcrion. The dual solurion consisrs of reshaping rhe signal, srdl on
rhe base of rhe prior growrh parrern knowledge, in order co subrracr,
or ar leasr co arrenuare rhe variarion in rhe amplirude of peaks and val-
leys. In signal rheory rhis is known as frequency demodularion (fig.
V1.l3 ).
a
c
" I
i Inal profile
Demodulated profile
Figure VI.13 - Growth trend removal: graphic illustration of the demodulation algorithm on a plaice (Pleuronectes platessa) otolith
image profile (a) using an a priori von Bertalanfty growth model (b). The demodulated (or anamorphosed) profile (c) presents
rings of similar width, unlike the original.
223
applied to otoliths larvae

1 wirh an
and to oroljths of adult Po!!ctchius virem
1991) and Thttnrllts dorsal fi n ray seccions (Troadec &
Antoine, pets, with a von Bertalanffy function, The aim
was Iy co derecrion and at the same time to esti-
mate the number of faint or due to bone alterarion, for
instance, The demodularion was followed a Fouriet transform and
the were lIsed to estimate the
tings by
1.3. CS quantitative analysis: from 10 to 3D

While
citacive
some cases a
presents and illusttates the basic parameters, our goa!
draw the reader's atrention to the existence of vatious methods
the same parameter.
1,3,1. Growth zone counting: from 1D to 1,5 profiles

systems came into use,
were reducing their localisation to a
when ring growth measurements were for some
work (\'Velleman & -.r.vt,,"rk' 1995; Troadec et ai, ,
the tools available still use profiles to detect growth zones
Why then are there such differences in their respec-
rive
One of the main reasons is rhat the human reader derecrs the 111
2D and locates them in 1D when most current deteccion software

carries our rhe whole process with lD information. 1'1.1 ,'v£"rt 1,,"1
if the zones do not vary roo mllch in if their contrast is
enough and if rhe filter patameters can be we
can expect ro obtain a detection rate of around 80%, In
we can say that if we are abJe to count rings then automatic sys-
tems will be Hnor roo bad",
When on a profile, we definitely lose any
infotmation and the will be very sensitive ro local
defaults that could be interpreted as peaks or In order
ro enhance the and to perception
of local (ontinui ty, some software now allows ro the dara
from several les and to combine them, This can be done in rwo
ways
that all pixels at the same distance from the
are combined, This way IS when we have
224
~ ~
Y~ g y ~ Ig
ORIENTATION = Directio n of the maj or inertia
ax is es timated from secon d order momenlS
A REA
A(X) = L g(x',YJ) 8=.!atan ~
2 ivf2x - M",
with ' g (r;,Yi) = I ifc X
with : M2> = _ I - I.(x, - MI,)2
g(x"y ,) = 0 if <l X A(X) x
M" =_ I - LCYi - Mly)2

A(X) x
MLr< = _1- L(X;- MI,)G'i - Mly)

A(X) x
~ ~
Y~ Y~
FOURIER DESCRIPTORS:
g PERIMETER = Length of X bou ndary Ig • With a polar re presen ta ti o n
(red points s ho win g an am bi gu ity o n a rad ial )
. Sum of the bo undary pixels in the different
direc tions
x L(X) = No + N90 + N45 + ,v 135
R(8) = Ao+L A"cos(n 8- <I),,)
,,= 1
.... . ....... .
2 . C roft on perimeter = sum of the bo unda ry pixel s • With elliptic descriprors (2 Fourier series
correc ted for diagonal tenns (a = square pix el size) corresponding /0 the projection
of th e shape on the x alld y axis)
.. '
L(X) = 4' x [ a X (No+ N9o)+ :h X (N 4 5 + N m )] cV
X, =Ao+L X"
,v
Y, =C...L Y"
,r- I =1
x
~ -+
Y~ g BARYCENTRI: = Centre of gravity Y~ g
GEODESIC 0lS1 ANCE
(')
dX (a,b) = are(a ,b) if a alld be X o
(A:j=( Ml x,Mly) with: 3
'C
co
dX(a,c)=+=
MIr-_I_Ixi ~
A(X) , e:
<I>
u; '
MI,-_ _I_ m
' , A(X) LY1
x Q.
'"
(]O
'"
~
'3
rv ~
rv
Figure VI.l4 . Image analysis: graphic illustration of some basic parameters used to measure and characterise objects on digital image s, o
'" ::J
a profile ser rhar is precisely perpendicular ro your growrh zones and

when rhe dara poinrs do nor presenr roo much curvarure , so rhar dara
are nor combined wrongly;
- afrer synchronisarion of rwo landmarks, e.g. nucleus and edge. In rhis
case, all rhe profiles are resized and dara can be combined more safely.
Dara from rhe differenr profiles can be combined by compuring sra-
risrical esrimarors like rhe average (Caillier et aI., 1996), bur rhe
median esrimaror, being more robusr ro aberranr dara, is usually pre-
ferred (Troadec, 1991; Welleman & Srorbeck, 1995).
Rings are assimilared in 1 D profile ro maxima or minima (peaks or
valleys). Smoorhing linear filrers are used in order ro selecr rhe main
peaks or valleys, srarring from a simple moving average algorirhm
(Cook & Gurhrie, 1987; McGowan et aI., 1987; Szedlmayer et aI.,
1991) and moving ro more selecrive Fourier filrers (Troadec, 1991;
Caillier et aI., 1996; Morales Nin et aI., 1998) or adaprive Top-Har
rransforms (Troadec, 1991) (fig. VI.l2f)
190
170
150
] 130
>-
5110
90
50~------ __- -______- - - -____ ------~------ __

o 50 100 150 200 250
Distance from nucfeus fpixel s)
Figure VI. IS - Acquisition of image profiles: comparison of different computational methods on a pouting (Trisopterus luscus)
otolith image (a): Single profile (blue arrow)' multiple-averaged parallel profiles (green box) and median of multiple synchronised
radiate profiles (red dot lines). Graphic representation (b) of each method showing that single profiles will miss some rings while
multiple profiles will tend to delocate or over-estimate ring width (TNPC software, Noesis).
When considering a peak (or a valley), we can iUlare rhe growrh zone
as being ar:
- rhe grey value maximum in rhe peak (or a minimum for a valley);
- rhe geomerric cenrre of rhe peak;
- rhe beginning of rhe peak;
- rhe end of rhe peak.
226
The exueme value method is the most widely used bur it is essential to
check whether the autOmatic location fits with your own percep-
tion. Actually, when we are faced with an homogeneous area we tend
to locate the at the centre of the bur if there is a
luminance maximum, we will be attracted it and locate the ring at
this extreme. speaking, the location of the and
end of the influenced by the filrer parameters.
All these methods that operate on a ring by basis can be consid-
ered a~ "local" to "global" methods that are based on a spec-
tral estimation in which the number is estimated on the whole
on a global basis. These methods can be used in certain cases
where the growth pattern does not vary or where appro-
has been
&
1.3.2. 2D and 3D CS shapes

of surface or
volume, as a feature of fish
history
viduals to be identified. In
between North American and Salmo
1987; Reddin & 1992) and to dis-
between individuals of wild and &
1990), as well as to otoliths &
Friedland & Reddin, for stock discrimination.
with a tablet but a camera is
nrl'tprrpd in order to circumvent manual errors. cs illumina-
tion must be in order to limit of
the CS from the background) to a threshold that will provide a
binary
np/"nrnnn<f'n into a sum of
sine and cosine waves with in harmonics. The

gross is described low frequencies, while the addi tion of
higher-order harmonics refines the CS The ourline of
the CS can be determined by an
Fourier coefficients from a polar co-ordinate rec)re;;en
the contour (Younker & or from Cartesian co-ordinates
with elliptical coefficients The latter
approach is to be preferred as it circwnvents the problem of
the centre of the and can be
the first to
227
Manual of fish scleroc hronology
3D information is usually used by a reader faced with a new spec ies .

The choi ce of the best sectioning plan requites the observation of cs
from different stages of growth, in different planes and following a
se ri es of grinding stages. Thi s is done until th e read e r is able ro
recon struct a m e ntal tri-dimensio nal shape dynami c of the cs. While
we wait for non-destructive methods such as computeri sed tomog ra-
phy to become available fot cs work (Hamrin et et!', ( 999) 3D tepre-
sentation is currently obtained by the alignment of se rial curs. These
methods are s till ted ious ancl thus are not yet widely used in age ing
labo ratories.
The Ctitical point about these rechniques (fig. VI.l 6) , is their ability
ro align the different e lements of the set of sli ces. The individual
steps involved are:
- embedding th e CS in resi n;
- cutting with a blade o r a rhtead saw. This step determines th e reso-
lurion on the z-axis;
- alignment mad e with sofrware vis-a.-vis a reference - the fiducial
poinrs - that can be obtained by dtillin g holes perpendicular ro rhe
g ri ndi ng plane (Bai ley et Cl!., 1995) or by a square embedd ed wi th rhe
CS in rh e resin (Ttoaclec & Fi chou , pets. dara);
- slice digitisation by a frame-grabbe r.
The image set can rhen be visualised as follows:
- a rough 3D represe ntation as a vol ume image, pixels becoming 3D
points called voxels. Cenain sofrwate p ac kag es allow us ro make dis-
tance measurements in 3D;
- a reconstructed view of th e outer edge and/or of the ring ed ges after
prior manual or automaric digirisation as described above for 2D shapes.
The reconsrruction sofrware can rh en provide .) D shape fearures.
2. Cognitive information processing: Are you reading or interpreting?
Req uiring a computer to simulate, eirher entirely or parti a lly, an ag e

estimation process implies rhe possession of a conceptual framework
clearly based on a precise rerminol ogy. Putting hi s fing e r on the
semanti c confusion still mainta ined roday by sclerochronologisrs in
th e undifferenti ared use of rhe terms " read ing " and "interprering"
Sych (197 4) proposed to di stin g ui sh rw o components in an age esri-
m atl on process:
• reading, which is the recog nirion of certa in structures by mean s of
a main a lphaber or a key ;
• interpretation, which is rhe comparison of s rruCtu res wirh events
in rhe life cyc le.
Thi s simple di srinCtion, which seems obvious ar firsr sighr, made Sych
rh e firsr to propose a logical ancl statisrical fram ework for rhe analysis
of calcified stru ct ures on rh e bas is of rigorou s dec isi on trees, which
228
Yaxis
MMS'"I
I
I
I
I
..- Silicon I
I
mould I
I
I
I
I
+---1- -1--- Plastic I
I
box I
I
I
I
I
I
I
c I
I I
L _ _ -I_
Stage~-~~~ 11
b -r--7I--------
I
I
I
I
I
I
I
I
I
I
I
I
I . I
: ~ It! I
r--- ---------~--~
I
-----------------~
~=--------
-----.--
---- ----
Figure VI. 16 - CS 3D reconstruction: description of preparation process for reconstructing CS in 3D from serial cuts. a) Inclusion
in a plastic box and embedding In a silicon mOUld, b) Serial cutting with a thread saw. c) Digitisation of the 1st CS slice on a
microscope equipped with a motorised stage (MMS). Four fiducial points (fp) are acquired and the orientation is computed. Then,
all the other slices are aligned In translation and rotation in reference to it. d) Visualisation of the image set with 3D volume ren-
denng software (BOB software !Brick of Bytes), AHPCRC).
allow uncercainties regarding rhe decision rules co be manag ed. In line

wirh a similar rhoughr process, Casselman (1987) proposed rhar che
dara acquisirion and interprecarion sreps should be clearly separared
and suggesred chac " . .. rh e fallacy in pasc approaches has been chac che
cwo components - dara exrracrion and dara interprecarion - could be
229
treated simultaneously ... ". Actually, the localisation of a growth ring

on a calcified structure does nOt imply anything abour its periodiciry.
Ir involves only a process of visual perception which can require more
or less long training. This growrh ring may subsequently be classified
as having a given periodicity (annual, daily, false ring, etc.). This is the
interpretarion phase which requires knowledge that has been gained
during validation experiments and in rhe process of previous inter-
pretations. The former is based on purely visual identifying criteria
(growth ring, nudeus, primordia, check, etc.), and can usually be ful-
filled very quickly for a given CS and a given preparation method. The
larrer is based on more or less complex logical schemes, integrating
information relared, among other aspects, to the species concerned,
the srock or popularion, and/or the catch zone, and combining instan-
taneous (or individual) and hisrorical information (fig. VI.17).
Although they may be closely linked, these two processes require
information of quite different natures (Troadec, 1992). Dealing with
them by computer involves two disrinct scientific fields:
• pattern recognition for visual perception;
• the cognitive sciences for interpretation.
Although currently available CAAGE systems are almost exclusively
based on image analysis systems, we can observe a growing interest in
the interpretation stage, which probably constirutes the greatest
source of variabil i ty (Morison et aI., 1998).
A first approach has been ro use objective criteria in order ro assign a
growth zone a given periodicity. Pointing OUt that growth zones are
deposited according ro a relatively regular growth pattern, Small &
Hirschhorn (1987) proposes a system based on fitting a growth model.
Avon Berralanffy growth model is fined for each calcified structute
ro a set of locations of digitised growth zones. The residual analysis
provides criteria for determining the validity of the growth zones.
This approach was then extended by rhe introduction of a constraint
in the reading process through the demodulation of the image profile
by a growth patrern (Troadec, 1991; Lagardere & Troadec, 1997).
Anothet approach consists of asking the computer ro manage only the
interpretation level of the age estimation process. Casselman (1987,
1996) developed a system for the inrerpretation of calcified structures,
CSAIS (Calcified structure age interpretation system), and has devel-
oped and refined a software, CSAGES (Calcified structure age-growth
data extraction software) (Casselman & Scon, 2000), that incorporates
the system and uncouples the acquisition phase from data interpreta-
tion. The software is thus based on:
- a data acquisition module: CSAS (Calcified structure analysis soft-
ware) that uses a digitizer inregrated with a compurer through a
keypad;
230
imenral ne urophysiology. \X/ hil e we do noc wish co be (00 swee ping,

we may say chac che re are chree cheories (Benzino u , 20(0):
• che cheory of "dire cc percepcion" advanceu by G ibso n be cwee n 195()
anu 1979 , accord ing co which che space-cime scruccure of che se nsory
sysce m implied in che vi sion , che so-called "opcical necwork " , is suffi-
c ienc co acco unr fo r che process o f percepcion. An y ince rprew cion, con-
scrucri o n o r repres e nraci on is ch e n regarded as pos r-pe rcep cive;
• che "Gescalc" rheory, which assoc iaces cwo proces ses, a primary "bor-
com - Llp " process, which cra nsfor m s and o rg ani ses che recin al image in
di screce unics ac cording co cheir geom ecri cal and dy na mic characccris-
cics, and a secondary "cop-d o wn " process, whi ch is non - vi sual an d is
di recced by concepcs, and which consi scs of assoc iacin g, by means of
progress ive ad juscm e nc s, chese "o bjecc ive com po ne nrs " w ir h cognicive
informarion pre-exisce nc in memory (fig VI.lHa);
• JascJy, che "con sCl"ucci vi sc" cheory, for whi c h pe rc ep ci on is a kind of
u nc on scious consrruccion of sig nifica nce, scarcing from inc o mpl e re
inform acion provided by rhe re rinal image.
The contributi01l of ne1lroscience ...

Visual pe rcep ci on chu s appea rs co be a co mpl ex process for which che
availa bili ry of an inco mpl ere re rin al im age is by no mean s ,tn obscacle.
In order co be able co ide nri fy an o bj ecc, che human vi sual sysce m ofren
ha s (0 ignore dara, co seek m eani ngs, g acher e/emencs, sepa rar e enri-
cies, co nrinLl e concours , suppl em en r forms a nd fill g"ps.
The Gesral c p oi nr of v ie w, accordin g co whi ch ou r visual sys re m is a
pacre m con scrucc ion machin e, re ce nd y rece ived ch e supporc of ne uro -
sc ien ce by showing chac ce rrai n re ri nal cell s rean selecrively <Lr a ve ry
early srage co d isco nrinuicies in luminou s inren s ir y. Parre rn rec ogni-
ri on involves ce llular gro upings in rb e g eni c ul are body a nd v is ua l co r-
cex (co nical columns), so m e of which cod e for loca l alignmel1Cs and
conro urs in a given v icinir y, an d orhe rs rhac cod e for che poiarici es of
co nrra sc a nd di scon rinui r ie s. Con ne cr ion s b er \Veen rhese groll llings
e nable us ro make form s em erge in o ur percep tion (fig. VI. I gb-d ).
And what ahOllt calCified strllctures in tbis ...

The p ercep ri on of primary pa rrerns in calcifieJ s cruccures in vo lv es
id enriD cari o n processes of mo re o r less cl osed concencric co nro urs char
prese nr a range o f concrasrs an d disruprion ill rh eir conrin u icy a nd 111
rh eir oprica l de n siry. All chese param e re rs chen va ry ac cordin g co che

indivi d ual, rh e srag e of d eve lopmenr, che geog rap hi cal a rea a nd rhe
spec ies Ii1v o lved . Pe rcep ri o n a lso includes rhe p roc essi ng of seco ncl a ry
parcerns , slIch as sca le Cl1lI7II/i to rm ecl by rhe narrowin g of ,il'm /i, or
oro lich nudei whos e conrours include a se r ofj!rill![)rdia, or g ro\Vrh p<1 r-
rern s made lip of rhe varying widrh of growrl1 zon es.
232
(a)
t
Ib) (c) Id)
Figure VU 8 - Visual perception: a) Influence of context and knowledge on shape perception and
completion. b) The perceptual the fragmented elements of each Circle in only
one Unit although those are In a noisy background. cl A figure made up of three com-
plete discs interposed one behind the other rather than of only one in contact with
two incomplete contiguous forms. dl means that miSSing parts of an
incomplete triangle are perceived as if they present
In conclusion we may say rhar age esrimarion on rhe basis of CS is a

complex process information of different natures
1992) and at differenr levels
and that artificial vision
understandable
we bel ieve that

and
of efficient CAAGE systems.
233
Manual of fish sc!erochronology
B. How a computer can help

in calcified structure analysis
Compurer vision systems have gradually come co be regarded as a
-
necessary componenr of the equipmenr of ageing laboracories, but this
has not always been the case. This debate on the urility of the com-
puters, heard many times during meerings and workshops, has opposed
colleagues involved in various "branches" of sclerochronology, the most
distanr often being those involved in microstrucrure analysis and those
specialised in the routine inrerpreration of otolith macrostructures. In
fact, the argumenrs of the one party lost their relevance once they had
been placed in the conrext of the work of the other. A CAAGE system
now offers access to a wide range of tools that any age reader must be
aware of. In addition ro its own image acquisition, handling, processing
and data storage functions, the computer is also an open door to other
powerful methods of analysis such as signal processing technology or
cognitive sciences (i .e., srudy of narural and artificial cognitive processes
including artificial intelligence, cognitive psychology ... ), which them-
selves are currently conrinuously evolving.
1. What can you expect from a CAAGE?
When many virtues are attributed ro the CAAGE systems: objectivity,

accuracy, precision, productivity, memorising, most of them are quite
simply ptoperties of any computer assisted system. But what really
happens when they are applied to age estimation problems) By
emphasising the debatable concept of objectivity, have we hidden
other properries that are JUSt as essential to the analysis of calcified
structures)
The systematic use of computers in sclerochronology starred in the
eighties in order to lighten the task of making repetitive measure-
menrs (mictostructures analysis) and extracting shape factors. Assis-
tance in morphometric data acquisition was, in practice, the first
motivation of "sclerochronologists" in their recourse ro CAAGE sys-
tems. Their other potentials for standardisation, precision or produc-
tivity gains or even data srorage, which were only exploited in rather
a scattered way, seem to have become essential only very slowly 10
laboratories working on routine srock assessmenrs.
1.1. An aid to quantification

The first CAAGE systems were the outcome of the association of a com-
puter and of a micro-densitometer (Ichiara, 1963; Van Utrecht &
Schenkkan, 1972) which allowed the record ing and analysis of optical r
L.
profiles. Then, the appearance of cameras allowed two-dimensional

information to be utilised (Mason, 1974), although the quantitative
analysis continued to be more or less limited to image profiling. Of
the quantitative information that is now available on calcified struc-
tures, we can distinguish:
- contrast information directly related to variations in optical density
(zones of growth, checks, (iratli, growth patterns);
- structural information relating to particular events (nttc!ei,primordia,
accessory primordia), of particular structural arrangements (crossing-
over, resorption marks, etc.) or reference positioning marks (reading
axis);
- shape information.
If CAAGE systems offer very efficient assistance in the acquisition of
contrast information, or assistance that is even essential for shape
quantification, the situation is quite different for structural informa-
tion, which is a more complex psycho-visual process, in which the
human operator is still more powerful.
The ways in which a CAAGE system can help are:
- in ptoducing a good-quality image with a high resolution and a high
signal-to-noise ratio;
- in enabling us to improve image quality (contrast enhancement,
noise reduction, etc.), tasks for which digital image processing offers
many solurions (see chap. VLA.1.2)
It also enables us to locate the growth zones on such images in a more
or less automatic manner and thus to count them more easily. Growth
zone localisation, associated with strucrural information such as the
nucleus, offers us immediate access to the morphometric dara of inter-
increment distances as well as to other fine characterisation aspects of
growth zones (intensity, contrast, frequency, etc.). The analysis of
these data by spectral methods enables us ro identify periodic phe-
nomena (Vasi/'kov, 1977, 1979; Geffen & Nash, 1995) and a prior
knowledge of one of the signal components lets us introduce con-
snaints into the age estimation process (Small & Hirschhorn, 1987;
Lagardere & Troadec, 1997). Similarly, the analysis of temporal signa-
rures associated with Bayesian methods allows incomplete growth his-
tories to be exploited (Ogle et at., 1996).
Image contrast enhancement also enables us ro obtain a 2D contour of
calcified structures and thus to quantify their shapes along with the
auromation of their measurements and their classification (Campana,
1987). This approach resulted in many studies of stock discrimination
using fish scales and otoJiths (Pontual & Prouzet, 1987; Campana &
Casselman, 1993; Richards & Esteves, 1997) based on the external
contours of the CS, a limitation which should disappear with the
recent development of algorithms involving deformable models (Ben-
zi nou et at., 1997; Troadec et at., 2000).
235
1.2. Objectivity or standardisation?

Objecriviry is rhe fearure mosr frequenrly menrioned wirh respecr ro
CAAGE sysrems (Fawel, 1974; Planes & Laval, 1990; Welleman &
Srorbeck, 1995; Caillier et al., 1996 ; Lagardere & Troadec, 1997).
However, rhis is an ambiguous concepr which very ofren hides a mis-
reading of rhe deep mechanisms involved ar rhe compurer level in arri-
Dcial vision (see VI.A.3 .2.4), and which requires ro be cleared up. By
definirion, according ro rhe Cambridge Inrernarional Dicrionary of
English, objecriviry is " ... nor influenced by personal beliefs or feel-
i ngs; based on real facrs ... ".
On whar does rhe objecriviry of rhe compurer in facr resr' Is ir in rhe
subjecriviry of rhe programmer'
According ro rhe algorirhm selecred, rhe compurer will derecr objec-
rively, bur more or less successfully, maxima or minima on an image
profile, or perhaps growrh zones if rhe algorirhm was designed in 2D,
and will finally derecr any false rings if a clear definirion is available.
Mosr algorirhms will rhen produce, in rhe presence of rhe same dara,
rhe same resulrs, wherher or nor rhese are correcr. The objecriviry of
rhe compurer is rhus srrucrured by rhe conjuncrion of our capaciry ro
correcrly describe realiry and rhe availabiliry of suirable marhemarical
conceprs.
In facr rhe "objecriviry " of compurers lies:
- in rhe reproducribiliry and rhe consrancy of rhe resulrs rhey prouuce,
limiring drifr bur also rhe capaciry ro adapr rhar is ofren necessary
wirh biological marerial;
- in our capaciry ro conrrol rhe informarion inrroduced inro rhe inrer-
prerarion process, eirher by informarion mas king (Morison et at.,
1998), or by rhe conrrolled inrroducrion of informarion and con-
srrainrs (Lagardere & Troadec, 1997).
Thus, rhe objec riviry of rhe merhods of compurer vision is quire rela-
rive. In rhe face of discrepancies of diagnosis berween human reader
and compurer, an experr will nor have ro abdicare his judgemenr and
ar rhe same rime, rhe novice will srill have carry our his rrain ing in rhe
company of experienced human operarors.
1.3. Accuracy and precision

The concepr of accuracy has an absolure characrer and makes sense in
sclerochronology only wirh reference ro marerial of a known age or ro
validarion experimenrs rhar permir esrimares of growrh mark period-
iciry ro be made. The accuracy of a CAAGE sysrem will depend
srraighrforwardly (1) on image qualiry and (2) on rhe qualiry and
availabiliry of rhe idenrificarion crireria of growrh zones as well as of
rheir implemenrarion in algorirhms. Some s rudies of auromared
merhods have rhu s noriced eirher a rendency ro underesrimare
236
Computer~assi$ted age estimation
or to overeStlmate some age

groups Troadec et tlf., or better
accuracy than that of the expert & 1997). A dou-
of resolution does not an increase in nprtnn""'~
All in fact on tbe adequacy of the
an underestimate of the zone number in old
wdividuals is often related to the decrease in the width of
the growth zones in the course of rime. On the contrary,
due (0 reduced of the
the recourse (0 the usual criteria of
widthllocation of the parrern, ete.)
of the human operator (0 focus his on
cs zones et af., 2000) or to
is still poorly simulated by current systems.
the of CAAGE systems to reduce
son tI/., 1998) by the operawrs through a standard ised pro-
cess that is in itself an essemial fac(O[ In the accuracy of the
age estimation process. recollrse to the compurer will
In an noticeable way, accidema[ errors such as
or calibration errors (Fawel, 1 Morison et a/., 1998).

it should be that even wday, in of the
continual in their CAAGE systems reach
the expert accuracy only when CS present highly contrasred
1995) and remain on rhe low side of expert
mance when difficulties.
Precision is of a system to
By lrs very narure, rhe use of a highly
standardised coded in the form of computer program, will
enable LIS (0 increase the precision of our estimates, The of a
CAAGE system can be defined in the terms:
• At the level of localisation and measurement of zones.
Although evaluated by users, the reproductibility of
when carried out cam purer, is very high wirh thar
of a human Opeta(oL
• At the level esrimation or the number zones. While
it enables us (0 the process in a identical way, it also
supports the of less variable estimates over time and
between of the results produced by CAAGE
systems with manual methods some authors iet et af., (on-
eluded char had similar level of while Others noticed
in faVOur of human ope'atars aI.,
1997)
237
1.4. Productivity
The productivity of a CAAGE system can be appreciated at various
scales of the age estimation process, either at the level of a specific task
such as the localisation of the growth zones, or at the level of the whole
cs analysis process, or even at the level of the organisation of a stock
assessment laboratory, by considering their capability for conserving,
exchanging and revising interpretations. Does the CAAGE systems
allow a real increase in productivity' And if so, how'
Time-saving is evoked by various authors (Cailliet et af., 1996). Planes
& Laval (1990) saved 50% on otolith microstructures, Watarai &
Igarashi (1990, 1992) saved 30% on salmon scales, Szedlmayer et af.
(1991) saved up to a ratio of 3.3, but none seem to have included the
duration of ring detection contro!' Existing algorithms are essentially
based on the processing of image profile. They allow entirely auto-
mated counting on very few species, i.e. reliable enough to avoid a sys-
tematic a posteriori control that can be a tedious task and time-con-
suming in the case of microstructures. In fact, all these authors were
working in different contexts: macrostrucrures or microstructures,
otoliths or scales, thin slice or whole samples , ete. This brings us to a
question that will be addressed later: viz., what sort of study are we
performing? Cailliet et af. (1996) consider that CA AGE systems do not
allow us to treat samples prepared by means of fast methods (burning,
whole sample) whereas Welleman & Storbeck (1995) and Ttoadec et
af. (2000) evaluate fully automated algorithms on whole otoliths.
Outwardly contradictory, these comparisons show that even though
all are working within highly standardised contexts, the former
worked on a deep-sea fish, Sebastes m/us, with high longevity and the
latter on a flat fish, Pleuronectes pia/ma, with a short lifetime and a high
rate of growth .
If digital technologies imply a high potential for gains in productivity,
especially for routine readings and otolith microstructures analysis
(batch digitalisation, "all day work"), they will be fully exploited only
via the development of more rel iable automation algorithms that will
limit human intervention and operate night and day if necessary.
However, such a development will also involve greater standardisation
of sample preparation and interpretation procedures, phenomena
which will be among the dominant tendencies in sclerochronology in
the coming decade.
1.5. Data and knowledge preservation

Information storage, whether temporary or long-term, is one of the
main advantages of CAAGE systems, from the simple capability of com-
menting about a cs on a video screen in a group (Morison et af., 1998)
238
instead of to follow one another on a

storage of measurements et at., 1
, 1998).
This ability to store rough or
any
the novice reader's
the temporal
allowing revisions of such
particularly to a quality insurance process.
available storage tools still present some defects:
after conventional (COR
stili does not provide the resolution of an average This
limitation will tend to with rhe improvement of frame-
grabbing hardware. digital cameras (1
already provide an close to that obtained rhe human
eye in the """,",I,pr ..
"The
oriemation or
operations are essential when three-dimensional information needs to
be throughout the thickness of the Most of rhese
limitations can be circumvented by the acquisition of
sequences or by the combination of data from different focal
This represents a huge amoum even if stored with a high com-
rario, and in any case means the loss of Direct
observation under rhe is still often necessary.
.. The longevity of image storage formats is not unlike thar of
convemional media. Formats are numerous, ranging from the well-
known TIFF format ro ]PEG, which is among the most In
terms of ratio. An EFAN round table (EFAN, 1997) recom-
mends for recourse ro formats for storage and
an ASCII format for associated data. In it will be a of defin-
mg che namre of informacion to be scored and che
duration of stOrage. The EFAN Cell "Information
1998, 2000) format
PtO, to be to
create a database of annotated accessible via the Web by all the
scientific community (http://www.efan.no/tro/refdbin.htm).This
has been some workshops
wirh use.
239
2. For what kind of study?
We have seen rhar rhe reasons for resorting co a CA AGE sysrem are
numerous and even rhough rhey srill have some defeers, rhe cosr of
rhese sysrems is no longer a real obsracle. Nevertheless, rhe various cri-
reria involved will nor carry rhe same weighr bur will depend on rhe
objeerives of rhe srudy. Do we need co auromare growrh zone counr-
ing, co memorise inrerprerarions , co be able co exchange dara wirh
orher readers' We usually disringuish age esrimares relared co one-off
srudies from rhose relared co resource assessmenrs, which are essen-
rially recurrenr (Morison et at., 1998).
2.1. One-off studies

These srudies aim co answer specific quesrions regarding rhe age and
growrh of given popularions, or (0 use CS as recorders in order (0
analyse and pur in remporal perspeerive cerrain paramerers (saliniry,
environmenral signarure, ere.) or evenrs (harching, migrarion, repro-
duerion, meramorphosis, ere.). The samples may be colleered for several
years and are rhen processed progressively or garhered for processing
ar rhe end of rhe srudy, very ofren by a single scienrisr.
In rhis rype of srudy, rhe need for very long-rerm scorage is less obvious
bur ofren concern micros([ueru res analyses which require (Ools rhar
allow good producriviry. When experience of age esrimarion on rhe
rarger species is non-exisrenr, a validarion srudy will be made, or rhe
scienrisr will rry co calibrare himself againsr orher readers. Validarion
srudies can be merged in rhe one-off srudies as rheir goal is co answer
a specific quesrion, i.e. (0 define rhe periodiciry of cs marks. In rhis
case rhe scorage of rhe resulrs in an arbirrary form is fundamenraL
2.2. Routine analysis

Rourine analyses consisr of esrablishing over a long period of rime rhe
evolurion of rhe demographic s([ucrure of an exploired popularion. le
is probably rhe mosr demanding rype of aeriviry in rerms of rhe accu-
racy, precision and srabiliry of inrer- and inrra-reader inrerprerarive
schemes. le obviously requires high producriviry in order (0 deal wirh
rhe large volume of samples involved. Generally speaking, rourine
analyses make use of several players: samplers, assisranrs, readers and
assessors. These should have relarively massive recourse co CA AGE sys-
rems, bur in faer rhe penerrarion of rhese sysrems has so far proved co
be s([onger among experrs involved in one-off research srudies rhan in
rourine laboracories. While rhey have mosr of rhe qualiries required,
240
as being below that of human

operawrs, deal wieh the of a few
seasonal growth zones,
could lead co called inco question and,
in order to be C<\AGE software
[he whole prore~ mem-
This will make up the framework of rhe rourine
laboratories of CO!11orrow, bur ie still remains to be
241
242
Otolith microchemistry
Cha r VII
H. de AJ Gcffcn
243
244
A. Introduction
Since the early 1970's there has been a growth in efforts to use the
chemical composition of calcified strucrures to ad dress a wide range of
questions in fisheries sc ience . This approach assumes that the chemi-
cal composition of hard ti ssues broadly reflects the physico-chemical
characte ri stics of the environment ro which fish are exposed.
Otoliths in particular have been referred ro as continuous recorders of
exposure to th e environment (Campana et al, 1997). Otoliths a re
metabolically inert , unlikely to be resorbed or reworked (Campana &
N eilson , 1985) and g row throughout the life of the fish, so their com-
position probably has the hig hest porential of all calcified structures
for carrying environmental information.
Recent years have seen a rapid development of th e range of applica-
ti ons of otolith microchemistry (OMC), broadly divided into those that
are concerned with fish ecology (reconstruction of the life hi sto ries of
individual fi sh) and those that are concerned with populations (dis-
crimination of fish populations, validation of age estimation).
Otoliths are exceptionally pure in compari son with other biogenic car-
bonates. Otol i th mi croc hemi stry is often interested in mi cro-scal e
variations In el ementa l concentrations and thu s depends on several
recent technical advances in analyrical chemisrr y and geochemisrry.
The decoding of orolith chemical information poses several problems:
i) OUt poor knowledge of the mechani sms of elem ent incorporation
a nd their systems of reg ulation , ii) the difficulty of assessing the
uncertainty (error probability) associated with the result of a particu-
larly compl ex m eas urement procedure because all steps which lead ro
this result, starting from the fish capture, are potential sources of m ea-
surement error, and iii ) the high COS tS of analyses. This has so metimes
res ulted in speculati ve interpretations and co nflicting conclusions.
How ever, advances in otollrh mi croc hemi stry continue to develop,
benefiting from a growing information base from on-going studies,
fr om new techn olog ica l developments, and from a growi ng under-
sta nding of the biomineralisation process.
The main goals of this chapter are ro offer an insight into the state of
the art of OMC, addressing rhose aspects of research thar have applica-
tions in fi sh ecology and fisheties, analytical procedures and uncertain-
ties in analytical processes. It large ly refers ro recent pa pers by Cam-
pana (1 999) and Thresher (1999) thar offer comprehensive overviews of
current kn owledge in fish orolith chemistry. Some basic chemical def-
initions and principles are reviewed in order ro provide read ers with the
background essential for understanding microchemistry applications.
245
Otolith composition and its telationship with physiological and envi-

ronmental conditions ate described, followed by discLlssion of the
various applications of microchemistry. The use of other calcified strLlc-
tures (scales, vertebrae, fin rays, etc.) is only briefly treated, as the
methods and applications involved in recem years have concentrated
primarily on the analysis of otoliths. The final section of this chapter
presents some of the methodological issues that should be considered
at the beginning of microchemistry studies.
B. Some basic chemical definitions

and principles
Dalton's atomic theory of matrer, which constitutes the base of mod-

ern chemistry, defines the ATOM as the simplest unit of matter capa-
ble of a separate existence (i.e. which cannOt be subdivided in particles
with the same characteristic properties), ELEMENT as substance with
only one type of aroms, COMPOUND as substance comaining more
than one type of elements, and MOLECULE containing two or more
aroms.
Although many subatomic particles are now known, for most chemi-
cal purposes the atom may be considered as consisting of a posi tively
charged nucleus containing prowns (positively charged) and neutrons
(neutral), and sutrounded by a cloud of negatively charged electrons.
The arom (and hence the element and its position in the periodic
table) is characterised by its atomic number (Z) which is defined as the
number of prorons in the nucleus and its mass number (A) which cor-
responds ro the number of protons and neutrons (collectively called
nucleons) in the nucleus.
Atoms with the same value ofZ but different values of A (i.e. dif-
ferent numbers of neutrons) are termed ISOTOPEs . Isotopes of the
same element have nearly identical chemical properties, yet they can
have very different nuclear properties, possibly including radioac-
tivity, magnetic characteristics and weight.
Variations in the relative abundance of the different isotopes of ele-
ments occur through different processes such as mass fractionations,
radioactive decay and anthropogenic activities (e.g. processing of
nuclear fuels, nuclear-weapons testing, etc.). Radioactive isotopes are
unstable isoropes that spontaneously disintegrate over time to form
orher isotopes. Stable isotopes do not decay to other isoropes but may
be produced by the decay of radioactive isoropes, and if so are termed
246
radiogenic. For instance , stromium has four naturally occurring sta-

bles isoropes: 84Sr (0.56%), 86Sr (9.86%), 87Sr (7.0%), 88S r (8258%).
The numbers in paremheses refer co the naturally occurring relative
abundance of these stable isotopes. 87Sr can arise from either the pri-
mordial nucleosymhesis along with the other Sr stable isotopes, or
from the decay of the radioactive metal 87Rb.
Stable isotopes of light elements such as H, C, N, 0 and S are often
measured in s tudies of trophi c ecology, temperature effects and
metabolism. There has also been recent imerest in the isotope chemistry
of heavy elemems (e.g. Sr, U, Pb) as environmental indicators (Ches-
ney et aI., 1998; Hobson , 1999).
Isotopic data is expressed as the relative difference of the sample from
a standard, according to the following notation :
(VII.I)
where hM and IM are the heavy and light isotopes of an elemem M, x
is the sample and std is the standard. A delta (8) value is reported in
%0 ('per mil') notation . A positive (negative) delta value indicates that
the sample has a higher (lower) heavy to light isorope ratio than the
standard. Several analytic standards are available for the differem
elemems . For carbonates of low-temperature origin, 8 18 0 and 813 c
values are reported relative ro either the PDB (Peedee belemnite) or
the equivalent VPDB (Vienna-PDB) standard. Water oxygen isotopic
values are usually reported relative to (Vienna) standard mean ocean
water (SMOW or VSMOW) for marine studies, or standard light
Antarctic precipitation (SLAP) for freshwater studies.
1. Stable isotope mass fractionation
Because of differences in acorn bond strengths between heavy and

light isocopes in molecules, isocopes fractionate between coexisting
phases during physical or chemical processes .
Fractionation occurs via either kinetic or equilibrium processes the
latter resulting in temperature-dependem isocopic fractionation. This
is the basis of stable isotope thermometry.
Fractionation of isocopes between two phases A and B is expressed ,
for a given temperature, by the fractionation factor Ct.:
(VII.2)
where R is the isocopic ratio such as 13C1 12 C, 18 0 / 160 , etc.
In term s of 8 values, this expression becomes:
(VII.3)
Values of a are generally very close to unity for most elements of
interest, typically 1.00X, and it is common to discuss isocopic frac-
tionations in terms of the value of X in %0 (per mil fractionation).
Furthermore , the per mil fractionation is approximately equal co
l03lnCt. since 103In (l.OOX) "" X.
247
Additionally, when values of both 8A and 8B are less than about 10,
the per mil fractionation, 10 3In<X, is very we ll approximated by the ~
value, defined as:
(VII.4)
A major reason for the interest in using the "103In<x" expression is
that, at biologically relevant temperatures « 200 0 C), lO ' ln<X is near-
ly proportional ro the inverse of temperature (l fT).
Interest in oxygen isotope records of marine carbonates as a rool for
paleotemperature measurement rose in the early 1950s, and Epstein
et CIf. (l951, 1953) provided the first paleotemperature equations
based on laboratory-grown bivalves. Isorope fractionation is often
referred to as being in equilibrium. In this context, equilibrium refers
to the energy state tather than to a stable isotonic situation . The equi-
librium energy state can be calculated directly from the strength of
the atomic bonds, which differ for the different isotopes. Because
temperature affects energy state in a predi ctable fashion, the isotope
ratios of chemical processes, such as precipitation, are calculable.
When the measured isotope ra tios in carbonate conform to predic-
tions (as illustrated in the above equations) the sys tem is said to be in
equilibrium .
2. Radioactive decay
Only a small proportion of isotopes are known to be indefinitely sra-

ble. All the others disintegrate spontaneously by processes broadly
designated as radioactive decay. This process transmutes the so-called
"parent " radioactive isorope into a more stable isorope , a so-called
"daughter", specific ro that parent. The process continues until a Sta-
ble nucleus is produced. For in stance, the 238U decay series undergoes
14 stages of decay reaction before the end product e0 6Pb) is formed.
Radioactive decay is a statistical process that depends only on the
instability of each particular radioisotope. The decay activity over
time can be expressed by the following exponential radioactive decay
equation:
(VII 5) At = AO .e- At
where A 0 is the initial activi ty of the parent isorope (i n becquerels or
disintegration.time· I), A' is the activity at time "t" and t.. is the decay
constant (in time-I).
The rate of radioactive decay is typi ca lly expressed in terms of either
the radioactive half-life, T, which is the time required for one-half of
any given quantity of a radioisotope to decay, or the radioactive decay
constant. They are related as follows:
(VII.6) T = ln 2ft.. = 0.693ft..
248
If the initial activity of a dosed system is known or can be esti-

the time since the system was formed can be estimat-
ed from VII.5. This is
When is not known, radiometric In some
cases by the so-called d methods, which
are based on the measurement of the ratio of two members of
is the once a paten t (P) has been

it a process that results
over rime in an increase in rhe of sho[[-
er-lived
(VII.7)
in which the second term is the residual

char was present ar c=O;
co the parem can thus be
VII.5 and VII.7:
(VILS) +
If the syscem is nOt state in which che

racio remains consram will be at a face which on the
and half-lives. Two situations can occur:
The first one, known as "rransiem IS the situation in
which [he balf-lives of P and D are of the same but

In that the
value of
occurs when the
than the half-life of D and the decrease
of the time scale. In that
VII.9 and the activi-
Ap(
(VII. 10) AD I = 1
In that the ratio increases with time, the

value of 1 on the half-life of
the
In the measuremem of the activity
an estimation of the time since the parem
isorope was in chis case into the carbonate The
half-lives of the parent and govern [he rime-scale
to which rhe method will be Due [Q the form
249
of (he decay law, (he precision of rhe esrimares, which depends on rhe
analyricai measuremem errors of borh parem and daughrer acriviry,
will decrease as rhe equilibrium scare is approached (fig. VII. 1). Ir is
worrh noring rhar rhe merhod is valid only if rhe sysrem meers rhe
crireria discussed below regarding orolirh radiomerric ageing.
10 12 14
Years
Years
2 t-----------------~~===-----__1
O~----~--------------
o __ ----~
10
Number of daughter half~lves
4 6 8 10
Number of daughter half-lives
Figure VII.I - Relationships of parent (red curve) and daughter (blue curve) achvities and activity ratio (green curve) as a function
of time. (•••• ) upper and lower estimates of activity ratio with a ±IO% measurement error; (....,) eHect of the measurement error
on the age estimate range.
a) Transient equilibrium situation. Modelled with Tp = 5.76 years and To = 1.91 years, the half-lives of 228Ra and 228Th respec-
tively and Ap arbitrarily set to 2. Insert: initial activity ratio set to 0.2.
b) Secular equilibrium situation. Ap was arbitrarily set to 2. Insert: initiat activity ratio set to 0.2.
250
C. What are otoliths made of?
OtOliths are of calcium carbonate (CaC0 3 ) crystallised on

matrix (Dannevig, 1 et aI., 1969). are
pure as has been shown a number of broad
elememal assays which demonsuated that the rotal
rities amounts to less than 1% of otolith (Edmonds et al,
. Thresher et al., 1 ProctOr et al., 1995, et at.,
1997). Recent studies have also that the otolith is
with the endolymph in which it bathes
Gauldie & 1998; Milton &
.... >ca",,!', that the fluid component may
the ultimate composition of
componems
these are critical to otolith growth and com-
In with blood endolymph is rich in K', relatively

low in Na+, has a total Ca concentration of 1-2 mM, and a
low protein content The is more alkaline than that of
the blood unlike what is observed Vercebrates
(Mugiya & Takahashi, 1985; Payan
components dis-
mechanisms are still poorly it is now recognised that the

endolymph play a pivotal role in calcium carbonate
ration and inhibition, as they also do fOf other processes of biominer-
alisation and isation (lnvolving non-I
matter) e.g. Trichet & 1995).
The otolith protein matrix was first analysed and termed "orolin"
et etl. The total contem of an otolith can be
divided in two components: water-soluble proteins and water-
insoluble (WIPs). In WSPs account for nearly half of
amino acids and have a

1993). They contain a
in the of accretion rate by aning as a calcification inhibitor
(Wnght, 1991 b). The role of WIPs is strllcmral (Cam-
pana,
251
_ Plasma (I)
100 _ Proximal endolymph (I)
Dislal endolymph (1)
Plasma (2)
_ Proximal endolymph (2)
_ D,stal endolymph (2)
g 10
.~
c
'"uc
o
U
prot Na Cl K tot Ca P04 Mg totC02
Figure VI1.2
Ionic, total C02 and protein
concentrations of plasma
and saccular endolymph The elemencal composicion of ocolichs has been broadly assessed in
in trout (I) and turbot (2), recem years for a variecy of species and environmems. Campana (1. 999)
Concentrations are given
in mM for all constituents
reponed a roral of 31 elemenrs rhar have been derecred in ocolirhs, nor
except for proteins, which including radioisoropes such Th and Ra. As anricipared, and due co
are in gJJ. Data are
from Payan et al. (1999).
improvemenrs in analyrical dececrion limics, more elemenrs have
since been dececred (e.g. Sc, Ti , V Geffen et a!. unpublished and Tl, Rb
Pomual et a!., 2000).
Figure VIl.3 shows rhe disrriburion across rhe periodic cable of ele-
menrs derecred in orolirhs. The mosr abundanr elemenrs in che
orolirhs of marine species are found in groups 1-2 and 14-17 of che
periodic table, which ref1ects ro some exrenr the relative elemenral
abundance in seawater. Transicion metals as well as lanrhanides and
actinides are less abundant.
The range of concentrations of otolith elements with respecr ro major
habitac types is summarised in figure VU.4. The dominance of cal-
cium carbonate means that Ca, C, and ° ace the major elements. Sr,
Na, K, S, N, Cl and P have been reponed in concentrations greater
than 100 ppm (minor elements) whereas all other elements are present
ar trace or infra-craces levels and can only be assayed by means of very
sensitive analyrical techniques.
le is worth noring that che elemental composicion depends on rhe
otolich CaC0 3 polymorphs. Alrhough acagonire is the normal crysral
morph for sagittm and lapiili, ic may be replaced, ofcen partially, by
vaterice (or more rarely calcire) in so-called abnormal "crysralline"
otolirhs (Car/scrom, 1963; G auldie et aI., 1993) The mechanisms of
replacement are not yet fully undersrood bur clearly resulr in different
252
Grol!P r- 1 2 3 4 5 6 _
7 ~_ 8 9 10 11 12 13 14
~_15 16 17 , 18l
'Period
Concentration scale in ilg.g- I
tiE
I
<I 10 lOO 1000 10000 Detected
2 He
Atomic number El emental symbol

2 ) Li 4Bc ~ B 9 F 10 Ne
Stable iso tope: _ Radio-i sotope:
atomiC mass atomic mass
3 11 Na 1% Mg
4 21 Se 22 Ti 29 Cu JO Zn 36 Kr
39 y 40 Zr 1 41 Nb
5
6 "L'
----;1"-----11-
" Hr
.
I
--
T, "w "R' "0. i ""
i
78 Pt 79 Au
t
7 103 L_r~I_04_ Rf I 105 Db.~ 1_06_S_g l I07_B_h_I_108HS I 109 Mt 1 110 I III 112 116 117 118
---'-- -~.- - " .. ~~~-----!
Lanthanoids
Actinoids
* 157 La
#
58 Ce
i ~
~;'~ pm ' " Sm I ~., i .. Cd I

"p, ' ''Am l"cm l "Bk i ·cr
M T-:-r: Dy , " Ho
-r-
~I-"-"'- i. Tm . '" Vb
"~OOFml "'Mdl "'NO
l-=:--1
._- \ I o
g
se
~.
n
an
::r
Figure VII.3 - Distribution ac ross the periodic table of the elements detected in otoliths. Atomic mass is reported for isotopes used either in stable isotope chemistry or radiometric dating. The '"3
~.
N
U1 concentration ranges apply to results from marine fish. Nitrogen is present in significant quantitie s as part of the protein component , but the absolute concentration is not easily quantified .
W -<
_ Marine spp.
1000.00
Freshwater spp.
_ Estuarine spp.
tlI)
100.00
""
.=;
c
0
~ 10.00
~
<=<1l
U
C
0
u 1.00
ro
<=<1l
E
<1l 0.10
w
0.01
• 11
~~~~~~00~0d~~~0~$~~q~~~~~~~
Figure VI1.4 . Summary of published otolith minor and trace element composition (mean ± 1SE) from three major habitat types.
Bars without error bars indicate element concentrations that have been reported in only one study. Calcium (not shown) was
reported as 38.02 ± 0.50 and 40.72 ± 1.82% of otolith weight for marine and freshwater species respectively. Data are from
Campana (1999).
concents of minor elemencs and trace elemencs: for instance rhe

varerite portion of a turbot orolith has lower concencrarions of Sr, Na
and K and higher concentrations of Mg and Ca than the normal
aragonite portion (fig. VII.5). Brown & Severin (1999) also reporred
decreases in Sr, Na and K in varerite oroliths, but did not provide dara
on Mg concencration. Thus, it is important to ensure that orolith sam-
ples collected for analysis are 'normal' aragonite, and crysralline
oroliths should be avoided for routine analyses. Micro-Raman spec-
troscopy can accurarely determine the presence and locarion of
changes in CaC0 3 polymorph (Gauldie et aI., 1997), and this infor-
marion can help to interpret sudden (small sparial scale) changes in
elemental composition.
Figure VI1.5 3500

Change in elemental
concentration in a partially
vaterite turbot otolith.
Measurements were
obtained using a Cameca
SX 50 WDS fitted with
five spectrometers
""
ab
.=;
c
0
.~
3000
2500
2000
SJ
-
-
- K
Sr
Mg
~
c
(Pontual unpublished data). <1l 1500
u
C
0
u
ro 1000
<=<1l
E 500
<1l
W
0
0 100 200 300 400 500 600 700

Distance from nucleus (Ilm)
254
D. Otolith elemental uptake:

how and where?
Although this question has been paid relatively litrie direct arremion
umi! now, it is crucial for applications of OMC to assess what the pro-
cess of orolith formation acrually represems.
Unlike orher biomineralisation processes, orolith formation is an acel-
lular process and is completely dependenr on the endolymph. The
cranslation of environmenral facrors inro otolith composition is a com-
plex process which involves four nested comparrmenrs:
- the external medium, where variations in abiotic facrors occur;
- the blood plasma which responds ro the external medium bur also
exhibits endogenous variations;
- the endolymph, which modulates the various signals and regulates
the formation of the otolith;
- the orolith itself, which inregrates and records a response ro all these
signals.
Our currenr state of knowledge abour each of these stages in oroli th
formation , and how each affects oroJith composition , is summarised
below.
1. Between the external medium (water and food) and the blood
plasma
The inorganic plasma conrem is mainly derived from the surrounding

waters, either by branchial uptake in freshwater fish or by assimilation
through the inrestinal epithelium in marine fish, which swallow con-
siderable quamities of water as pan of their osmoregularory process
(Simkiss, 1974). Uptake processes depend on a large number of fac-
rors, including chemical elemenr propenies (see e.g. Phillips & Rain-
bow, 1994). Schematically, uptake of major alkaline meral ions such as
Ca, Na , Cl, and non-merallic ionic species such as chloride, sulphate
and phosphate occurs through active cranspon pumps and is highly
regulated. Trace metal uptake depends on [he rela[ive ambienr ionic
concenrra[ions, and may be species- and environmenr-specific. The
uptake process is regarded as passive, not requiring expenditure of
energy. It depends on [he affinity of water-soluble ions with organic
ligands (e.g. metal-binding proteins) presenr in [he membrane of [he
exchange surfaces.
Elemenral uprake also depends on abiotic characteristics of the envi-
ronmenr such as pH, salini[y, dissolved oxygen, and temperature
which determine the concenrra[ion of free ions available for uptake.
255
Along wirh uprake from surro u nding warers, some elemenrs, espe-
cially essenrial micronurrienrs and conraminanrs, are ar least partly
supplied by rhe di et, as has been shown by several studies offish nutri-
tion a nd ecoroxicology (see e.g . Bijveld s et al. , 1998; Cooley &
Klaverka mp , 2000). At the presenr tim e, it is difficult ro generalise
about the incorporarion parhways of all elemenrs. Orolirh Sr, in par-
ri cular, has been shown to respond to assi milation from food and warer
in juvenile shad Alosa sapiclissima (Limburg, 1995 ), juvenile rilapia
Oreochromis nilotiCtts (Farre ll & Campana, 1996) and juvenile G trella
elevata (Gallahar & Kingsford, 1996) but only from water in red drum
Sciaenops ocellattts (Hoff & Fuiman, 1995).
2. From the blood plasma to the endolymph
Very few studies have exam i ned rh e relationshi ps berw een blood
pl as ma and endolymph chemi srry. Following pioneering works on
otolirh biomineralisation by Mug iya and co-workers (Mugiya, 1966;
Mugi ya & Takahas hi, 1985), Kalish 0991a) examined rhe effects of
physiology and environmenr on otolirh chemisrry by analysing sea-
sona l variarions in otolirh composirion (Ca, Sr, Na, K, S) in relation to
var iarions in rhe chemisrry of rhe blood plasma and rhe endolymph.
Bo th ioni c conrenr and various meraboli res such as prorei ns were
analysed . In combination, rhese derermine rh e proporrion of free ions
exchangeable rhrough the saccular epithelium. Resulrs indicared rh ar
rh e physiologi ca l (reproductive) sratus of rhe fish was rh e driving fac-
tor in rhe seasonal variarion of rhe otolith Sr/Ca. Srarvarion slightly
decreased plasma Ca co nce nrrarion, and m odifi ed rhe acid-base equi-
lib ri um in rhe blood plas ma as well as in rhe end o lymph, bur rhe ionic
conrenr ofb orh fluids was similar in starved and feeding fish (Payan et
aI. , 1998) Edeyet et cd. (2000) showed rh ar plasma and endolymph
composirion varies during rhe da ily cycle in Scophthalmm maximllS, but
ioni c gradienrs wirhin rhe endolymph (Payan et aI., 1999) are ma in-
rai ned . There are no published data on rhe conrenr of trace ele menrs
in rhe end olymph, and rhe transporr m ec hanisms for various eleme nrs
rhro ugh rh e sacc ula r epirhe li u m are srill being debated. Given rhe
complex ce llular mapping of rhe saccular epirhelium, including dif-
ferenr types of ionocyres, (Ta kagi , 1997 ; Pi sa m et al., 1998), ir is likely
thar active and reg ulared ionic rranspo rrs occ Llr rhroug h rhis i nrerface.
Kali sh 0991a) inferred from rhe correspondence berween blood
plas ma and endolymph Sr/Ca rhar Ca and Sr may be transpo rted into
rh e endolymph by a paracell ulat roure . Mug iya & Yoshida (l995),
working on isolared sacmli., concluded thar rhese elemenrs were rrans-
ported by a rransce llular roure in CaYassim aurata .
256
3. From the "'nrlnl~'mr\n to the otolith
The extenc to which variations in endolymph composition ultimately

affect the of the otolith has not been established
yet and the scarce data available on this subjecr deal with calcium
and a few minor ions. Although there are obvious
between the of endolymph and that of the otolith
(Kalish, 1991a; Payan et aI., the correlation between the
two media may be not as high as had been Data ftom
Kalish (l991a) indicate that seasonal variations in endolymph and
otolith did not match for Na, K and
similar
showed that the increase in the disral K concentration was
reflected in the distal otolith K/Ca ratio. However the difference in
Na/Ca between the and distal sides of the otolith could not
have been explained
Such studies pose various both due ro the characteristics of
the endolymph and the orolith and to methodological limitations. In
the first the resolution differs for the measuremenc of
endolymph and orolith in tbe endolymph can
be measured instantaneously, diurnal variations
et aI., 2000) ID the orolith are over
which depends both on the diameter of the analytical probe (see
and otolith rate. Second methods of
endolymph composition have usually been restricted ro
total ionic content, while it is the concentration of free ions
(i.e. not linked to that is the relevant infot-
mation in tefms of otolith element methods of mea-
chemical composition may also be limited by imer-
ference for some elements of interest.
processes have not been studied in
adsorbed
whereas elements such as Na,

surface in the interstitial space
nothing is known about the elements that could be bonded to the
",VU""'"", apart from the elements that make up the amino
0, N, P, S). In general, more information is available

about the' of cations than anions. In the past the incor-
of divalent cations into the mineralised portion of the otolith
257
has been assumed to be by substitution, whereas monovalent cations

are thought to fit into inter-crystalline spaces. However, as the mea-
surement of trace elements becomes more reliable, the existence of dif-
ferent mechanisms of incorporation will have to be acknowledged and
reconciled with models of otolith composition. From this perspective,
the current lack of data on the elements associated with the protein
component of the otolith is especially problematic.
4. Elements that are likely to vary depending

on environmental availability
This question is obviously of major concern in OMC. Campana (1999)

identified elements that are likely to vary depending on environmen-
tal availability, on the basis of arguments which are briefly sum-
marised here:
First, distribution coefficients De between water and otolith are use-
ful indicators of elements that are highly physiologically regulated
(i.e. discriminated for or against at one or more of the interfaces: gills,
intestine, saccular epithelium, otolith). The distribution coefficient is
De = (element/Ca)otolich I (element/Ca)wacer with element and Ca refer-
ring to molar concentrations.
Very low distribution coefficients « 0.05) are observed for elements
such as Na, K and Cl, whereas Dsc is about 0.14 and, for many trace
elements, the distribution coefficient is greater than 0.25 and may
even apptoach 1.
Secondly, comparisons of published data on water, blood plasma and
otolith compositions (normal ised to Ca) for freshwater and marine
species lead to inconsistent relationships for Mg, Cu, P, and Na. The
consistency was greater in the case of elements such as Sr, Zn, Pb, Mn,
Ba and Fe, indicating that the relative environmental abundance of
these latter elements may be well reflected in the otolith. Campana
(999) also noted that Li, Cd, Ni and other less abundant elements
may well respond to environmental availability.
This classification remains somewhat speculative, and awaits further
investigation. As noted by Thresher (1999) the distinction between
minor ions that are physiologically regulated and "unregulated" trace
elements may be problematic, given that some trace elements are
known to be involved in fish metabolism as essential mictonutrients,
or are toxic (or both, depending on the concentration level). For
instance, Zn is known to be an essential micronutrient that is involved
in the formation of fish bone and cartilage, becoming toxic at high
concentrations. Cadmium, which is also present in the environment ,
is a toxic metal thought to interfere with Zn metabolism. The intesti-
nal assimilation of both elements has been shown to differ, possibly
258
because of the presence of carrier-mediated lO
the intestine at., 1 It IS
not for annual pattern of Zn

concentrations observed in Salvelinus otoliths reflects
in environmental or fish metabolism (Halden et at.,
2000).
259
E. Applications of otolith microchemistry
1. Patterns of migration and environmental history
1.1. Salinity
1.1.1. Migration behaviour as revealed by Sr/Ca ratio
Seawater Sr and Sr/Ca exhibit a relatively small (2-3 % ) spatial vari-
ability with surface water slighdy depleted relative ro deep ocean and
highest surface values found at high latitude and upwelling areas (Vil-
liers, 1999). Although highly variable depending on geology, weath-
ering and hydrographic conditions, freshwatet Sr and Ca concenrra-
tions are much lower (about 60 pg/ I a nd 1.510 4 pg/ l respective ly
versus 8000 pg/l and 4.2 105 1lg /1 in seawater). Tbe Sr/Ca variability
(of about a facror 5) between fresh and salt water has supported the use
of otolith Sr/Ca ratios in the investigation of the migration strategies
of a number of species. Sr/Ca ratio can be measured using various tech-
nigues, some of which can provide high temporal resolution (fig.
VII.5 and chap. VILG)
Speci es that undertake diadromous migrations during their life cycle
have been extensively studied. Anguiliformes (Anglliffa spp.) exhibit
a clear decrease in Sr/Ca ratio when they migrate from the marine
environmenr ro freshwater (Casselman, 1982; Otake et af., 1994;
Tzeng et af., 1997). However, comparisons of the Sr chronology with
metamorphic checks on the orolith have indicated that the initial
decline in Sr/Ca is not related ro the habitat as such bur ratber is asso-
ciated with th e leprocephalus-glass eel transformation (see below).
Such chronologies have also provided information regarding the rate
at which fish enrer the estuarine nursery grounds.
Close relationships between otolith Sr co ncenrration and ambient
salinity have also been found in Salmonids and have been used ro dis-
tinguish between anadromous and non-migrarory individuals (Kalish,
1990; Halden et a f., 1995; Babaluk et af., 1997). Some cases of resi-
dency have also been idenrified in striped bass Morone saxatifis popu-
lations previously thought ro display a nadromous migration
behaviour (Secor et a f., 1995b; Secor & Pi ccoli , 1996).
OroJith Sr concenrration has also been used ro infer the down-estuary
movements of juvenile Afosa spp. (Limburg, 1995, 1998), age-and
size-specific coasral dependency of adult Morone saxatifis and spawn-
ing migrarions of differenr species (Secor & Rooker, 2000).
Field studies have bee n confirmed by laborarory vaJidations. For
instance, Sec or et et!. (1995b) showed that Sr/Ca ratios in the oroJiths
of juvenile M. saxatifis were positively related to ambienr salinity.
260
These amhors also showed that the otoliths of fish to various

sa[inities also showed tlucruations in the Sr/Ca ratio that
to the satini ty et af.,
Sr/Ca ratios retlect the ambient Sr/Ca ratio rather than per se or
absolute Sr water concentration. This may why studies that
over a narrow range of variation Fowler et
af., failed co establish a clear between otolith Sr/Ca
and ambiem salinity.
do not fracrionate through

and assimilation. This hypothesIs was first validated on
nde Satmo sCllar et al., et a/.
(2000) showed that food contributes to the of
that incorrect identification of the ori-
of individuals may result when are based on
water source & \Veber, 1999).
the remperarure and sal of water of juvenile
Bn?1Joortia patrontlJ, et af. (998) found chac otolith
did reflect the water buc was not affected by
water temperature. concluded from their
results that the utility of Sr ratios a indicaco[ of
is limited ro low salinity environments « or over wide
ranges of
1.1.3. Stable '''V'.VI.I''''

Water masses are often characterised differences in oxygen
ratios and fish movements between water masses can thus be traced
the oxygen catios of the oroliths. Both temperature
tion affeCt the otolith oxygen ratio. In
marine environments, the range of water ratios is narrow, and
most of the variation in orolith oxygen ratio is due to temper-
ature differences VIL6a). freshwaters vary
in oxygen ratio because of the sources of these waters. The
otolith oxygen ratios of fish from freshwater
and from coastal waters affected freshwater
influenced by both temperature and the oxygen ratio of
the waters VII.6b). Accurate temperature
tions from these environments concurrent of water co
261
measure rhe environmenral isorope rarios. However, for migrarion

srudies, relarive values of isorope rarios may be sufficienr. For example,
differences in isorope rarios can be used co disringuish berween
migraring and residem fish (Nelson et af., 1989; Kalish, 1990; Norch-
core et af., 1992).
In pracrice, orolirh oxygen isorope rarios have co be measured from
bulk samples (from dissolved whole orolirhs or cores), so ir is difficulr
ro rrace small-scale movemenrs and migrar ions in i nd i v id uals.
a Water 8 18 0
·1.2 .(J.8 ·0.4 0 0.4 0.8 1.2
3.8
Norlh Atlantic
Western At/antic
i\outh Atlantic
Anfarctlc
North Atlan re
3.6
.f?
;;,
~
0
0
3.4
_ Effecl oflemperature
Effect of water (over marine conditions)
3.2
0 10 20 30
Water temperatur e (OC)
b Water 8 18 0
·16 ·12 ·8 -4 0
3.8
............ North Atlantic

Wes lern At/anlic
South Atlantic
North Atlantic
Antarctic
3.6
Lake Baikal ''-,
.... ............
laurentian Great Lakes
.f? ........
;;,
~
.............
0
0 .......
3.4
- Elleel 01 temperature
_ _ _ _ _ [ lIeel of water (over fre shwater
and marine conditionsl
3.2
0 10 20 30
Water temperature (OC)
Figure VI1.6 . Otolith oxygen isotope ratios (otolith ( 180) in relation to water temperature (solid
line, lower axis) and in relation to water isotope ratio (waterolSOl.
a) Across representative marine environments as indicated.
b) Across representative freshwater and marine environments as indicated.
262
Developments in the use of microdrill s allow discrete samples of

otolith material to be removed along tracks that follow growth zones.
Up ro 20 discrete samples can be taken within each annulus with the
aid of computer-controlled micro-milling systems (Wurster et aI.,
1999; Weidman & Millner, 2000).
1.2. Temperature
1.2.1. Sr/ Ca ratios as indicators of individual thermal history?
Early geochemical studies predicted that increasing amounts of Sr
s hould substitute for Ca in inorganic carbonate as temperature
dec reased (Kinsman & Holland, 1969, in Gallahar & Kingsford
(1996), and Sr/Ca ratios have been measured in coral skeletOns as a
recording thermometer (e.g. Smith et aI., 1979). With regard to
otoliths, numerous field and laboratOry experiments have been per-
formed, but these have provided conflicting conclusions. The rela-
tion ship between SrlCa ratio and temperature has been reported as
either negative, positive, non-existent or inconsi stent (tab . VIL1).
Table VII.l. Some reported effects of temperature on Sr/Ca ratios (see also Secor & Rooker, 2000).
Species Life stage Experiment TOC Sr/Ca Comments Reference
~f(T)
A rripis Imlla Juvenil e Lab-rea red 13- 22 -+.?' No clear relationship (Kalish, 1989)
F"ndldllJ Larval Lab-tea red l 6-3 2 .?' .... Adju sred linear model (R.dtke &
hm roc/illlS Morales-Nin , 1989)
CII/pea barengllS Larval Lab -reared 6·13 .?' .... Adjusted linear model (Rad rke el al., 1990)
CII/pea harer/gm Juvenile Lab·reared 2· 18 .?' .... Adju sted hypetboli c (Townse[ld tI aI., 1992)
mod el
GadllS /lIorIJlta Larval Lab·reared 5·14 .?' .... Adju sred exponenrial (Tow nse[ld el aI., 199 5)
model
Haenllllotl Adult Field 1l-30 -+.?' Inverse relation sh ip (Sadovy & Severin,
plll/nieri betwee[l Sr/Ca and 1992)
body g rowrh rate
EPim-phclllS Adult Field 17·30 -+.?' Inverse relat ion shi p (Sadovy & everin.
g llllCl!liJ between Sr/Ca and 1994 )
boo )' g rowth rare
Sciclenops Larval Lab reared 2[· 34 .?'.?' Adjusted model (Hoff & Fuiman, 199 5)
ocellalllS Juvenil e (Sr/K, Ca , Na);
al so in vesrigared dier
and salinity
Pagrm major JuvC[lile Reared 21· 29 .?'.?' Also inves tigated (Arai eltd.,1 995)
(sea farms) Fe, Mn, Zn
Gire/la elevala Juve[lile Lab reared 19 & 28 -+.?' In consi stent effecrs of (G allahar & Kingsford ,
increas ing an d decreasing 1996)
rem pe rarure~ also
investigared Sr-enriched
warer and di er
",IOI'one saxalilis juvC[liie Lab rea red l5 &25 -+.?' , .?' .... Inconsistenr effect of (Secor el at., 1995b)
T with varying sa liniry
263
Kalish (989) early expressed doubts about the universal temperature

dependence of the Sr/Ca ratio in fish oroliths, suggesting that the
endolymph Sr concentration may vary seasonally and with age in a
given species rather than respond directly ro temperature.
Townsend et al. (992) argued that at extremely low temperatures fish
become increasingly unable ro physiologically control the penetration
of Sr into the endolymph by the concentration of calcium-binding
proteins in the plasma. These authors also noted that the slowing of
growth and metabolism in winter provided supporc for the hypothe-
sis of physiological interference processes on the incorporation of
strontium. The latter hypothesis was supported by Sadovy & Severin
0992,1994) who did not find any consiscent temperature effect bur
related the SrlCa variation ro that of body growth rate.
The apparent inconsistency of a general temperature effeer has been
"explained" by several authors as the resulc of the regulation processes
of strontium incorporation (and more generally on elemental incorpo-
ration). In view of the potential dependence on multiple endogenous
(species, life stage, age, somatic growth, sex, reproductive status) and
exogenous facrors (temperature, salinity, "s tress" , diet), and their
interactions, it appears to be much more complex than previously
thought.
1.2.2. Stable isotopes

The 8 18 0 of marine organisms has been widely used in che geological
sciences ro estimate temperature conditions during the life of organ-
isms. The use of fossil and living coral and Foraminifera is a standard
rool in determining climate variation over geological periods and the
literature on the subjeer is enormous. Recent papers provide examples
of che wide scope of these studies, including analyses of oscillations in
El Nino events in the Pacific using reef-budding corals (Wellingron
& Dunbar, 1995), global temperature changes, based on biogenic
silica (Shemesh et aI., 1992) and estimation of warming in inter-
glacial periods in the Aegean, based partly on fossil Foraminifera
(Aksu et al., 1995).
The applicarion of these techniques depends on the assumption that
the fractionacion of the oxygen isoropes during the precipication of
biogenic carbonate is governed by predierable quanrum energy equi-
librium conditions derermined solely by physico-chemical properties.
Because the chemical bonds of the various isotopes have different
strengths, temperature affects the ratio of these isoropes as they par-
ticipate in chemical reaerions, in this case as che isotopes of an element
precipitate into calcium carbonate. The 8 1R O increases as rhe temper-
ature decreases, since the 18 0 bonds are stronger and less thermal
energy is available co release these isoropes in chemical reactions.
264
There are cwo imporranr pcocesses chac may affecr oxygen

and rhus che temperarure estimates, from carbonate.
of "vital effens" which encompass
differences in [he
"WJh'"'' processes rhac potenrially aHen the
differences have been noted, When
are (lnribured ro bio-
chemical and mecabolic facrors that result in modificarion of rhe i50-
"r,><pru"rI in calcium carbonate, Variations in
rate, for example, may cause In kinetics and

incorporation of carbon dioxide in addition co the reservoir
of marine dissolved i carbon which is normally utilised
calcification, Under these circumstances the record
by a function of temperature
variation and will depend in parr on faccors which may
be It is therefore vical co test the of
equilibrium isorope rioning, and to be able (() lest
the relative influences of individual metabolism (or
rare) and temperature. Ir is also
peracure differenr carbonate materials such as
may differ in their concenrrarions (Grossman &
The second facmr that affects the isocope rario of carbonates is the iso-
cope raoo of rhe water (see Salinity and source of water are
the sources of variation in water oxygen ratios.
marine waters vary over a narrow range (-0,30
values of than those of mid-oceanic
relative to SMOW (more nega-
fresh waters show
greater for example: the value of Lake Baikal is 15.8
and the Laurentian Great Lakes vary between -6 and -9.
The use of aspects of the of extant
fish been based surveys of field -collected
often from a wide range of habirats (Devereux, 1967; Mulcahy
ettti., Nelson et al. , 1991; Iacuminetal., 1992;
Meyer- Rochow et al, 1 aI.,
1
mosr studies on fish have found a rela-
between remperarure and oxygen rarios, Carbon iso-
influenced by ambient
temperarure Kal 1991 Radrke
et at., 1 1998; Thorrold et aI., 1 1999)
However, few studies have controlled or monirored
rant fac(()[s that can introduce added such individual
rate or the ratio of the water in rhe
265
ranks . In a carefully comrolled experimem, Thorrold et al. (1997)

derermined rhar oxygen isorope rarios were a reliable indicacor of rem-
perarure in Micropogonias tmdulatus, and were relarively independem of
individual g rowrh rare.
A larg e number of published equarions relare warer remperarure and
oxygen isocope rarios in carbonares (rab. VII.2 and fig. VII. 7). These
relarionships are derived from differem raxa and somerim es differem
polymorphs, and rhe primary di fference berween rhem is rhe fi((ing of
polynomial v. linear models. There are also differences in imercepr and
in som e cases also in rhe slope (fig. VII.7). Few srudies have compared
g roups of spec ies over a wide range of remperarures. Experimenral
work has only been carried our on rwo marine fi sh species (iHicropogo-
nias tmd,ilatlls, (Thorrold et al., 1. 997) and GCld"s morhltcl, (Radrke et at.,
1996, 1998» and on e freshwarer species (Arripis trtttla , (Kalish,
1991 c), and rh e remperarure ranges in rhese srudies do nor overlap. Ir
is rherefo re difficulr co generalise abour rhe exacr relari onship berween
remperarure and rhe isoropic rario of fi sh ocolirhs. Ir is more likely rhar
rhe relarionship is curvilinear, alrhough rhe published pol ynomial firs
are derived from Inverrebrares and nor Fi sh.
All rhe ava ilable experimenral ev idence indicares rhar a reli a ble rela-
rionship exis rs berween rh e ox ygen isocopic composirion of ocolirh
aragonire and enviconmenral condirions experienced by individual
fish, even rhough rhe dererminarion of absoJure remperarure depends
on rhe equarion used (rab. VII.2). There are many applicarions in
which relarive changes in rem perarure are valuable, and rhe accuracy
of rhe remperarure esrimares is rherefore nor ar issue. H owever, where
accmare remperarure esrimares are required, more experimemal dara
is needed co berrer d efin e rhe exacr form of rhe relario nship berween
orolirh oxygen isoropi c rari os and acruai warer remperarures.
266
Table Vll.2 - Publi shed equations relating water temperature and oxygen isotopes ratios in carbonates .
Taxa CaC0 3 Temperature relarionship References
polymorph
M olluscs (Epsrein
aragonire et aI., 195 3)
Nor specified Calcire
forami n i fera Aragon i re TOC = 20.6 - 4.:34 (0 1BOcarb - OI ROw>t) (Gwssman
1986)
& Ku ,
NO[ specified Calcire TUC = 16.0 - 4.14 (0 1 Ocarb - 0180 war ) + 0.13 (0180carb - 0l SOwar)2 (Anderson.
1990)
Aragon ire Toe = 20.52 - 3.067 (oJ 80carb) (K alish ,
1991a)
Fish (Tho[wld
el aI., 1997 )
Fi sh (Radrke
el aI., 1998
Figure VII.7 24 - - - Kalish 119911

Comparison o Grossman & Ku 119861
of the temperature .~ - - - Cra ig 119651
estimates obtained from Q) Epsteln el al. (953)
0-
otolith oxygen isotope ratios o Anderson (990)
by using different published ~ 20 Radtke et al. (998)
isotope-temperature o§
relationships shown '0
in table V11.2. '0
E
g ]6
"0
Q)
ro
E
"5;
Q)
~ 12
ill
0-
E
Q)
I-
8-+----.----.----r----r---.----,---~----._--_r--_.
0,00 0,40 0,80 1,20 1,60 2,00

&18 0 - otolith oxygen Isotope ratio
2. Metabolism and ontogenic events
A number of faerors imeracr ro affeer rhe composirion of fish orolirhs.

These fa crors complicare rhe imerprerarion of composirion and have
somerimes led ro erroneous conclusions abour rhe relarionship
berween environmemal condirions and orolirh composirion. Orolirh
composirion reflecrs rhe combined effecrs of endogenous processes
such as developmem and reproducrion, and exrernal condirions asso-
ciared with habirar, behaviour and dier change. The following secrions
267
disc uss the influence of physiological processes on the availability and

incorporation of e lements into the oto lith.
Metaboli c rate is a major factor determinin g otolith growth, and for
thi s reason may influen ce otolith composition through the control of
the rate of incorporation of inclividual elements. The metaboli sm of an
individual fish will al so determine its exposure to the elements in the
external environm e nt by influen c ing food consumption and the
exchange rate across the g ill s and epithe lia. These factors may link
metabolism and otolith composition in a general way, so that otolith
conce ntrati ons of different elements will vary between individuals and
a lso within individuals over seasonal cycles. Several studies of metal
concentrations in fi sh otoliths have noted increased concentrations in
the otoliths of faster growing fi sh (Papadopoulou et al., 1978; Prora-
sowick i & Kosior, 1988), which may indicate that higher metabolic
rates are linked to higher rates of incorporation of some elements.
The best eviuence for the influence of metabolism on otolith compo-
sition comes from studi es of Sr/Ca ratios. Fluctuations in Sr/Ca were
initially interpreted in terms of seasonal temperature variations, but
evi d ence has acc umulated that links SrlCa ratios more closely with
i nd i vidual metabolism. Invers e relationsh i ps have been reported
be twee n SrlCa and somatic growth rate (Sadovy & Severin, 1994;
Fried land et aI., 1998), suggesti ng thar more srrontium may be incor-
porated into the orolith during periods of lower otolith protein sy n-
thesis and reduced accretion rate.
There is direc t experimental evidence that metaboli sm is a factor in
determining the (51 3c ratios in fi sh otolitbs (Kali sh , 1991b; Thortold
et aI., 1997). Unlike (5 18 0 , (51 ?C does not always show a close rela-
tionship to temperature and it is often close ly related to individual
fi sh s ize and growth rate. It has been suggested that so me of the
otolith carbonate may derive ftom metaboli sed carbon sources (entering
the plasma as CO 2 ) and thus relate to fish metabol ism rathet than to
ex ternal conditions.
Many of the changes observed in otolith composition have been charac-
terised as ontoge netic variations. For the mos t part, it has yet to be
determined whether variations in composition are direc tly linked ro
d evelopmental events, and thus are strictly speaking ontogenetic, or
whether the variation is a functi on of fish size or age . Cyclic variations
in otolith composition have been observed in life history transects, and
the amplitude of these variations is often lower in the outer secti o ns of
th e otolith. This pattern is certainly a function of fish age; perhaps, for
example, if adult fi sh move to minimi se changes in temperature. Size-
related change s, age-related c hanges, and stage- (development)-
related changes are o ften close ly coupled in fish, and it is therefore dif-
ficult to differentiate between purely ontogenetic pa tterns and
268
Otolith microchem;stry
occur with
should have clear link to
Some variations in orolith both
and size-related, For variarlOn due co
macuracion and has borh size and
componencs.
than
In other cases, sllch as

seem co 6sh size, There are
ofren trends 111 cbe concentrations of different elements across
bur co avoid confuslOo chese should nor be treated as
unless are lInked co
seasonal ilucruations should Doe be confused with oncoge-
oce these may be induced external condi-
2.1. Metamorphosis
In some of flsh, the rranSlLlon between the larval and
increase in increment width in

I'(!Jtmt(./ and
drastic are 110( believed to be relaceo to habirat Orake

et al. (1 (997) assoCIated [he decrease in orolich ratio [0 chat
ofehe the breakdown of
which
cion fcom coasraI waters into estuaries, if similar were not also
observed in the owlirhs of individuals held under constam
condirions. Fowler et cr/. (1995) described Ji1
between otOlirh cores and in fish held in constant
condi[Jons.
in habicat associated with
o(Oli[h Shlfts in dle(
this is
physi o logi cal cha ng es during meta morphosi s have ye t to be

addressed , whether experimemally or in the field . Processes such as
ca lcifi cation and developmem of rhe circularory sysrem can limir rh e
el em e nrs and con cenrrarion s avail a ble for in corpo rari on imo rhe
orolirh. D evelopmem of enzyme sysrems in respo nse ro dier shifrs may
iniriare comperirion for various meral s thar ma y have been IO corpo-
rared imo larval orolirhs .
2.2. Reproduction
Two aspeees of reprodueeion have rhe poremial ro affeee rh e composi-
rion of fi sh oro iirhs rhroug h endog enous ph ysio logi cal processes. In
female fish gamerogenesis invol ves the rranslocarion of m aterial for
yolk in rh e developing ova. The process of vi relloge nesis requires cal-
cium and rh ere is an increase in rhe calcium-binding p roreins pro-
du ced ( in p arcicul ar vitellog enin ). Th e effecc on the o colith is co
reduce the ava ilable concemrations of calcium and divecc protein syn-
thesis. The spawning checks observed in the ocoliths of adult female
fish ma y be the visibl e result of this process, although litde work has
been done co systematically dete rmine the soutce of these fearures . In
terms of ocolith composition , f1ucruati o ns in the ratio of minerali sed
co organic co mponencs would be expecced, a nd there may also be
accompanying shifts in the in corporation rate of ([ace elemem s.
Both male and femal e fish lay d own fat deposits that can be ucili sed as
energ y scores both for gamerogenesi s and for m etabolic requiremencs
during the repr oducci ve season , especially if there is no feedin g .
Because man y elemencs can be readily incorpo rated inco fany tissues,
the mobilisation of these reserves can induce or accencuate season al
f1uee uati ons in these elemencs in the ocolith.
Few auth ors have direcd y investigated the efFeees of reproduccive
acciv ity on ocolith composition, despite the impacc this could have on
the planning of surveys and the imerpretation of population results
(Thresher, 1999). Fluctuations in the maj or orolith elemems, Ca, Sr, Na,
and K, have been imerpreted as indications of reproduccive accivity in
seve ral scud ies (Fui man & H off, 19 95 ). Kali sh (1 9 89) corre lated
changes in gonadosomati c index with blood plasma concenrrati ons of
calcium-binding proteins, Ca, and Sr in A rripis trtttta and linked these
co f1ueeuati ons in ocolith Sr/Ca ratio. Experimencal wo rk on Poecilia
retiCtllclta (Th reshe r, 1999) sug gesred that increases in orolith Sr and
N a we re ofte n associa red wi t h spawning ( brood d evelopmenc )
epi sodes. However, in these and other species rhe variability among
individual females is hig h and it is d ifficult to generalise abour the
exaee effeees of reproduccive accivit y on specifi c chang es in orolith
compos ition. Because rhese changes are a funccion of endogenolls pro-
cesses , it m ay be that in d ividual va riability is inhetem and that cyclic
270
changes in some elements (Sr, Na) could provide more information

about individual s (for example, about reprodu ctive effort) than about
populations.
3. Age estimation
Fish age estimation using otolith chemical compositi on does not com-
pete with conventional age estimati on m ethods, but has rather been
devel oped with the aims either of valid ating the latter when they are
not easily applied (e.g. long-lived species) or of producing a n ag e
range to he lp develop conventional ageing criteria. Three kind of
methods have been investigated:
- radiometric methods based on natural radioisotopes disequilibria;
- nuclear weapon s testing 14C tracer ;
- cyclical variations in otolith elemental composition.
3.1. 21OPbj226Ra and 22BThj22BRa disequilibria

The two nuclide pairs of interest in otolith radiomecric dating are
210Pb;2 26Ra a nd 228Th j2 28Ra, which are parts of the natural 23 8 U and
232Th decay series respectively (tab . VIJ. 3). Both pairs involve iso-
topes of radium that oc c ur naturally in freshwater a nd seaw ater.
Radium is a calcium ana logue , following similar metaboli c pathways,
and thus can accumulate in cal cified structures . 21 0Pb and 228Th are
also present but in lower abundan ce. All have very low co ncentra-
tions and hence activity, which causes ptoblems for the precision of
m easurements.
Both pairs may be considered as simple daughter/parent decay pairs
since the intervening isotopes in both cases are very short-lived (tab.
VII.3).
Table VI1.3 - Fish otolith radiometric dating: radionuclides of interest

from the 238U and 232Th decay series with corre sponding half-lives.
Dashed lines indicate when intervening radionuclides are not shown .
23 8 U series Half-life 232Th series Half-l ife
2:l SU 4 .49 109 yea rs 232Th 1.4 1L0 1O yea r
22R R a 5.7 6 years
226Ra 1622 years 228Ac 6.1 3 hours
222Rn 3.82 d ays 228Th 1.9 1 year
21 Bpo 3.05 minmes
214Pb 26.8 minures 208Pb 5mble
214Bi 19.7 minU(es
214po 164 microsecon ds
210Pb 2 1 yea rs
206Pb St ah le
271
The 210Pb/226Ra method, whi c h follows the sec ul at equilibrium

sc heme , should theoretically be applicable ro age es timation over 100
years (five times the 2JOPb half-life) . Actually, current levels of ana-
lyti ca l precision makes it m os t suitable (i.e. with an acce ptab le un cer-
tainty) within the range of 0-50 yea rs (Campana, 1999) The limiting
facrot is now the measurem ent error of 21 0Pb since the d eterminari on
of 226Ra acrivity In otoliths has recently been imp roved (Andrews et
(If. , 1999b).
228Ra a nd 228Th hav e similar half-lives so that the it activity ratios
vary with time acco rdin g ro th e tra nsi e nt equilibrium scheme.
According to Campa na (99 9) the app li ca ble age-range is 0-8 years
(see also fig. VIII ).
The main difficulty in radiometric dating is caused by the continuous
growt h of the orolith throughout the life of the fi sh. If whol e o coliths
are used, continuous deposition of new m ate ri a l must be taken in ro
accou nt and t he radiometric eq uatio n reformulated usin g a mod el that
accounts for the increase in mass ove r time . Such equations, whi ch
were firs t d er ived by Benn ett et et!. (982) in the ir pi o neering work on
fi sh radiom(~tic age ing, were subsequ en tly correc ted and ada pted co
various growth models (Ca m pa na et a!. , 1993; Kimura & Kastelie,
199 5) . On ~ he other ha nd, assum ing th at th e core has roughly the
same age as the fish (plus the time between coll ection and ana lys is) ,
the Batema n eq uat io n can be directly applied when only core material
is analysed. This method w as first employed by Campana (990 ) and
should be prefe tred wh en it can be used.
Three assumptions are fundam ental ro radiometric ageing:
l. Once d eposited, radionuclides do not m igrate within the oto lith
and losses (gai ns) of an y radio nuclid es of interest, including inter-
vening radi on uclides, only occur throug h the decay (in growt h) pro-
cess. Tn orher words, the orolith ac ts as a closed chemical system .
2. The initial acrivity of daug hter/pa rent pairs in tbe otol ith is low,
idea lly cl ose ro zero and ca n be est imated .
3. Both parent and daug hter nu c lides are incorporated into th e orolit h
a t a rat e th at always maintains a con stant ratio co the rate of mass
increase of the orolith .
The question of "closure" is the most diffi cult co answer and has been
strong ly debated throu g h theoretical a rguments in severa l papers.
Except for the work of Gauldie & Cremer (1998) , whic h demonstrated
the poss ibil ity of diffusion of 222 Rn o ut o f oro lit hs of Hoplostetblls
atlClnticlIJ, there is a di stinct lack of experimental ev idence that would
validate the assumption of closu re for th e radionuclides of interes t in
oroliths. ]n poi nt offac t, a sig nifi ca nt loss of222Rn during 226Ra decay
should result in lower 2lOPb activity and hence lower age estimates.
However a num be r of works have res ulted in o ld rad iom etric ag e es ti-
mates that roughl y corroborate the estimates m ade using convention al
272
Certain methods of orolith srotage and

been shown to affect the stability of fine-scale
chemical distributions wirhin the orolirh
Procror & radiometric
JSSlle internal roscud
the core method.
The second (i.e. from an
nuclide or any incermediare
be satisfied if ocoliths of young fish can be
llsed co estimate InitIal ratio. If the core
method is used tbe "reference should be of the same age the
adult core and be available for and locations.
The third necessary when whole orollths and an
otolltb model are used. It the rate of
is an process
thar the otolttb mass
rare is known or assumed, which a circular argumenc if a
model is bujJt lip on the basis of Clmw//IJ-based ages chat have
co be validated (West & 1 Klmura &
1995)
has been Llsed of
nor oroliths can be for
convencional ageesrimatlon (Smith al,1991; al,1993)
the associated with the Ra assay (since 15
this method could have
ID
such
et cl!., 1990; Kastel le e/
l'en?ICO.fll.f (Stewart ct!., 1
(Andrews et cd., l'vlacmrOl'lm nOVC!l:zficllldiae (Fenton et c1l., 1990)

and spp. (Milton ai, 1995).
3.2. Atomic bomb radiocarbon chronometer

the dis-
when le collides wJCh cosmic
back co I 5
radiometric method (based
on lIse for age validation purpose see
Kalish, 1995). However, natural atmospheric radiocarbon levels have

been modified by human activities since the beginning of the indus-
trial revolurion (fossil fuel combustion, nuclear power plants) and
were particularly srrongly affec ted by atmospheric nuclear weapon
tests during the 1950s and 1960s. In less than ten years the atmo-
spheric concentrations of 14C doubled after remaining fairly conStant
for several thousand years. Since the end of the testing period, this
concentration has been falling (fig. VII .Sa) as the radiocarbon, in its
oxidised form 14C0 2 , exchanges with the other CO 2 reservoirs. As a
result the upper layers of the world's oceans have undergone a signifi-
cant input of radiocarbon which is clearly visible in 14C time-series
derived from measurements of either seawater-dissolved inorganic car-
bon (DIe) or by using proxies such as banded corals (e.g. Druffel &
Linick, 1975) or bivalve shells (Weidman & jones, 1993). Analyses of
the spatial distribution of 14C and its temporal 3D variations (fig.
VII.8b,c) through mixing and advection provide invaluable informa-
tion in various field of research (carbon cycle, global oceanic circula-
tion, etc.). Kalish (1993) demonstrated that the bomb 14C tracer was
also incorporated into fish oroli rhs, and suggested using it as a date
marker. Analyses were performed on orolith cores of New Zealand fish
whose ages had previously been estimated by conventional age-reading
methods. The reconstrucred orolith 14C time series showed a striking
phase coherence wirh thar of nearby corals (fig. VIL8d) and rhus con-
firmed the anrlltltts-based age estimate for those fish . Exrernal 14C
references are not required if a suitable collection of young fish from a
given species and location can be used ro establish a reference curve
(Campana, 1997).
Looking at the chronology of upper ocean bomb radiocarbon contami-
nation, orolith s of fish with presumed harching dares between about
1960 and 1970 are most suitable for validation srudies, although later
years (1970 ro the present) may be used if the decreasing rate of 14C
in the region of interest is suffic iently rapid (Kalish, 1995). Nore that
14C age esrimates provide minimum ages since potential contamina-
rion of the core by adjacent younger material would increase the 14C
sample concentration if the fish had hatched during the period of
increasing signal. This does nor apply if rhe decreasing signal period
is used. The synchrony of the bomb- 14C contamination of the upper
layers of the world 's oceans has been well established, although rhere
are substanri al spatial variations in the magnitude of the phenomenon
(fig. VII.8b). This is why this approach is mOSt useful (or rather, less
problematic) for species whose early life stages, at least, inhabit rhe
surface mixed layer (Kalish, 1995). For species living below this habi-
tat, it remains applicable at least ro moderare depths (Kali sh et al.,
1997), provided that a correction factor for 14C-derived fish ages can
274
be estimated usi ng existing data o n the penetration rat e of bomb

radiocarbon , as thi s has proved ro be highly variable, depe nding on
location (fig. VII.8c). With regard ro estuarine species, the bomb 14C
signal has been shown to lie intermedia te in phase a nd mag nitud e
between atmospheric and marine carbonate time series (Campana &
Jones, 19 9 8) . This is du e to more int ense and rap id water-air
exchanges in shallow, well mixed locations with strong riverine input.
200
150
100
7
;; 50
-",
·50
1960 1965 1970 1975 1980 1985 ·100

Ye ar 80'S 60'S 40' S 20' S 0' 20'N 40' N 60' N
a b
o 160
200 • FW coral ~: a "
...., ,
AI 'I I
120
o HerOll lsJ;md c.oral
_ 400
80 • New Zealand ftsh ()lolit hs
S
cS 600
0.
~ j'
;; 40
~ 800
<J
r
0 ~
1000 ,!!
1200 ~~-+--~-.~~~~~~r--r~.-~~
·40 !, 00 '(~oI
I t, ,,\,' ", '
·80
1910 1920 1930 1940 1950 1960 1970 1980 1990
·100 ·75 ·50 ·25 0 25 50 75 100 Date of calcific ation (years AD,)
c M 14 C(%~1 d
Figure VI1.8 . The bomb 14C chronometer.

a) Atmospheric hi story of t\1 4C in the northern (20 0 NI and south ern (20 0 S) hemi spheres showing the large change due to the
atmospheric testi ng of nuclear weapons. From Broecker et al. (1985).
b) Surface values from Eastern Pacific GEOSECS (1973) section compared to WOCE sec tion P17 (1992) data collec ted along
135°W. Value s in the temperate zones of both hemispheres fell while those in tropica l and equatorial latitudes rosed . From Key
(199 7).
c) 3D changes In the t\14C (WOCE P17: black asterisks · Eastern Pa ci lic GEOSECS: grey Xs) between 1973 an d 1992. From Key
(! 997)
d) t\ 14C (± lSD) of Pagrus auratus otolith aragonite from New Zealand over the period 191 8 to 1990. t\14C measurements from
hermatypic coral s sampled in Fiji and Heron Island, Australia are presented for compa rison. From Kalish (1993) with permission
from Elsevier Science .
As pointed out by Campana (1999), in estuarine and d eep-sea habitats

the orolith radiocarbon sig nal may be obscured ro some extent by tbat
parr of the carbon which is metabolically derived (one rhird according
ro Kal ish (1991b) if the fis h feed on prey whose 14C content is dif-
ferent from that of the surrounding water.
275
Such studies require th e us e o f accelerator mass spectro m e try (AMS), a

hi g h-preci sion a nd very ex pen s ive technique. Thi s is probably one of
th e reason s why appli ca ri o ns in fish biology h(lve remained relatively
few despire its in d ispurabl e adva nrages co mpared, for instance, to
rh e re larive ly poo r precision of radi o m erric ag eing.
NOte that as ide ftom fish validation studies, radi ocarbo n data may
provide information th at he lps to elu c idate qu es tions in fish biology,
such as th e loca tion o f early-life habitats (Kalish, 1995) M oreover, by
usi ng known-age or va lida ted- ag e fi sh , otolith l·jC tim e se ries can be
valua ble tools in other disciplines.
3.3. Other potential chronometers

Ea rly studies s ugg es ted rhat variarions in e lem e nts and ele ment rati os
co uld be used to ver ify fi sh ag e , if th ese variations were causecl by
cyclical seaso nal factors SLlch as temperature . Diffe rences in Ca co n-
ce ntrati on, coi nci di ng with opti cally different zo nes , w ere used to
indi ca te seasonal cycles in eel o toliths (Casse lman , 1982, 1987). Sr/Ca
ratios were a lso s ugges te d for thi s applicati o n (Racltk e & Targe tt,
1984) but furrher s tudi es have shown that th ere is too much un cer-
tainty a bout th e rel a tionsh ip between o tolitb Sr and e nvironment a l
cond itio ns to m ake tbis a useful ch ro nom eter. However, Th o rrold &
Shutdeworth (2 000) recently obse rv ed cycli cal v(lriari ons in Sr/Ca
ra tios that close ly matched the partern of va lid ared annu a l rings in
Pogol7iaJ crorniJ otoliths.
Osc illati o ns in th e concentrations of o th e r elements hav e also bee n
observed. Fo r ins tance, Halden et Cl!. (2000) interes ting ly observed an
oscillatory pattern of Zn in Stilve!illllJ CI!jJirllls otolith s th a r closely
marc hed tbeir opti cal annual stru Ctures . Sucb sig nals may ptove to be
su i tabl e c b ronometers in some si tu at ions, th ough the i r use should be
carefully inves tig at ed first.
\'lVeid man & Millner (2000 ) measured oscillations in 81 ~0 across cod
oro liths by drilling discreet samples for IRMS a na lys is. The number of
flu ctuations obse rved corresponded well to co nventi o nal age de termina-
tions, suggesting rhat 8 180 transec ts could be used for ag e estimati on
in sp ecies that ex peri ence regular annual temperature flucru at ions.
4. Stock-population discrimination
--------------------------------------
The m a jor appli cation tha t has driven most of the d evelopment in
o tolith microchemistry is s toc k di sc riminati o n . In numerous locati o ns
a ro und th e world spawn ing or feedin g sroc ks of fi sh mix an d for
fi s heri es assess m ent and legal p urposes it is necessary to id e nrify anc.J
quantify rhe rela riv e proportions o f the individual co mpon ents of the
276
stock. Even for it 15 often desirable to be able ro

idemi that may comribure to the total
Stock discrimination differences

but is based on differences in eiememai
or at least
pan of their lives in different environments.
Stock discrimination studies have
menral of the whole orolith
Vll.G.l.l.l) and of areas of the otolith
VII.G.1.1.2). Information from whole dissolved
oto\iths can often be collected more and with less risk of coo-
but in many the information
in the inner of the otolith and can
Two lines of research are to fur-
ther develop stock identification One is rhe continued
refinemem of tools and protocols to allow the efficiem and
of more elements; the second is the of
thar will allow the most efficient
tion of stock components based on the smallest number of elements.
It is that the selection of and data
method will vary with the since in some locations
differences be caused by a few minor elements (Sr, while m
others the differences may be minor and determined primarily the
presence or absence of (face and u] tra-trace elements. Thresher (
9) lists Sr, Ba and as the most used elements
for successful stock delineation. Otolith microchem has been
in stock identification studies for that occupy a
of different environments, both coastal and oceanic. The
of studies have cpr",rrl"d successful discriminations of groups of
bur it is not known whether rhe can be universally
or whether unsuccessful stock discfIminarions are simply under-
There were no detected differences indicative of
ca] dIfferences in the otOlith of a southern ocean
(lV!acrltronltJ taken from different
(Kalish a!. ,
when more
differences may
for fish sIze (either et et!',
277
in Thresher (1999). Preliminary srudies have been performed in many

pans of rh e world, us ually focu ssing on co mm erc ially imporram
srocks wirh good pri or knowl edge of rhe componenrs mix (Campana
et af., 1995), or using a pre-derermined ourli e r popular ion for com-
parison (Secor & Zdanowicz, 1998) . Auscra lian researchers have
moumed larg e-sca le srudi es of popularions (Edmond s et af., 1991 ,
1992 , (999), and have incorporared mi crochemi srry imo rheir sran-
dard srock assessm enr mer hods.
Orolirh composirion can also be used ro id e mify changes in habirar in
individual fish, ele menra l fing erp rinrs being used as narural rags. For
fish rhar move berween environmenrs o r warer masses rhis informa-
rion ca n be used ro ((ack movem enrs and analyse srock mixing. Dif-
feren ces in rhe composirion of orolirh cores have also bee n used ro
idenrifY spawning and nursery g round s as a firsr srep roward evaluaring
rhe relarive conrriburions of rhese componenrs ro rhe aclulr sroc k.
Much of rhe work on idenrifying nurse ry grounds is preliminary, and
has been resrricred ro d emonsrraring rhe applicarion of rhe principle.
Differences in orolirh compos iri on have been derecred in juvenile
Cynoscion regalis, (Thorrold et af., 1998b), juvenile Afosa sapidissima,
(Thorrold et af., 1998a) and juvenile Sofea so fea, (Ponrual et af., 2000)
from differenr es ruari es. O xyge n and ca rbon isorope analysi s has been
used ro disringuish be rw een m igrarory and non-migrarory individu-
als (Norrhcore et a f., 1992) and berween salmon from differenr naral
screams (Gao & Beamish , 1999). The differen ces in composiri on can
be used ro idenrify rhe source of individuals conrriburing ro rh e adulr
popularion, provided rhar rhe ch aracreri sa rion of rhe spawning or
nursery g round s and rhe an a lysis of rhe mixed gro up occur over a
period of rime during which rh e environmenr chemi ca l characrerisri cs
remain srable. This poinr is discussed in rwo recenr full-scal e ap plica-
rion s, rhe firs r based on ana lysi s of rhe core of juvenil e Termalosa tofi
(Milron et a f., 1997) and rh e second on rhe analysis of whole ocolirhs
of Gadus morhtta (Cam pan a et af., 2000) .
Orolirh microchemisrry dara is usually analysed using mulrivariare sra-
ris rical rec hnigues for srock separa rion . These permir rhe inclusion of
large numbers of elemenrs (or isoropes) and presenr proced ures for guan-
rifYing rh e imporrance of differenr elemenrs ro srock discriminarion.
Principle componenr analysis (peA) is a family of rechniques rh ar pro-
vides sy nrheri c represenrari ons of vasr dara sers, usually by m ean s of
g raphi ca l vi sua lisari o ns. The relarive proximiry of vari ab les (here
orolirh elemenra l/isoropic concenrrarions) is inrerprered in rerms of
correlarion, while rhe proximiry of individual s is inrerprerecl in rerms
of g lobal simiJariry of observed values. Disc riminanr analy sis is a
famil y of rechniques rhar are desi g ned ro classify (i.e. ro ass ign ro pre-
exisring classes; here, g roups of fish ) individuals rhar are characreri sed
by a number o f num e rical variables. Th e mosr commonly u sed
278
method, linear di scriminant anal ys is, is both d escript ive and pred ic-
tive. The discrimin atory power of the disc riminant functi ons should
be assessed by m ea ns of boots trap p ing or jack- knifing techniques
whi ch ptovi de accurate es tim a tes with confide nce intervals. Max i-
mum likelih ood-based methods m ay be more sui table fo r class ifyi ng
samples fr om mi xed-smck origin in cases where di scriminant anal ys is
lacks suffi cient di scriminato ry power.
5. Other applications
5.1. Habitat characterisation through elemental fingerprints

Differences in otolith com positi on of course prov ide va lua ble infor-
mation about fi sh populatio ns, but it may also be poss ible to m ake
cert ai n inferen ces abo ut ha bi ta ts a nd en v ironments from oto l i th-
derived information. Omlith microstructure has bee n used m indicate
habitat quality, using otolith increment w idth as an indi cator of fi sh
g rowth rate (Szedlmayer & Conti , 199 9). Our und em anding of how
vari ous ele ments are in corporated into the otOlith is still in compl ete,
and it is unlikely that the concentratio ns of individu al elements can
be used directl y ro indicate conce ntrations in the environment. How-
ev e r, vari ations in elemental co ncentratio ns across the otolith ma y
ind icate sho rt- term fluCtuati ons in th e environme nt and m ay dis-
criminate between stable and dyn amic enviro nments. There m ay exist
key elements tha t can be used m trace specifi c wa ter m asses a nd id en-
tify the fre q uency of incursion inro a habitat occupied by a fish popu-
lati on, especially when tha t pop ulation is known m be sed entary or
mig rating . Barium, for exa mple , is associ ated w ith ocea ni c waters ancl
its p resence in the otOlith could reco rd exc hanges be tween coas tal and
ope n ocean envi ro nments. Al ong wirl, Sr, as mentio ned above, other
elem e nts (o r isompic ratios) such as Pb and U may al so p rove to be
valua ble ind icatO rs of seawa ter or freshwater incursions.
5.2. Mass marking

Among the m e thods th at have been developed m m ark o mliths and
m o re ge nera lly fish calcifi ed stru c tures (c ha p. IVA . 1. 2 ), radi o la-
belling with the 85Sr radi oisotOpe and marking with pa rticular ele-
m e nts d epe nd speci ficall y on O MC techniqu es . The fo rmer was
em ployed by l ehtO ne n et af. ( 1992) m mark newl y hatched an adro-
m o us Coregontts favaretf.ls before release into the fi eld in ord er to stud y
the timing of their d ownstream mig ratio n.
Immersion o f Oncorhynchus kisutch fry in wate r enriched with la n-
thani des (la, Ce and Sm) resulted in de teCtab le levels in bony tissues
as late as 10 mo nth s after the treatm ent (Ennevor & Beam es, 1993;
Ennevor, 1994 ).
279
Manual of fis h sclerochronology
Immersion in a srronrium chloride (SrCI 2) solurion has also been pro-

posed as an inexpensive and relarively environmenrally safe merhod of
ragging large numbers of small fish wirh good success. Orolirhs of
marked sa lm on fry <oncorhynchlls keta and O. nerka) presenred clearly
idenrifiable Sr mark afrer 21 monrhs of rearing in freshwater (Schrod er
et Cif. , 1995). The porenrial of this method has recenrly been evaluated
as a means of marking the d orsal spine of juvenile PClgms al/ratlls (Pol-
lard el al, 1999).
From a large-sca le mark-recaprure experime nr , Clear et ttf. (2000),
using injecrions of SrCI 2 , proved that increments a re deposited
annually in the oroliths of Thllnnm maccoyii at least unri! the age of
13 years.
Depending on the experiments, various an alytical methods of Sr
measurement w ere used: backscarrered e lectron microscopy, EDS ,
WDS and ICP-MS (chap. VILG). Although useful for so me fisheries
research ex periments, SrCl 2 is not approved for use on food fish in cer-
rain counrries, lim iting its widespread use.
5.3. Analysis of fossil biogenic carbonate for paleoclimatology

and paleoecology
Fish bones , teeth and oroliths have been analysed for geological, pale-
onrological and archaeological stud ies. Oxygen isorope ratios in fossil
otol iths (Devereux , 1967; Patterson el aI., 1993) and fossil fish teeth
(Kolodny et a!., 1983) can provide information about paleorempera-
tures. Smith & Patterson (1994) and Parrerson et al (1993) not on ly
es tim ated temperatures from fossil oro liths, but also recons trucred the
seasona l temperature variations experienced by fish populations.
One aspecr that is debated among paleonrologists and geoc hemis ts is
the accuracy of applying experimenrally derived isorope-temperature
relationships (tab. VIf2) ro fossil material. During fossilisation, dia-
genes is can occur, which can transform aragonite inro the more stable
CaC0 3 polymorph calcite. Severa l srudies have attempted ro repro-
duce this process experimenrally, and thus derive conversion facrors
(Yoshioka et aL., 1985; Nelson et a!., 1986). The effeCt of diagenesis on
oxygen isorope ratios is inconclusive, but when aragon ite transforms to
calci te there is a release of Mg and Sr (Yosh ioka et a!., 1985).
5.4. Pollution history and anthropogenic influences

Water qua li ty moniroring programmes and studi es of the impaCt of
environmenral contamination rely on a co m bi nati on of measutemenrs
of the physical and chemical environmenr and the biota. The impaCt
of env ironmenra l contamination on organisms can be measured at
ecosyste m, community, population, and individual levels. Unfortu-
nately, moniroring and assessment require long-term studies, and
deteCting environmental co nt am in atio n is difficult in a reas where
280
there are no base-line dara. Chemical analysis of fish owlith

tion may otTer significam to our (0 derect current,
recent and historical levels of and ar tbe same time to

assess the impacc of contamination ar rhe individual and
levels. Environmemal contamination affecrs (he
vidual and this effeer propagates [0 levels of
fish and the whole communlty.
to reveal critical informaeion about rhe envi-
ronmenwl of fish, rates of fish from dif-
terem environments, and rhe effects of sub-lethal exposure
to environmental contamination. Alrhough fish movements and
may make it difficulr [Q juSt where an individual
was the record of concenrrations in rhe otolith may
evidence about how long ie was In with local popula-
rhe power of the In
poiming evenrs and locarion is enhanced. Historical
collections of otolirhs may also reveal the record of environmemal con-
tamination over of several decades.
metal concentrations have been measured in the wbole dis-
solved moliths of a number of fish at coasrallocations.
In reflect d iffer-
ences in environmental concentrations of metals . .For the
conrem of zinc in rhe otoliths of fish from Greek coastal waters was
near [Owns witb industrial of this mecal (Papadopoulou
merals have also been in otoiirhs ofBaicic
Gctdus mOl'hua 1988) and Scomberomoms '<tt)alla
et af., 1989). More recent studies have used ICP-MS and LA-
lCP-MS co temporal in the exposure of fish to ambro-
concaminarion (Dove & 1998; David Mi
CSlRO, pet's. comm.).
In addition to this field
show tbat metal concencrations in fish otolitbs
exposme concentrations £If., 1998). work to
date indicates that the a reflec-
tion of their in the environment. The distribution coerTi-
cient of each element differs (Milton &
Bath at., borh because of chemical charaneristics and
metabolic rements and responses ro toxic elemems. Environ-
mental condJ(ions sllch as or temperature can the
bioavailabi and of metals and other toxic elements into
the fish. Once taken up, the different elements,
merabol ised, or derox i fied via various
this affects how much is available for into the otolith.
Toxins that are s(Ored far tissue may become available when fat
281
reserves are mobilised, and this may induce a time-lag between

exposure and appearance in the otolith. The chemical characteristics
of individual metals and conditions in the endolymph may cause dif-
ferential precipitation or inclusion into the otolith. In addition, the
interactions of metals and proteins are very complex and it is probable
that many elements can be incorporated into the otolith via protein
components. This aspect has received little attention to date, but may
be inferred from studies of mineralised tissue in other taxa.
282
283
F. What about other calcified structures?
The need co assess "rhe exrenr co which org ani sm s possess narural rags ,
ch emo-p rinrs, rh e dura ri o n d u rin g whi c h rhes e a re re ra ined a nd
wh erher new p rinrs are acquired" was earl y recogni sed (Cal apri ce ,
1971 ) and subsequ enr invesri ga rions were underra ken on rh e chemi-
cal com p osirion of skeleral rissu es such as scales, d eithrtlm, and verre-
bral cenrres .
Alrhoug h rhe ocolirh may be rhe m osr frequenrl y used cs in eco log i-
cal and fish eri es srudi es, analyses involving sca les and bo nes are ofren
perform ed in order co address cerra in quesri ons. Anal yses o f scales a nd
fin rays m ay be rhe only viabl e m erh od of sam p ling end ang ered or
valuable fish (rec reari onal fis heri es , Salm onids, srurgeon, erc.), where
individu als cann o r be sacrifi ced or where orolirh ex rrac rion would
des rroy rhe value of rhe specimen.
Borh srrucrural descri p ri ons and rh e p ro cesses of minerali sarion of
rhese srru crures are beyond rh e sco pe of rhi s chaprer (bur see chap. II.B
and II.C and e .g . Francillon-Vieillor et al., 1990). From a chemical
poinr of view, all skeleral ri ss ues consisr of orga nic and min eral co m-
ponenrs . Bri efly, rhe org anic marrix is m ade of collageno us com po-
nenrs and non -co llage nous componenrs (proreogl ycan s, phosphopro-
rein s, phospholipids, erc. ). The mineral phase consisrs of " impure "
hydrox yaparires (CaS(P04)30H) conraining carbo nare, fluorid e,
whi ch subsrirures for rh e OH ion s in fluo roaparire , and min or and
rrace consriruenrs (M g, Si , Sr, Mn , Ba, N a, erc.).
Ir is worrh noring rhar , unlike orolirhs excepr in very unusua l sirua-
ri ons, rhe skeleral ri ss ues are involved ro vari ous d eg rees in rhe conrrol
of bod y homeosras is rhroug h rhe srorag e and release of calcium and
p hosphorus salrs, and rhus und erg o resorprion and remod ell ing pro-
cesses. Suc h p ro perries have ro be raken inro accounr when inrer-
prerin g rhe chemi cal com pos iri on of rhese bon y parrs.
The ran ge of ap pli carions ap p roxi marely reflecr s rhar of orolirh com-
p os irion ana lysis (rab . VH A ).
Ecoroxi colog ical research and po lluri on m oni ro ring of aquari c sysrems
have also made use of srudi es of fis h scale and bone composiri on. The
lirerarure on rh is rop ic is abund anr and undoubredl y represe nrs a
so u rce o f va luable informa rio n on m ec hani sm s of elemenra l or
racli onuclide uprake from warer or food and on d ifferenri al body ri ss ue
disrriburi on.
284
Table VI1.4 - Examples of studies of CS chemical composition (the literature cited is far from being
exhaustive).
Ape lication Species Analysis cs References
Migratory parrern M. Jaxa lilis Sr Scale (Couranr & Chen, 1993)
AripenJer IrctmmonlanllJ Sr Pee roral fin ray (Veinott el Cl!. , 1999 ;
Vei norr & Evans, 1999)
Salinity B revo(iY/ iCl pell rol1l(J 8i Sr lsoSr Vertebrae (Chesney el ClI., 1998)
Seasonal variarion ThllnrlllJ Ihynnm Elememal Venebrae (Calaprice, 1983)
composi t ion
Esox I"rim Ct! Cleilhra (Casse lman , 1974)
Differenriation Salmonids Elemenral Scale (Lapi & Mulliga n, 1981)
berween groups of fi sh compos iri on Verrebrae (Yamada et a!., 1987)
SwmberoTT/orllJ cavedlCl 1)\5N Bone collagen (Roelke & Cifuenres,
1997)
Diet sou rces and I)\ ' C and 8 \5 N Scale (Esre p & Vigg, 198 5;
rro)2hic pos ir ion Wainrigh r el a!., 1996)
Chem ica l marking Salmonids Sr Opercu lar bone (G uilloLl &
de-Ja-Nouee, 1987)
Scale (Snyde r el al., 1992;
Mulliga n, 1997)
Pagrlls aI/ra/lis Dorsal spine (Pollard el a!. . 1999)
Paleosa lini ries Shark , 7Srl 86 Sr Toorh enamel (Schmirz et a!. . 1997)
Meral pollurion Sah di/1/IJ "ljlinllS Pb Opercula (Koeck el a!., 1996)
Marine fi sh Se, Cr, Co, Fe, Scale (Papadopoul ou &
Zn, Cs, Ag Mora; topou Iou -Kass; ma r;,
(977)
Sha rk s Cd, Mn, Zn Venebrae (Vas el a!., 1990)
PIlIldllllJS beteroc!illJJ Zn Scale (Sa uer & Warabe , 1989)
285
G. Microchemistry:
methodological requirements and issues
1. Analytical techniques
Numerous analyrical rechniques have been used in OMC studies. As a

maner of facr no single merhod can fulfil all rhe requiremenrs of rhe
wide range of quesrions co be addressed. The decision as co which
rechnique is mosr appropriare depends on rhe applicarion, rhe aspecr
of composirion co be measured, rhe sensiriviry required and rhe level
of sparial (remporal) resolurion needed. These considerarions are ill us-
rrared by a block diagram linking main applicarions, measuremenr,
resolurion and rechniques (fig . VII.9). Concurrenrly, some characreris-
rics of major analyrical cools are provided in cable VII.5. In rhe fol-
lowing secrions, we summarise rhe basic principles and capabiliries of
rhe mosr imporranr rechniques wirhour arrempring co cover rhe com-
plere capabiliries or limirarions of each one.
1.1. Elemental composition

Analyrical rechniques are broad ly divided inro "surface" analysis rech-
niques which provide composirion informarion ar specific locarions or
spors on rhe ocolirh , and bulk rechniques which provide global com-
posirion informarion, usually inregrared over rhe whole ocojirh. Some
rechniques measure rhe concenrrarions of several elemenrs simulra-
neously (mulri-elemenr rechniques) while orhers are only capable of
measuring rhe concenrrarions of one elemenr ar a rime. The seJecrion
of rhe mosr appropriare rechnique for a parricular ocolirh applicarion
depends on rhe hyporhesis co be resred , as rhis derermines rhe sparial
resoJurion required and rhe eJemenrs co be assayed. Crireria sllch as
limirs of derecrion (LOOs) in reJarion co expecred concenrrarions, par-
ricular requiremenrs regarding absolure concenrrarions (as opposed co
elemenraJ rarios or reJarive differences) and rhe accuracy of rhe derer-
minarions wirh respecr co approved srandard reference marerials
(SRMs) musr be raken inro accounr when seJecring rhe appropriare
merhodology.
Table VII.5 - Analytical techniques used in OMC and their main characteristics.
Technique Acronym Type Information LODs Corrunents
Spatial resolution
Energy Dispersive EDS S - Elemenral analys is > 1000 ppm Multi-element analysis
Spectrometty (associated (or ED-EM) - X cartography - x: 0.5 ~m to [0 pm Surface conditi on crirical
with SEM or TEM)
Wawelength Dis pe rsive WDS S - Elem ental analysis 100- 1000 ppm Good for light elements
Spectrometry (or WD-EM) - X cartography - x: 0.5 pm to iOpm (Na, K .. .) wirh Sr and Ca
MorlO or multi-elemental
Surface condition crit ical
Secondary Ion Mass SIMS S - Elemental analysis < [ ppm (0 l OO ppm Surface condition critical
Spectrometry - Isotopic analysis - x: 0.1 ~m to 0.5 mm
- Depth profiling -z: I nmtolOnm
Particle-induced X-ray PIXE S - Elemental analysis = I ppm No t appropriate
Emi ssion - x: 10 pm to I cm for light elemen ts
- z: 1-1 0 I'm
Laser Ablation LA-ICP-MS S - Elemental analysis =I ppm Bes t comprom ise between
Inductively - Isotopic anal ysis - x: 5-20 ~m ranges of elements detected
Coupled Plasma - z: 5-20 pm and sensitivity
Mass Spectrom etty Multi-element analysis
Surface condition not critical
Aromic Absorption AA B - El emental anal ys is 0. 1 ppb to 500 ppb Mono or multi-elem ental
Spect rometry (flame, (flame) Bes t suited for lighr elements
furnace) 0005 ppb to SO ppb
(furn ace)
Aromic Emissi on AES B - Elemental analysis > 5 ppb Mulri-element analysis
Spec trometty (name , arcs Less vulnerabl e to matti x or
and sparks) signal interferences than AAS
Induct ively Coupled ICP-AES B - Elemental analysis d ppb to 30 ppb Multi-element analysis
Plasma-Atomic Emission (or ICP-OfS) Less vulnerable to matrix
Spec trometry or signa l interferences
than AAS
Inducri vel y Coupled ICP -MS B - Elemental analysis ppt ro ppb Multi-element ana lys is,
Plasma - Isoropi c anal ys is Some elemenrs subject rO
Mass Speu romerry interferences (e.g. Ca, Na .)
Bes t comprom ise between
ranges of el ements derected
and se nsi rivity
Thermal Ioni sa ti on TIMS - Isotopic analysis High preci sion and accu racy
Mass Spectromerry for isorope-ratio measurem entS
Need for efficient chemical
separation of the target
analy te
Isotope Rario Mass IRMS B - Stable isotopes Automatic micro-sampling
Spec trometry (H, C, N , 0, S) can give better spati al
resolution (10 ~m )
Limired by sample
size considerations
S: surface technique; B: bulk technique; SEM: Scanning Electron Microscopy; TEM: Transmission Electron Microscopy; x: spot sam-
ple area; z: sample penetration depth For bulk techniques, LODs refer to solution . They must therelore be multiplied by the dilu·
tion factor to reler to solid material.
287
Age validation Stock Migration Ontogenic events
Application identification and metabolism
& I \ /J~
. stock discrimination
Temperature Salinity Metamorphosis Reprod uction Metabolism
. nursery identification
\~
\ s:
'"
<Xl
<Xl
w
~
<=
!!C
\ <:2.
'''''''-'''''''' \
'--'- \ '"=r
'--,- ~ ~ '"
(">
iD
Aspect of g
21"Pb/226Jla 8omb ''C Isotope ratio of
composition 228ll1f'"Ra Sr!Ca 0180 Minor and trace elements Sr!Ca, ol3C =r
Zn he~ elements g
measured eg ' SrI'"Sr o
0"
/
OQ
'<
Fine temporal resolution Coarse temporal resolution

(surface techniques) (bulk techniques)
a spectrometry AMS IRMS + mlcrodrlllinl Low sensitivity High sensitivity High sensitivity Very high sensitivity
Techniques y spectrometry WRMS (surface techniques) (Ca Sr Na K Mg) LA-ICP-MS SB-ICP-MS and mass resolution
T1MS SIMS (surface techniques) WOS PIXE ICp·AES T1MS (bulk techniques)
EDS SIMS AES HR·ICp·MS (bulk techniques)
HR-lA~CP-MS (surface techniques)
Figure VI1.9 . Synthetic block diagra m showing main applications of otolith microchemistry. For each application, the aspect of composition that is measured is shown, along with the rele·
vant analytical techniques. Solid arrow: methods in general use; dash and dotted arrows: more rarely used methods; dotted arrow: method not recommended; bold text: most commonly
used techniques.
The of p"< r, rp,,, I

elemental composi cions in terms of element
throughout the microchemistry lirera-
tip<nlrp"ti
or
the machines. In it
may also be reasonable to examine the relationships between elemen-
tal ratios, especially where the ratio is (0 be influenced
specific conditions. However, element con-

cenrrarions as element
and even
.1.1. Surface
A wide range of analytical utilises the interaction of a pri-
mary energy beam (photons, ions) with the
The imeraction results in the emission of beam
or ions) whose characteristics on the nature, S[fUC-
Of the insrrumems (see rab. VII.)
used Hl OMC, EDS and WDS
both use a electron beam whereas PIXE uses proron beam.
All these ins(fumems analyse the emitted X-ray signal.
multi-elemental number of e1emenrs (hat can be
with WDS on the number of spectrorneters the
normally non-destructive and have very good
resolution. Their LODs, which are often
differ by several orders of tab.
are derived from mass spec(fornerry (MS). MS

separate ionised aroms or molecules from their differences
ratio (mlz) and thus can be used ro measure either
or elememal The first step in an con-
gas phase ion from a material. This is done in
a primary ion beam accelerated and focused on the
surface of a material. Nore that SIMS allows depth by con-
tinuous, of che surface material. In LA-ICP-MS, the
excitation source OCP for inductively plasma) is argon
at very remperarure that efficiently excites and ionises atOms.
JCP-MS was first to operate on liquid The recem
coupling of a laser ablation (LA) in the far UV now
the of discrete on the surface of solid
(rough powder, secrions or thin
These techniques work with otOliths rnounred on slides or in
epoxy blocks. Some are very sensitive ro surface
WDS, SIMS, These therefore require a highly polished,
mirror-flat surface with no defects" Other techniques such LA-ICP-IvlS
can be used with little sectioning and
More derails on VULe
289
1.1 .2. Bulk analyses

Bulk anal yses in O MC have used eith er aromi c specrromerri c methods
or MS methods. The former are based on rhe inreracrion of e lec([o-
magn eric radiations with matte r. Aromic abso rpti o n spectrometry
(A AS) exploits the absorptio n of lig ht ro measure rhe concenrrario n of
aro ms in a gaseous phase . The lig ht source is usuall y a hollow-ca thod e
lamp made of rh e el emenr to be m eas ured so rh at rhis tec hnique is
ge nerally mon o-e lem enral. Th e sam p le is d ried and vapo rised (ro
obtain free aroms) in a fl ame or a graphite furnace. The former only
acceprs soluri ons whe reas the latter can a lso accept so lid s and is a
much mo re effi c ient a tomi ser. Ato mic emi ss io n spectcometry (AES,
a lso call ed OES: Optica l emi ss ion spec tcoscopy) de termines rhe ele-
m ental concenr ra rion of sol uti o n samples by meas ming the optical
emi ss ion of excited acoms. Vari ous exc itatio n sources m ay be used,
including fl am e Ot indu c rive ly coupl ed pl as ma (I C P-A ES) wh e re
aromi sarion is much more effici ent. A major advanrage of AES com-
pared co AAS is rhat it is a multi-elemenr technique.
Because of its advanced capabiliti es (sensitiviry a nd less inrerference,
exce pt for a few e lements; see below) le p -MS is di splacing the o rher
techniques for rhe dererminari on of rrace and ulr ra trace elemenrs. It
can be ope rated in borh standa rd and iso tope-diluti on (ID -ICP-MS)
mod es , th e la tter provi d ing un parall e led m eas ure m e nt acc uracy.
Recenrl y developed hig h-resoluri o n HR -ICP-MS improves sensirivity
even furrh er and reduces inrerfe ren ce in compari son with convenri onal
qu adrupo le ICP-MS. D erection powers < 1 p pg are claim ed b y manu-
facrurers fo r non- Inrerfered isoropes .
Bulk analysis is generall y perform ed on soluti on sam p les ob rained by
d issolving cl ea ned a nd dried o to lith s in an ac idi c so luti o n afte r
weig hing (see chap . VII.G .2 and VIII.C for more derails).
1.2 . Stable isotope measurement

Mass specrromerry rechniques are used ro m easure stab le isoropes b ur
rhe exacr analyri ca l merh ods depend on rh e isotope be ing analysed .
HeaVi er elemenrs such as Sr can be measured using either sur face or
bulk tec hniques (LA- or SB - ICP-MS) bur lig hter elemenrs such as C, N
and 0 are measured using Iso tope rati o mass specr ro merry (IRMS). The
vaporisin g source used in ICP - MS aromises rh e eleme nts, but rhere is a
risk thar only some of rhe lighrer and m ore volarile e lements are chan-
nelled inro rhe MS detector, leading ro und erestimates of th eir con-
centrati o ns.
The p rocedure for rhe routine anal ys is of o tolirh C and 0 isotopes by
IRMS requires the carefu l di sso luti on of the otolirh in a sea led system ,
in order co capture evo lved CO 2 , The gas molecul es are ionized and
srripped of a n e lecrron , cau sing eac h m olec ule ro be positi ve ly
charged. The charged mo lecules pass unde r a magn et rhar separates
290
them to their mass. Faraday collectors then measure the

of each beam of ions of mass after have been sepn-
the magnet. gas mass ratios are measured a
reference gas calibrated with (0 an international standard.
Mass ratios should be corrected for effects in accordance with the
procedures of (1957) and rprn",pr~
tape fractionation between reaction
with phosphoric a ftactionation factor «X) of 1.0
(Friedman & O'Neill, I
Prior to ""rA",r otoliths should be treated to remove
components since the contains C and 0 with different
ratios. The can be removed It is also
possible to exploit to remove
orol bur this has not yet become a common pte-treatment tech-
for analysis
materials are 0.05%oand 0,07%0 for carbon and oxygen
For oxygen, this to 0,2°C for the
estimate of temperamte range.
IRMS may be with automatic devices that operate
with ultra-small carbonate by an au(O-
mated device dis-
~'Hnn,If', across sectioned oroliths (Wurster et aI., 1999).
(SIMS) and laser
a temperature range of ::: uses gentle

to resolu-
tion as
0,08%0 (r
soon offer a
otolith ratios, Fm these surface it may be necessary to
pte-treat the otol iths or before (0
remove [he protein components,
1.3. Radionuclides
A number of methods can be used (0 measure radioacrive DU'lVljC~
depending on the mass, type involved and accuracy
desired. Methods that have been used in otolith radiomerric
include alpha spectroscopy, gamma specrroscopy, thermal ionisation
mass spectrometry (TIMS) and accelerator mass spectromerry (AMS),
291
Manual of fi sh scl eroc hronology
Alpha specrrosco py and gamma specrroscopy are based on rhe rype of

d eca y of radionuclide s by parricle emission (eirher an ex parricle,
whi ch is a helium nucleus , or a ~ parricle, which is an elecrron, or less
ofren a p osirron ~+ ), as well as high-energy ph orons called gamma (y)
rays .
The ex parricles e mirred from a g iven nuclide all have rhe same energy
or are divided inro a few m onoenergeri c groups berween abour 4 and
6 MeV Alpha-emirring radionuclides are ofren quanrified by alpha
specrroscopy. This rechnique requires elemenrs ro be chemically sepa-
rared before analysis in order ro minimi se inrerferences berween d if-
ferenr nuclides emirring ex parricles, whose energies can differ in a
rang e near rhe resol urion of rhe derecrors used in alpha specrromerers.
Like ex parricles, gamma rays are monoenergeric, and d ecaying gamma
emirrers provide gamma specrra wirh well-defined energy peaks rhar
are ofren used ro qu anrify specific nuclid es.
Wirh reg ard ro orolirhs, 21 0Pb is generally assayed by ex-specrroscopy
rhrough irs ex-emirring granddaughrer 210pO (T)!2 = 138 days), w hich
is wirhin 5% of secular equilibrium wirh 210Pb for orolirhs analysed
one or more years afrer collec rion (Bennerr et af., 1982 ; Fenron et Cif.,
1990).
Orolirh 226R a has been measured eirher direcrly by ex-specrcoscopy
(Fenron et cd., 1990) or by rhe so-called 222 Rn e manarion rechnique ,
222Rn (T 1/2 = 3.8 2 days) being used as a proxy for irs parenr 226Ra
(Benner r et af., 1982).
Alpha specrroscopy has also been used co m eas ure 228Th, while 228Ra
has been assayed by gamma specrcoscopy using rh e g amma energies of
irs daughrer produer 228Ac (T 1I2 = 6. 13 hours).
Nore rhar prior ro rhe analysis, sa mples mu sr be submi rred co rh orough
cleaning in ord er ro re move any exogenous co nraminarion , and su bse-
quenrly co chemical separarion processes. Derailed procedures m ay be
found in Fenron et a f. (1990) for 210PbP 26Ra analysis and Smirh et af.
(991) fo r 228Th/ 228 Ra analysis.
In order ro improve rhe precision of radi oc hemical ageing of orolirhs,
an ion exchange separarion rechnique of Ra and TIMS analysis has been
recenrly used (Andrews et a f. , 1999b). In comparison wirh conven-
rional merhods ir has been proved ro be more accurare and precise, less
rime- consuming and requiring less calcified marerial.
Due ro rhe exrremely low narural abundance of 14e (rhere is one 14e
arom for 10 12 l2e acoms) and rhe inabiliry of convenrional MS co dis-
ringuish berween 14e and rh e very abundanr nirrogen isocope 14N ,
o rol irh 14e measuremenrs require rhe use of parricl e AMS.
292
2. Data
Several issues to data have been raised

developmem and increase in applications of OMC. It is to
realise that there are many seeps in the process of obtaining informa-
tion about fish orol ith composition and at each step there are factors
that affect the of the data, These issues
affect the of since they concrol the
to each individual fish or
measuremenc is a incerest to
co some of the procedures recommended for
lytical is available from the Eurachem/Cirac
Group (Ellison et cd., 2000), this IS for more
it is a source of useful methods for
data in OMC studies.
2.l. Potential sources of uncertainty

le is clear from the discussion and
the of OMC is of recent
There is not yet sufficient information available co
all che and processes that can alter the composition of fish
moliths. Some of the is due to processes that are
still understOod. Other factors act the process fish cap-
ture, and VII.lO). The or
range of error, has nor yet been quantified for all of these
some can be manipulated to minimise rhe error. Not all of the
has been quantified for elemencs that are of interest in
OMC, although there now some evidence that elements bonded co the
material or adsorbed onto the surface are less robust (i.e. more
affected the than oche-rs. The
rions refer to the steps ind icared in
refer to the listed from each step.
2.1. L Sampling
The strategy employed for
tenrly result in problems in
be made to ensure either selective or
on the The method used to collen fish is also important
293
because capttlre conditions may exacerbare srress-induced or post mortem

changes in rhe endolymph. When rechniques such as rrapping or gill
nerring are used, rhe inrerval berween fish caprure and rerrieval may be
sufficienr ro alrer rhe concenrrarions of some elemenrs.
2.1. 2. Fish storage

Somerimes fish are srared afrer collecrion unril rheir oralirhs can be
exrracred and analysed. The storage method has been shown ro a lrer
oralirh composirion. Significanr differences in rh e concenrrarions of
N a and K were observed berween oralirhs of fish srored frozen or in
erhanol (Milron & Chenery, 1998; Procror & Thresher, 1998), and rhe
puriry of rhe srorage medium may also be imporranr. The effecr of stor-
age duralion is unknown. Mosr srudies have res red srorage merhods
over weeks or monrhs, bur many applicarions require rhe analysis of
samples rhar have been srored for much longer. There should be a
limir ro rhe amounrs of individual elemenrs rhar can leac h our of, or
be inrroduced inro, rhe orolirhs during srorage, bur rh is is likely ro
differ berween elemenrs. The effecr of srorage merhod and durarion
also varies wirh fish size and spec ies , si nce rhe size of rhe orolirhs and
rheir posirion in rhe head will affecr rhe rare of exchange berween rhe
orolirh and exremal med ium, or from rhe fish rissuedu r ing defrosring
or srorage in erhanol.
2.1.3. Otolith extraction

Duri ng orol i rh exrracrion rhere is a dang er of (Ontamination of rhe
ocolirh, especially when using mer a l di ssecrion cools and paper
envelopes for srorage. Likewise, sltrface deaning afrer exrracrion needs
ro be consisrenr because of rhe need ro remove any adhering rissue.
The orolirh may also be conraminared by elemenrs (parricularly Na) if
rhey are cleaned using bare hands. The effecrs of conraminarion can-
nor be quanrified, because rhey vary wirh each orolirh and acrion, so
plasric or ceramic rools should be used ro ex rrac r orolirhs and rhey
should be scored in inerr conrainers. Orolirh composirion is ofren m ea-
sured afrer age reading , and somerime orolirh readers use differenr
m ed ia ro enhance rhe rin gs. Ocolirhs are somerimes immersed in warer,
glycerine, or oil and rhese can conraminare rh e orolirhs or mobili se
various elemenrs, changing rh ei r sparial disrriburion. Mosr orolirhs
are srored dry before OMC analysis, borh for ease of srorage and because
of rhe porenrial of srorage fluids ro mobilise orolirh elemenrs. Desi{ca-
tion also adds an unquanrified error since rh e prorein porrion of rhe
orolirh can disinreg rare roger her wirh any accompanying eiemenrs,
and eiemenrs disrribured in inrer-iarrice spaces may be displaced.
294
Sampling Otolith extraction Concentration
measurement
Instrument operating conditions
CRM & matrix effects
Surface quality
Contamination
Calibration models (computational effects)
Sampling strategy
Surface cleaning
Instrument drift
Capture conditions Immersion
(pre- and post mortem
changes in endolymph composition) Blank correction & LOO
Assumed sto"ichiometry
Otolith
composition
Fish size Weighing precision Pseudo-replication
Purity of reagents Handling of outliers
Immersion Instrument drift

Method (ethanol purity.
freezing/defrosting
conditions ... ) Contamination Chemical interferences
Handling of values <LOO

o
g
5'
3
n"
Fish storage Otolith preparation Data analysis Cl
n
-:J"
ro
3
N v; "
<D
en Figure VII . 10 - Potential sources of uncertainty associated with the process of analysing the chemical compo sition of fi sh otoliths and other CS . ~
2.1.4. Otolith preparation

O(Olith contamination is almost unavoidable of
Owli(h cores or that have been cue
must be carefully selected ro minimise contamination (see

Even if water used for and
immenion itself may cause mobilisation of some elements
Na). Oto11ths are porous, and fluid
capillary action. It is important to ensure the used so
as nor (0 introduce contaminants. Some comain
ethanol or oils, and papers and can include
levels of AI wetl a~ other metals. Glass is a source of as well as
S and and can contaminate the otolith surface. The resins used for
should have low levels of trace and it is often
the resin otolith samples in order to esti-
mate concemrations or potential levels of contamination.
Gloves should be worn preparation in order to avoid
contamination from skin salts and oils.
Bulk results that must be normalised to the exact
nrPFlrmn thus has a direct and
effect on the calculation of elemem concentration.
2.1.5. '"n,nrlclt'An measurement

The most difficult part of OMC is the measuremem of (he elements in
the fact that most
cations to undersrand some of physics
and chemistry, and sometimes to learn (0 opera re machines that are
designed for use in other sciences. Biological also rend to
these machines to their limits of "prt()rrn,~
centrations of the elements of interest are as is the amount of
available and the need for spatial resolurion may be great.
nor apparent to new users.

can be used ro most of the measurement error, bur
such studies have been
lllstmment conditiom have a
mem of element concenrrarions. For for electron
qualiry of the data depends on the interaction between beam diame-
ter, rhe and the of the excirarion current and
sition time. The sensitivity of bulk analyses is affected dilu-
tion rates and the number of elements measured, and also varies
with the element or isotope measured. In all cases the instrument set-
used represent a between the needs for
accuracy, and of analysis.
296
One of the major sources of in measuremem results from
The of rhe error can be estimated

that include different CRMs, ancl the availability of new CRMs for
otoliths should alleviare rhis problem, Even with suitable CRMs,
matrix need ro be controlled or taken into accoum, There are two
types of effects to be considered in OMC. The otolith matrix
cakes in the determinarion of otolith composition inco
account because the array of elements and their relative con-
centrations that are associated with the protein is not known, Some
prococols for bulk will dissolve the so that
both and components are measured, Other
tion "ghost" that represents differem
amounts of the The effects of the protein
marnx on the measurement of elements by surface methods are harder
co but they include variations in the quality of the surface
(har alrer the of the Mauix effeCts also mean the ana-
associated wirh elements within com-
In ICP-MS and WDS the detection of different elements in the
otolith is complicated the predominance of
tfibured these mCltrix can be controlled by
where the analytical blanks and standards are also in a com-
solution, This is easier to achieve for SB-ICP-MS than
for LA-ICP-MS or WDS, where there are no suitable CRMs. The quality
of the orolith surface is an important consideration in sur-
Only LA-ICP-MS is insensitive to the
(but the surface should still be free of contamination), Cracks
in the surface of the otolith cause extra of electrons and thus
introduce errors in the detection of elements and the of
their concentrations.
The translation of the machine-measured signal into concentrations of
elements on a calibration mode! based on the measured
from the analytical standards. The fit of the calibration lines should be
checked before (0 ocolith otherwise COJJ1-
will introduce measurement errors.
instrument is a significant problem in these techniques,
The term refers ro or sometimes in the sensi-
of the machine over the course of one or more consecutive analy-
sis sessions. There are several methods used (0 minimise the effects of
drift on the determination of orolith The usual
is to try to over a short time period, Sometimes a sub-
of owliths can also be re-measured at imervals w
instrument drift and correct for it later calculation stages,
Randomisation of samples ro ensure that drift errors do not sys-
affect any particular group.
One significant source of error in measurement is the analytical blank.

The of the analytical and procedural blanks affens the blank
correction used instrument software to calculate concentrations. The
blanks are also for calculation of the LODs, and any contami-
nation or matrix effects will result in lowered Blank cor-
values for element concentra-
tions. These are to concentrations below the LOD.
In WDS the limits anPlllfl7! (LOD) are estimated for each element from
the standard deviation of the when the standatds
are measured. For solution-based ICP-MS, the limits of detection are
estimated from the standard deviation of measuremenrs on
blanks to the solution that is used to dissolve
The estimations to those of solurion and have to
be corrected a dilution factor to refer to the otolith i.e. to
the LOD in terms of (= pp m) of otolith. To no agree-
ment methods of estimation of the limits of detection for
LA-/CP-MS has been and several methods have been
- estimation from measurements on the argon gas (i.e. wirhoU( abla-

tion);
estimation from measurements on standards NlST 612 &
610);
estimation from measurements on standards.
Each method leads to a differem estimate of LODs bur none of them
because of the lack of matches with the otolith matrix.
The first one is the least 'conservative' since it does not take into
accoum the of ablated material, which influences
LOD values.
Whenever the amount of otolith material varies
between spots (as in laser or is nor
concentrations are usually relative to some constant measure.
In stable the IRMS measures ratios in the sam-
ple and compares this ratio to that in the standard (PDB, SMOW, erc).
In LA-ICP-MS rhe usual merhod is to normalise the COUntS of the ele-
ments with tespect to simultaneous countS of calcium. When calcium
concentration is not calculation of elemental concentration
IcnW!'!letJC1l for calcium within CaC0 . If
3
otoliths are as pure the proportion of calcium 40%.
H
The difference from the "true value will affecr the estimated concen-
trations of the otolith
dances. any in the distribution of calcium
within the otolith will result in under- or over-estimation of the true
element concentrations.
In OMC there are many sicuations in which (he concen-

trations measured are close to the LODs, and methods of treatment of
valuej' below the LOD muse be decided before statistical The
Iireracure unfortunately conrains few details
about either the calculation ofLODs or the treatment of values that fall
below the LOD. It is necessary to report how the LOD has been calcu-
how the results falling below the LOD are treated sta-
and what percentage of the results for each element fall
below the LOD, This should be separately for each group of
Differences in these values between sessions can obscure the
real comparisons between groups and produce rrends m
the data.
There are several methods for with analytical results below the
LOD:
- i nel ude all the values which are below the LOD in calcu-
lations of means to represent individual fish or otolith areas;
set values below the LOD to 0, °in calculations of means to
represenr individual or otolith areas;
set values below the LOD to the LOD, the LOD value in caleu-
lations of means to represent individual or otolith areas;
- enter values below the LOO as values;
convert aU values of elements which have measurements below or
near (he detection limit ro data and use
statistical methods,
In some applications it is (0 ignore elements that have many
values below the LOD. This is useful for improving data quality when
the results are subjected (0 multivariate in stock separa-
tion In all cases, (he occurrence of values below the LOD
must be reponed for each as:
.. percentage of all points below LOD;
• percentage of individuals with below the LOD,
Statistical methods that include the of the proportion of
below the LOO should be characterise popu-
lation and individual responses.
The values obtained for element concentrations are only reliable if the
measurements are free of or chemical lnf,,.-h,,,,,,"rp( Interference
can artificially elevate the calculated concentrations or obscure their
measurement. The presence of interference can usually be detected
the concentrations calculated for different of a sin-
element. If the ratios between the concentrations in the
sample to the naut/'at,!V then those data
values can be as free of interference. It is helpful ro be
299
familiar wi(h likely sources of inre rfere nce before beg inning an OMC
scudy. Speerral inrerfe ren ces in X-ra y emi ssion lines are published for
WDS and EDS, while sof(ware in newer ICP-MS ins(fum enrs o ffer s rhe
capabiliry o f chec king likely inrerfe ren ces (wirh assoc iared quanrifi ca-
ri o n) fo r eac h iso cope o f any given e leme nr wirh compound s of simi-
lar nominal masses. In ICP-MS, spec rrosco pi c inrerfere nce is mosr
likely co occur wirh isoba ric io ns (e.g. 87Sr and 87 Rb), re frac cory ox id es
(MO+) and a rgon polyacomic io ns (ArX + wirh X= :H,C,O,N ) formed
from (he arg on plasma. Wi (h rega rd co (he la rrer s icuarion , rhe case
rhar is ofren di sc ussed is rhar of inrerfe rence berween 40 Ar1 60+ and
56Fe+. Vari o us ArX + ions may inrerfere wirh rhe various Fe isocopes,
alrhough co differenr ex renrs, complicaring rhe m eas uremenr of (his
e lemenr by rhi s rechniqu e. Wherher an inre rfere nce will be separa(ed
from an isocope of inre resr depe nds o n rhe ins(rume nr mass resolurion
whi c h has been considerably im p roved in recenr years.
As m e nrioned above, instmment drift can produce OMC da(a (fends rh ar
are aerually a reefaers of rh e analysis. Some rec hniques use a ucomaric
sof(ware co correcr for ins(rumenr d rifr before producing rhe final co n-
ce nrrarion valu es. le is use ful co examine rh e conce nrrarion dam before
sra(isrical analysis in order co dereer (he presence and influence of
ins(rume nr drifr. The dam values for each e le menr should be plocred
sequ e nrially ag ai nsr rhe o rder of m easuremenr. If rhere are (ime rrends
in rhe dam, i( is somerim es poss ibl e co derermine and coneer for rh e
drif( usin g a regression model fir co meas uremenrs of rhe blanks or
srandards mad e ar (he sra re , middl e, and end of eac h session.
Prior co srarisrical analysis , rhe d am should be examined and seieerion
crireria ap pli ed co exclude olltfiers , or aberranr a nalyrical reslllrs . Cer-
(a in dam poinrs should be re jeered fro m fureh er an a lysi s, especially
wh e n rhey res ulr from irreg ulari(i es and fraerures in rhe orolirh sur-
fac e, rh e inclus ion of resin in a SPO(, ere. In WDS analysis, measure-
menrs are so m erim e inadveree nrly made ar fraccures or scrarches if (he
visllalisa rion an ac hmenr has low m ag nifica( io n or if m eas ure menrs are
mad e along pre-se r radi al (rac ks. Aberranr poinrs in WDS can easily be
idemified by rh eir Ca values , and rhe res ulrs for all ele mems m eas ured
ar s uch SPo(S should be re jecced. In LA- ICP-MS fraccures and res in a(
(he edge of rh e o(Olirh may inadvene nriy be included wirhin abla(ion
poinrs becaus e rhe exac( ablarion volume vari es and cannor be con-
(fo iled. These po ims should be ide nrifi ed by rhe values of ab undanr
elemenrs such as Sr or Mg. Conrinued improvemenrs in laser (ech-
nology may reduce rhe errors in ablarion poinrs. The ide nrifica(ion
and re jecri o n of aberranr poinrs s hould be ba sed on crireri a rh a r
depend on how far rhe resulrs devia(e from rhe average. Local areas of
co nramin a rion ma y also produ ce abe rranr p o ims rhar should be
rej ec (ed in (he same way.
300
Otolith microchemi stry
When suspect data points ate the result of analytical problem s they
should be removed before statistical analysis. Occasionally, anomalous
data values that ca nnot be attributed to analytical problems are
observed. This can happe n in multi-element determinations in which
the concentrations of all other elements for this measurement are "nor-
mal " and one element is "abnormal". In some publications these out-
liers are removed from statistical analysis, but there is no real justifi-
cat ion for thi s.
There is a danger of psettdo-replication errors in analysing otolith com-
position data. OMC is expensive and time-con suming and the re is
often press ure to reduce th e number of samples analysed. In some
stuclies SPOtS measurecl along an otOlith transect are pooled to repre-
sent a single value for an individual. Likewise, several spOts meas ured
in one area of the otOlith are often analysed statistically as ind epend ent
replicate samples of thar individual. Values measured along a transec t
are usually not independent because of age- or size-related trends in
composition. Independ ent replicate measurements for an individual
fish should ideally be taken from different otoliths. A random sub-
sample of transect measurements can be lIsed for statistical analysis ro
avoid the problem of co-d ependence. Thresher (1999) discusses other
pseudo-replication ptOblems associated with sampling strategy and
fish handling.
2.2. Accuracy and preciSion of analytical tools

Very few stud ies have focused on methodolog ical copics. Gunn et af.
(1992) reviewed the principles of using Electron probe micro ana lYSIS
(EPMA) for otOlith composition analysis (Ca, Sr, Na, K and S). They
provided the first quantitative comparison of the detection capabili-
ties of EDS and WDS and concluded that EDS is not sensi tive and pre-
cise enough to provide suitable data for otOlith composition analysis.
They discussed the effects of WDS operating conditions (beam area
(A), accelerating voltage (E), current intensity (1) and acquisition
time ) on data quality and concluded that a beam power density
(BPD = E . I tA) of 3 flW.pm- 2 is an acceptable compromise. Toole &
Neil se n (1992) also di scussed the effect of measurement errors associ-
ated wi th Sr and Ca quanti fication by WDS on hypotheses drawn up
from otolith SrtCa ratios.
More recently, an international otolith composition experiment
(Campana et aI., 1997) compa red the potent ial of the most popular
surface techniques (EDS, WDS, LA-feP-MS and PIXE) and examin ed
differences among laborarori es using similar ins trumentarion. The
resul ts showed thac:
• there is no single instrume nt to be preferred for elemental analysis.
Na and K were measured by only E MA (EDS 1Flla.:fl'
BlJRot.~itc!~
Centre de Br I
8ft 70 - ~ ~O(l ,J I_ :/;7P t- eol
--- --. -- .~
elements were measured with LA-lCP-MS or PlXE. As expected , EDS

showed the highest LOD and the poorest precision;
• accutacy, precision and LODs differed among laboratories using the
same instrument. Such differences may be partly due to laboratory-
specific operating conditions. This makes it difficult to compare
published data and indicates that inter-calibration is needed for pro-
jects involving several laboratories;
• the development of certified reference materials (CRMs) for otoliths
is critical to the further development of elemental analysis and is also
a prerequisite for comparisons of published data. Actually, geological
CRMs that are often used in OMC do not match the otolith matrix
(aragonite and proteins) nor do substitutes that have been proposed for
probe analysis such as otolith powder fused into glass beads (Campana
et al., 1997) or pressed carbonate pellets (Perkins et al., 1991; Pearce
et aI., 1992). A CRM for solution-based analyses, prepared from Lttt-
Jantts sebae otoliths, has been announced, with certified values for Na,
Mg, K, Ca, Sr, and Ba and reference values for Cu, Zn, Cd and Pb
(Yoshinaga et aI., 2000).
302
303
304
Preparation and observation techniques
Cha r VIII
Preparation
and observation techniques
305
306
As has been seen in the oreliths and skeleton

bones are used to estimate the age of fish in years, seasons or
of cs steps that may be com-
such studies it is neces-
sary to know the constraints on available time and costs, and
the final and expected results (chap. HI). The choice of an
age estimation method for a given involves on an
CS ete.) and prepa-
ration method sections, thin sections,
ete.) for that CS. will help the reader to choose from
among the multitude of techniques available.
In any the choice of method for cs may also
depend on the size of the e.g. direct embedding of larval oreliths,
or on the type of final analysis required, e.g. and polishing
otoliths for microstructure studies and "clean" extraction and prepa-
ration for studies. In the latter rype of
are primarily governed by the need
re reduce
The aim of this the reader's choice in the direction
first by flow cham to help
an extraction method for a
a and
an observation method. The selection of both the method
and the observation to be employed should
also reflect the demand for demonstrable assurance
control measures (McCurdy et af., 2000) (see also Web site
307
Manual of fi sh scleroc hronology
A. Aid to decision trees

H . Troadec, H. de Poncual
The primary aim of rhis sub-cha prer is ro p rovid e rhe reader wirh an
overview of rhe su bsequenc descriprions of ex rracri on , prepararion and
observari o n p roc ed ures. Ir is also incended ro g ive rhe reade rs direcr
access ro rhe relevanc procedures according ro rheir individual g oals
and consrraincs . The rask of ourlining rhe decision (fees was complex
and exhaus ri ve represencarion of rh e diffe renc ways ro process eac h CS,
in so far as rhi s is possible, would ha ve resulred in a huge and unusable
n ow ch an. For rhi s reason, we have chosen ro redu ce rh e scope of rhe
dec ision rree ro rhe principal CS processing procedures and ro primarily
rarge r a public of nov ice users. These decision (fees were based on rh e
book marerial and cons rirl!(e rh e uni que link berween rh eo ry and
pracri ce. Ho wever, CS experrs m ay cons ider rhem incompl ere, ove r
sim p lifi ed or eve n wrong as counrer-examples can read ily be found in
rhe lirerarure, w hile novices ma y even have difficulcy in a nsweri ng
som e of rh e q uesri o ns. We ho pe rhar ar leasr rh is s ub- chaprer will
guide novice readers in rh eir choice of a CS, specially when lirera rure
is poor, and accelerare rheir CS p rocess ad jusrmen r.
Trees are composed of:
• leaves char are represenred by recra ngular boxes and cor respond ro
proced ures described in rh e foll ow ing sub- chaprer. The mulrimedia
version has hyperre xr links be rw een each leaf and rh e relevanr sub-
chapreL Iralic rex r indicares oprional proced ures;
• concrol branches rh ar are represenred by diamond shaped boxes and
conrain condirions permirring orienrarion of rhe ([ee run when a node
is reac hed . Mos r of rhe conclirions re lare ro srudy aims, bur some will
re quir e a CS prepararion ro be made before che quescion can be
answered, (e.g. , "Ring conrrasr enhancemenr needed r ).
A cree run fini shes when an en d leaf is reached, rh e prorocol being
rhe colleccion of che procedures idenrifi ed during che run . When
en co uncering a mulciple choice branch, or a quescion n ode chac
appears ro be ambiguous, refer ro rh e lireracure and /or yo ur p erso nal
experience in order ro compare che differenr solucions. If yo u incend ro
inrroduce a cec hni cal innova rion (i.e. vary ing a preparaci o n procedure
or using a differenr CS), you should assess che relacive benefic prov id ed
by chis cechnical improvem enr versus your capa bilicy ro compare yo ur
resulcs w ich previous scudi es. Finally, if you ince nd ro use several
observac ion cechniques, re me mber ro ch eck chac all rh e prepararion
proc edu res are comparibl e and rhar rhey can be adequarely comb ined
ar rhe differenr srages, before beginning rh e work.
308
The firsr deci sion-nee uses very general crireria ro selen an appro-
priare CS , i.e. orolirh , scale or bony rissue. The remaining rhree rrees
focu s on rhese CS and on rheir differenr processing m erhod s.
Temporal
resolulion required' Daily ----------~
No
Seasonal
Yes Yes Are scales
usable'
No
fISh survival
> ---Yes
required'
No
Old Reliable re sulls

individuals? No No-- from ololilhs Yes - -- - ---1
or scales?
Yes Yes No
Low bone
Yes
remodelling'
No
309
large fish
w :s;
o '"co
:0
9<.
S-
w
n
No ro
(3
'"
::y
"
::0
Q.
0
I)Q
'<
No
Yes
Seasonal
Daily
< 1
Yes
1
ClJT OR BREAK OTOLITH
,-------Ves------~
No
Improvement
Ves of increment contrast
needed? Temporal resolution ;>------- Seasonal
required?
Daily
Light microscopy
2 WHOLE OTOLITH
Age and growth Chemical

composition
Clupeid
or Scombridae? ____------ Ves
No
Improvement of increment
Ve s Embed otolith
contrast needed?
No
light microscopy
311
Manual of sclerochronology
CONTRAST ENHANCEMENT
Oiotith embedded? >---- Yes --------"""
No
Staining
long term
conservation?
No
Embed otolith
312
MICROSTRUCTURES
Yes--+<:::-
Yes
No
Mounting
with polyester. Mounting with
epoxy resin or thermoptastic glue
No thermoptastic glue
High quality
of surface state required' > - -- Yes - - - - ,
No
What information Chemicat

Age and growth
required? compositIon
< l~m
1
Etching
1
Light microscopy SEM
313
~anual of fish sclerochronology
5 MACROSTRUCTURES
Routine work Multiple

Individual embedding No
e.g. demersal species? Yes--
embedding
Multiple
Sectioning sectioning
Mounting
Mounling
High surface
NO
quality required?
Yes
Grinding and polishing

(manual or automaticl
Age and gro\\1h What information Chemical

required? composi tion
Contrast enhancement Microchemistry

Yes
required?
3
No
~
Light microscopy
Preparation and observation technique s
Use bone preparation methods:

No BONE
Tree
Yes
r-- - -- --No Long-term storage?
Yes
Routine work
or high-definition image? Yes ------~
No
Ac etate cellulose
Slide mounting imprint
Light microsCODY
Verification
needed? , , -1L-__ S
_E_M_ _---'
31 5
High commercial value
Yes species or high remodelling No Light microscopy
process'
w s:
m '"c:>
~
S-
t;;.
:::r
V>
<"">
ro
No
Ye s o
<"">
le.g. Osteichthyans) :::r
le.g . Chondrichthyans) o
:>
o
(5
ao
'<
Annulus reduced
to AGL'
No~ No -+-<::.:: Yes
Yes No
Yes
Embedding
No---- .'11
Yes
Yes ~
B. Extraction and conservation

J. Panfili
The stages of extraction, handling and conservation of cs are of prime

importance for the success of the subsequent stages of the analysis to
be undertaken : age estimation , microchemical analysis, ete. Inappro-
priate conservation techniques can cause irreversible degradation of
the samples. All the planes of extraction, preparation and observation
described in this chapter refer ro the classical orientations of living
symmetric organisms: sagittal, frontal and transverse planes.
When CS are being collected and conserved for later studies of their
composition (microchemistry) it is important to red uce as far as pos-
sible any sources of sample contamination. Gloves should be worn to
prevent contamination by salts and oils from the skin. Metal dissec-
tion tools are also a source of contamination and extraction should
therefore be carried out using plastic or ceramic tools. Storage affects
the composition of otoliths and potentially other CS. The samples
should be stored in inert containers (not paper envelopes) and should
not be stored in conserving fluid. As far as possible, the extraction and
manipulation of samples for microchemistry studies should be carried
om und er clean conditions and if possible in a laminar flow cabinet.
1. Storage and preservation of whole fish
The method used to conse rve the whole fish influences the preserva-
tion of cs. For example otolith structure can be altered by inappro-
pri ate conservation techniques, as post mortem degradation or dissolu-
tion inside the labyrinth of the internal ear is a possibility (Brothers,
1987). Thi s is generally the case for any internal CS.
It is bette r to extract calcified material from freshly killed fish at any
stage of their life history. Extreme care should be taken with larval and
juvenile stages as their otoliths are smaller and more liable to degra-
dation (Brothers , 1987). If conservation is necessary, this may be done
(1) by freezing or (2) in alcohol. Several alcoholic solutions have been
mentioned in the literature bur we recommend the use of95% ethanol
(at lower concentrations some risk of deterioration remains). One
should also check the volume of alcohol relative to the volume of the
fish in order to avoid dilution of the effective concentration. It is of
course strongly recommended to avoid acid solutions such as formalin
for conservation because of the calcareous nature of the otoliths or
other structures and the possibility of secondary destruction
317
(Williams & Bedford, 1974) . Even buffered formalin should be

avoided (Brothers , 1987 ).
2. Scales
Th e sc ales are the easies t structures t o be ex tract ed. Th ey ca n be

removed direc tly bur careful ly, even on living fish, with forc eps (fig.
VIII. B.!). The survival of individuals aft er scale removal is not a prob-
lem (scale loss is a natural phenomenon). The area from which the
scales are sa mpled mu st be chose n carefull y: the shape of the scale dif-
fers from one area ro another within individual fish , so the sa mpling
site must be stand ardized . This si te may also differ from one species ro
another (Werd er & Soares, 1984; Bag liniere & Le Louarn, 1987). The
first experiment on any new species should compare vari ous sampling
locations before choosing those that are least rich in regene ra ted sca les
or in vari ations in shape (Paul, 1968; Werder & Soares, 1984; Al-A bsy
& Carlander, 1988). Sampling locations are usua lly in the latero-dorsa l
area (e.g. under the dorsal fin) or an atea where the scales are protected
from damage (e .g. under the pectoral fin s for some species). Some loca-
tions a t which the scales are modified, such as the latera l line, should
be avoided. Several scales should be ex tracted at the same location for
the sake of futur e comparisons. The main ad vantage of scale ex traction
is that the fish can be kep t alive during sam p ling .
Afte r ex trac tion, scales should be cleaned. The cleaning operation is
fairly simple , ranging from direct conservati on with mild cleaning
(e.g. wiping in absorbent paper ) ro ultrasonic bath cleaning . The bath
may contain distilled water, normal water, potash, sodium peroxid e or
tr yps in. The duration of imm ers ion in th e act ive so luti on mu st be
controlled ro avoid partial destruction of th e sca les .
Scales are usually co nserved dry in labell ed vials or envelopes. Scales
a re normally hydrophilic ti ss ues and dry co nservation distorts their
o rigin al shape. When a scale that has been srored for a long time is to
be studi ed it is sometimes rehydrated (e.g. for large or thick sca les for
some spec ies), normally in water and without the need for any par-
ti cu lar precaution s ro be taken. Scal es are also often conservecl after
mounting between m icroscope slides (chap. VIII .C1 ).
3. 0toliths
Oraliths should be extracted from new ly killed or d ead fish, or after

conservation by freezing or in alcohol. If necessary, it may even be pos-
sib le ro use o rolith s extracted from cooked fish, providing that suit-
able precautions have been taken ro ensure that the orolith structure
has not been altered by the cookin g process. Extraction is rela tivel y
318
Figure VIII.B.1
Scale extraction from
a Salmonid (Saimo trutta),
using forceps,
in the latero-medio-dorsal
area. A, anterior; D, dorsal;
P, posterior; V, ventral
(photo © Ifremer
O. Dugornay).
simple in large fish, depending on species and/or individual size, bur

is more difficulr in larvae and juveniles. Obviously, prior knowledge
of rhe cranial and vesribular anaromy of a given species wi 11 faci li rare
orolirh removal. Usually only rhe larger orolirh is exrracred and srored
for subsequenr srudy (rhe sagitta for non-Osrariophysean and asterimts
for Osrariophysean species). The procedure is more complicared when
we wish ro exrracr all rhe orolirhs, due ro rhe small sizes of rhe 2nd
and 3rd pairs: in such cases a binocular microscope is essenrial.
In a recenr review of descriprions of rhe inrernal ears of carrilaginous
fishes, Lychakov et a/. (2000) poinr our rhar rhey do nor have orolirhs
like bony fishes. The rerm "orolirh" is rherefore resrricred ro bony fish
species. Age in Chondrichrhyans is esrimared using parrs of rhe inrer-
nal skeleron.
When removing rhe orolirhs for rhe firsr rime from a given species,
one should be aware of rhe orienrarion planes of rh e marerial under
s(Udy. This is very imporranr for rhe subsequenr srages of prepararion
and descriprion. All orolirh images should incJ ude an indicarion of rhe
orienrarion.
3.1. Extraction from large fish

Excepr in very small species, orolirhs can be exrracred wirhoU( rhe
need for magnificarion (e.g. by binocular microscope). There are four
main rechniques of removal, rhe choice of which depends on rhe plane
of rhe cranium secrion:
• fronral head secrion;
• rransverse head secrion;
• sagirral head secrion ;
• rhrough rhe gills, venrral cranium secrion.
319
The frontal head section is universal and can be used for any kind of
fish cranlum
method is
modifications. The
bur range from for small individuals to electric bone saws
fish tunas, swordfish, and kitchen knife is
The section must be taken carefully in order to avoid
cutting into the internal ear and the otoliths, After the
cranium section, the owliths are usually removed with forceps, Care-
ful removal may permit extraction of the whole semi-circular canal
system, the three owliths, but only the
O(olirh is extracted.
3,1,1, Frontal head section (fig, VIILB.2)

A frontal section of the part of the cranium is normally
rhrough the dorsal part of the eye, to the major axis of
the and at the outer of the opercular bone
VIILB,2a), The upper part of rhe cranium is removed
The brain is carefully removed from rhe cut cranium
VIlLB.2c, c', d, The cavities of the inrernal ears
rhe semi-circular canals are rhen visible from above: are
rioned at rhe bottom of the cranium in the part of the
brain cavity and lateral to the main axis of rhe fish, The otolirhs are
removed wirh e} This method is used
in laterally (most highly evolved) and in flatfish,
3,1.2. Transverse head section (fig, VIILB.3)

A transverse senion of the head is made at rhe posterior parr of the
the posterior parr of rhe pre-
a'), The whole head is cur off and the
from part is Looking from the back at the head rile
posterior part of the brain bulb) is rhen visible in the upper
part of the cranium and the twO caviries of the inrernal ears are located
laterally and below it. The brain does not normally have to be
removed, The otolirh;; and the semi-circular canals are directly
removed with VIIJ c'), This method may
training, depending on the of the head, in order
to be sure of the level of the cur: there is often a risk
of the otoliths, It is used mainly for such as tuna
and also for Iliformes,
320
a a'
b ~ ____ ~ b'
c c'
d d'
e e'
Figure VIII.B.2 . Otolith removal with a frontal head section. a, b, c, d, e: small species (Vinciguerria nimbaria, Photichthyidae,
4 cm SUo a', b', c', d', e': medium-sized species (Trisoplerus luscus, Gadidae, 25 cm SU. a) Lateral view of frontal section with
scalpel blade. a') Dorsal view of frontal section with knife. b, b') Views of frontal section after removal of the dorsal part of the
cranium and muscles. c, c') Location of brain (white arrow) before removal. (d, d') Otolith location after brain removal; white
arrows indicate location of internal ears; black arrows show sagittal otolith. e, e') Sagittal otolith extraction with forceps; white
arrows indicate the location of the internal ear cavities; black arrows show sagittal otoliths. A, anterior; D, dorsal; L, left; P, pos·
terior; R, right; V, ventral (photos© Ifremer O. Dugornay).
321
3.l.3. Sagittal head section (fig. VIII.B.4)

A sagi ttal section is made passing through the middle of the head
(fig. VIII.BAa, a'), from the mouth until at least the end of the oper-
cular bone. Due ro the [atero-centtal position of the internal ears in
the cranium the section must be done precisely. A transverse seCtion
of the head in the opercular area then permits separation of the tWO
half heads. Each half head is treated separately. The brain is removed
ro locate the entire internal ear (fig . VIII.BAb, b', c, c'). The oroliths
and semi-circular canals are carefully extraCted from the ear, at the
a a'
b b'
c c'
Figure VIII.B.3 . Otolith removal with tran sverse head section. a, b, c: small species (Vinciguerrio ;,;;;;bo,ia, Photichthyidae, 4 cm
SU. a', b', c': medium-sized species (Trisopterus luscus, Gadidae, 25 cm sU. a) Lateral view of transverse section with scalpel
blade. a') Dorsal view of transverse sec tion with knife. b, b') Sagittal otolith extraction with forceps after removal of brain. c, c')
Sagittal otolith extraction; white arrows indicate location of internal ear cavities; black arrows show sagittal otoliths. A, anterior;
D, dorsal; L, left; P, posterior; R, right; V, ventral. (photos© Ifremer O. Dugornay)
322
back and beneath the brain This method is

mainly used in with
makes it easier ro extract the three otolirh as rhe anatomical
position of the is shown precisely, this a useful
method for unfamiliar
3.1.4. Through the ventral cranium section (fig. VIII.R5)

The IS our from rhe ventral face of the fish head
VIILB.5a). The are visible and the branchial arches are CLlt on
The ventral structure of the neurocranial bones
appears after all the surrounding tissues have been
cleared away. The bullae are locared in rhe medio-Iateral parts
of the neurocranium. A small cur imo the external part of the bulla
opens the internal ear, from which the main otolith the
can be removed VIILB.5c). If the is pushed inside
the brain it is very difficult to extract it without all the cra-
nial bones and the whole brain. This method is particularly
suitable when there is a need ro conserve the aspect of the
head after rhe otolith for in commercial
destined for the market. It is therefore suitable for any kind of non-
only for the extraction of the
Some other semi-automatic methods for sampling otoliths with drills
in fish markets have been improved and are now suitable for
tam commercial such as the Scombridae
3.2. Extraction from small fish VIII.B.6)

This descripuon concerns all fish and stages with lengths
of less than 2 cm, i.e. and/or larvae. The extraction
must be done in an . medium (we recommend 95% ethanol
bm some authors use water, glycerine or The use of 95%
ethanol prevents erosion of the otolith structure. The
removal is normally done in the same conservation medium is used
for rhe whole individuals.
be Llsing
a d binocular) with polarised
They are known to be refringent to
them to be disci from the
VIII.B.6a). Otoliths are removed and
needles 150flm co tease apart the
are then cleared of surround-
the disseccing needles.
appropriate
The most delicate step
323
a a'
b b'
~_&liO _ _ _• C'
c
d _ _ 5.._.,jjjJZ.:O~::U_1II d'
Figure VIII.BA - Otolith removal with sagittal head section. a, b, c, d: small species (Vinciguema nimbaria, Photichthyidae, 4 cm
SU. a', b', c', d': medium-sized species (Trisopferus iuscus, Gadidae, 25 cm SU. a) Dorsal view of sagittal section with scalpel
blade. a') Media-dorsal view of sagittal section with knife . b, b') Views 01 brain location (white arrow) in hall-head. c, c') Brain
removal with forceps inside ecch half-head. d, d') Sagittal otolith extraction with forceps; white arrows indicate location of inter-
nal ear cavities; black arrows show sagittal otoliths. A, anterior; D, dorsal; l, left; P, posterior; R, right; V, ventral (photos©
lfremer O. Dugornay).
324
a ____
b
Figure VIII.B.5 . Otolith removal with ventral cranium section

through gills: Gadus morhua (Gadidae). a) Ventral view of opercu·
lar aperture and gill arches sec\lon with knife. b) Ventral view of
base of cranium (shown by knife). c) Sagittal otolith extraction
after cutting of cranium; white arrow indicates location of internal
ear cavities; black arrow shows sagittal otoliths. A, anterior; L,
left; P, posterior; R, right. (photos© Ifremer O. Dugornay). c
is handling. If the oroliths do not need any furrher preparation, the

best method is to embed them direcrly in resin (chap. VIII.C.2.2.3).
They are handled by sucking up rhem direcrly into a drop of liquid
resin; orherwise otoliths must be handled dry for srorage.
The same procedure can also be employed for removing otoliths from
embryos in eggs. Firsr, rhe embryo is exrracted from the egg and then
rhe otoli rhs are removed as described above for larvae.
Secor et af. (1992) described rwo orher merhods for exrracting very
small fish otolirhs; bleaching and embedding, but rhese need much
pracrice and rime, and finally they are less successful: we do nor recom-
mend rhem. Bleaching consisrs of immersing the marerial in sodium
hypochlorire and exrracring rhe otoliths after tissue lysis. Orher pro-
rein enzymes such as trypsin (Rojas-Belrran & Vincent, 1993) have
also been used, bur the success of rhe removal procedure seems ro be
variable and it is not possible to confirm rhat there has been no ero-
sion of the protein content of the otolith. The embedding method
requires the individual fish to be completely dehydrated and then
embedded in synthetic resin (chap. VIII.C.2.2). The handling of rhe
fish is facilitated and it can be cut (sectioning and/or polishing, chaps
VIII.C.2.3 and VIII.C.2.5) in any plane and particularly at the otolith
level. This method is nevertheless very complicated and needs careful
325
training at each step. Due to the thickness of the saws ancJ the rela-
tive size of the owliths, it is often very difficult w reach them in the
brain area.
a b
Figure VIII.B.6 . Otolith removal from larva e under binocular microscope and polarised light: Vin ciguerria nimbaria
(Photichthyidae). Otoliths are clearly located with their refringence against the polarised light. a) Dorsal view 01 anterior face of
a larvae; S, sagitta; L, lapillus; Scale bar = 500 ~m (photo J. Tomas). b) Otolith extraction using dis sec ting needles; cranial bone s
and the brain are removed before isolation of the sagitta (yellow arrow) and lapillu s (red arrow); Scale bar = I mm (photo©
Ifremer O. Dugornay).
3.3. Otolith cleaning and conservation

3.3.1. Cleaning and handling
Oroliths must be cleaned before srorage in order to remove any
adhering remnanrs of the mamla and vestibular tissues after dissec-
tion. After drying, any remains of such tissues would preclude good
observation of whole owliths or good quality embedding in synrhetic
media. The easies t way is w clean them during or immediately after
extrac tion. Mechanical cleaning is done by teasing away the ti ssues
with fine wols such as forceps and dissecri ng needles with the owlith
in a liquid medium (e.g. water or ethanol) (Secor et aI., 1992). Owliths
can be simply cleaned by wiping the m with absorbenr paper. Reaction
cleaning can be done by immersion in dilute bleach (10 to 100%
sodium hypochlorite) for a given tim e (a few minutes to several hours),
following which it is important ro rinse them several times with water
and/or ethanol, and then dry them.
Handling does not pose a major problem for large owliths (> 500 jJm):
they can be manipulated with forceps a nd/or by hand. Smaller
otoliths « 500 jJm) are much more difficult to handle, especially
those from larvae, which have diameters of only a few microns. Small
owl iths are also ex tremely fragile and ca n be crushed with very slight
326
pressure. We recommend tbe hlcure use of these oroliths

before any protocoL Secor et al (1992) list four tech-
for handling and otoliths:
- remove the orolith with a small amount of the
Transfer the liguie! with the otolith to a clean
slide or storage
shunt the 0(01 ieh over to a clean area of the medium and
let it After drying press a down on the top of the owlith
and transfer it to the storage conrawer (perhaps a binocular
up dried owliths with a werred needle and place
or invertebrate to transfer
otoliths,
The handling of very small otoliths nevertheless If
no further examination of the whole molith needed, we recommend
It in the media (chap, VIJI,C.2A.1), The otolith
can be sucked inw a drop of medium (resin) at tbe end of
the medium
water or ethanol): there the media as most resins
are The otolith inside the drop of resin is pur
into anmher of resin on a cover slip. allows it
to be observed on both sides and/or to be anmher tech-
3.3.2. and conservation

The best method of otoliths is to store them
which are ro "tissue

culture can be individually handled or stored in
special supports The microtubes are also rigid to protect the
otoliths from fracture to their concavo-convex shape, which often
if they are stored in earlier recommended

(Williams & dark storage areas are probably
1987),
Some authors mention conservation in a medium such as
which can be useful for and small otoliths. The concentration
must be at least 95%. We have also mentioned direct
media (see but care must be taken not to make the moum too
the distance of the may be very
sho[( at (Brothers, 1987).
4. Other skeletal parts
4.1. Extraction
All rhe or her skeleral parrs belong ro rhe inrernal skeleron and musr
therefore be extrac red by dissect io n. However, th ere is a difference
between the "ex terna l" bones that belong ro the fin s (spine, fin ra y)
and are eas ily extracted, and the "inrernal" bones that frame the body
(opercu lar, verrebrae, cleithrttm, etc.). Th e c uning rools used vary
according ro the size of the fish bur range from sea l pels for small ind i-
viduals, throu g h various types of knives, ro elect ric bone saws for
la rge fish. Fin ray removal starrs with the separarion of the target ray
(generally 1s t a nd /o r 2nd) from irs neig hbours wirh a knife (fig.
VIII.B .7a) and conrinues when the surrounding muscles at the base
of rhe ray are cut in order ro extract the whole piece (fig. VIII .B .7 b).
The bo ne structure musr be extracted carefully in order ro conserve
its integrity fo r furrher analyses. Removing intern al bones (e.g. ver-
tebrae) is al so easy but the dissecti o n is more complete: the muscles
and th e tissues surrounding the ta rge t bone are first carefully
removed (fig. VULB.Ra) and then rh e bones are cur ar rhe leve l of
their a rticularions and ex tracted in their enrire ty (fig. VIILB .8b). If
the dissecring planes are uncerrain, ir is berter ro take rhe bones wirh
the muscles tha r are anached ro rhem, even if this means cleanin g
every rhing afterwards.
4.2. Cleaning and conservation

Cleaning of skeleral parrs is an essential precursor ro good qua li ry con-
servarion. The inreg ument and the muscles arrached ro the bones are
usually removed by hand after a period in a bath o f boiling water,
diluted bleach, or potash or rrypsi n. We recomme nd rhe simples t
m ethod, thar employs a barh of boiling water. The durarion of the
immersion musr be controlled in order ro prevenr overhea ting of the
skeletal e le ments, and may range from a few minutes for sm a ller
pieces to several hours for the la rg esr pieces . Progress should be
checked ar reg ular intervals.
Conservation is very s imple and is ge nerally performed dry. For all
bones, srorage in paper envelopes can be recommend ed because mosr
bones are simulraneously flexible and resistant, while the paper of the
envelopes allows some "ventilarion" of the ri ssues, but they can also
add some very severe contamination for larer mi crochemistry analysis.
Storage in plastic packing often leads to the appearance of fungi which

may subsequently destroy the bone. For dry conservation, care should
be taken to ensure that the tissues are not attac ked by specialized
insects : insec ticides or naphthalene may be added to the envelopes.
For the most fragile samples alcohol can be used as a fixative, in order
to avoid acid fixers (Meunier, 1988). Ethanol between 70 and 95% is
recommend ed for this purpose.
b
Figure VIII.B. 7 - Fin ray removal from large fish: white tuna Thunnus alalunga (Scombridae). al Dorsal view of first fin ray separa-
tion with knife. bl A transverse section of the skin is made before and after the fin ray. The fin ray is extracted ensuring of the
integrity of the bone (photos@ Ifremer O. Dugornayl.
b
Figure VIII.B .B - Vertebrae removal for large fishes: extraction of caudal vertebrae in the white tuna Thunnus a/a/unga
(Scombridael. a) Sectioning with knife and removal of skin and muscles above the last vertebrae. bl A section is made between
two vertebrae and the vertebral bodies (arrows) are extracted after sectioning the vertebral rays (photos© Ifremer O. Dugornay).
329
330
c. Preparation of calci'fied structures

w.] McCurdy,] Panflli, E] Meunier, A.] Geffen, H. de Ponrual
1. Scales
Scales do not normally need (0 be prepared for observation of thei r

growth marks. However, as they are hydrophilic strucrures, dry con-
servation will evenrually result in tissue dehydration and deformation.
It is therefore preferable (0 prepare them on a suppon in order (0 pre-
serve their original shape.
1.1. Direct observation

Unmounred scales can be observed direcdy under a low-power
binocular microscope or compound microscope: they are observed
either dry or after immersion in a rehydration and clean-up bath (e.g.
water, alcohol 70% (095 % , glycerol with water) (chap. VIILD.l).
1.2. Slide mounting

The simplest mounring method consists of locking the scales between
twO microscope slides (fig. VIII.Cl). The scales are first rehydrated
(in water or dilured alcohol) and then placed direcrly on a referenced
microscope slide (fig . VIII.Cla). A second slide is placed on (OP of the
scales and fixed (0 the first slide with adhesive tape (fig. VIII.Clb). Ir
is essenrial (0 thoroughly check the stfllcrural inregrity of the slide
unit and reinforce it with additional tape if necessary. The preparation
is therefore both immediately usable and s(Orable. If the scales are
large, it is possible that subsequenr dehydration will deform them and
a b
Figure VIII.C.I - Scale mounting between two slides. a) The scales (red arrow) are positioned on the referred slide (yellow arrow).
It IS preferable that the scales are wet in order to be able to deform (to compress) them more easily. b) A second slide (yellow
arrow) is deposited on the preceding one and comes to compress the scales. The two slides (yellow arrows) are fixed together
with adhesive paper (photos© Ifremer O. Dugornay).
331
exen press ure on rhe slides, causi ng rh em ro move apan slig hrly. Wi rh
rime rhe scales will rend ro slip from rh eir supporr. Ir is rherefore occa-
sionally necessary ro ensure rhar rhe uncaped edges of rh e slides are
well sealed by using eirher adhesive rape or fl ex ible gl ue (e.g. sil icon
masri c).
1.3. Cellulose acetate impression

An alrernarive m erhod of preparing scales is ro make ce llulose ace rare
impression of rhe exrernal face (fi g. VIII. C.2). Ce llulose ace rare is a
relarively flex ible plastic rhat allows hard elements to be imprinced by
applying mild press ure. The scales are placed between twO slides of
ce llul ose and the unir is passed through a jewe ll er y p ress (fig .
VlII.C. 2a). The unir is rhen wirhdrawn and rhe scales removed (fig.
VIll. C. 2b). The acetate slide thar was direcrly in concact wirh rh e
ex terna l face of rhe sca le presenring the cirmli, rerains the imprinc of
these ciYClIli (fig. VIII. C.2 b). Th e ace tare slide is th en direc rly observ-
able under a low-power binocular or compound mic rosco pe and can be
preserved in th ar state for m any years.
a -=-___....I--'-"'---"' b
Figure VIII.C.2 . Scale impressi3n on acetate peel. a) The scales (red arrow) are positioned between 2 acetate peels (yellow
arrows). The whole is pa ssed between the two rollers of a press (e.g. jeweler press). b) The external face of the scale (red ar row)
is separated of acetate (yellow arrow) and its print rem ain s visible (yellow arrow) (photos© Ifremer O. Dugornay).
332
2. Other structures
2.1. Simple preparations

Some CS, e.g. bone, can be observed
nal conserved state and others
e.g. cleithmm and otoliths. None of these
the CS to be embedded to observation. These calcified
structures are typically observed either
observation medium (chap. VIILD.l). a
cation device with additional illumination or a binocular
2.1 L Whole calcified structures

For some from flatfish and cereai n
it is to count the (mm"i wirhout extensive
Individual otolith:; are immersed in warer, alcohol or oil
and viewed ei ther transmitted or reflected (chap.
VIILD.1.1.2 and VIII.D.l.I A range of oils are llsed co or
reduce the reflected form the observed surface of CS. Historically,
(hese have included clove oil and immersion oils. Some oils sllch are
Creosote are no use as are considered to be
Oils with known hazards must not be used for this purpose.
"Baby oil" is often used as a oil otolith readers. A cold light
source should be used to reduce the of the oil
observation.
2.1.2. otoliths
Otoliths may be broken in twO che a or
cutters. The broken owJiths may be mouoced in Plas-
ticine" M clay) and oil baby
for observation under incident
lared within a clear
observation cell.
2.1.3. Simple for other structures

Some bones are observed without fJ,,,,,:<c<v<,,e.g.
can be viewed or after immer-
eicher transmitted or reflected
without prYlhp"iri
from fish or verrebrae. The secrion or the slice is then
observed, dry or wet, under a binocular
333
2.2. Embedding and impregnation

2.2.1. Embedding media
All embeddin g m edia should be scored in a cool d ark place and used
in accord a nce wirh rh e m a nufa crur e rs' i nsrrucri o n s. Hig h-r rans-
pa rency polyes rer res ins have a shorr shelf life and sho uld be purchased
in sm all qu anriries as rhey can become opaque as rhey d ereriorare. The
principal em bedding m edia and rheir applicari ons are li sred in rable
VIII. C. 1, cepci nred fro m M osegaard et al. (1 998). The mosr u sed
embedding m edi a for rhe nexr srages of preparari on are rh e hard an d
irreversible synrheri c res in s (pol yes rer, ep ox y, erc.). These urilise a
caralysr ro polymer ise rhem (fi g . VlII.C.3): d epe nding on rh e med ium
se iecred, mi xing is by weig hr oc by vo lume. For exam p le, cara lysr is
used ar 1-2 % b y weig hr of poiyes rer res in (e.g. 2S g of cl ear cas ring
polyes rcr resin and 0. 2 g of caralysr).
Table VIII.C.1 - Standard mounting materials (from Mosegaard et Cl/. , 1998).

Material Uses Advanrages Disadva nrages
P o lyester r es in - hi g h-qu ality - mulri-purpose - nO Il-rev ersible
(PR) perma ne nt fi xing - mode rare op ti ca l cla ri ty - var iabl e serring qua lity
- embeddin g - g ood grinding/ - slow sening ( 12 -2 4 h)
an d surface g rinding polisbing prope rri es - rox ic
- lig ht stable - low sbrinkage
- ca ta IYSt req u i red
Epoxy res in - high-qu a lity - ha tder th an PR - non-revers ible
pe rmanent fix ing - hi g h opri ca l cl ari t y - moderate/s low se tt ing
- e m beddin g - li g ht stabl e - rox ic
and surface g rinding - low che mi ca l conramin ari o n - expensive
- mini ma l shrinkage - ca ra lys t required
- wid e rang e of appli cat ions
Tbermoplastics - high-q ua lit y - fast se rring ( m inures ) - heat needed (70 -1 60 °C)
reversib le fi xi ng - eas il y re moved - devel ops gas bu bbl es
- embed d ing - h ig h opti ca l cl ari ty - modera te s hr ink age
and surface g rinding - non -roxic - hig h Si come nr
- good g rindi ng/
po li shing properries
Supe r G lue - rap id se tting , - easy ro use - hi g h S, Pb co nre nr
(cyanoace rare) high srreng th fixing - sets wirhout hear - ca n de tach from slide
• surface g rindin g or ch em ical hardeners
uv d e nral g lues - rapid se tting . - se rs with ou t hea t - very expe ns ive
big h strengt h fi xing or c hemical harden ers - opt ical cla rity nor know n
- surface g rinding - no n-roxic
Wax - q ui ck tempora ry fix ing - fa st , - soft
- em bedding o nl y - cbeap, - poor op tica l properties
- easil y re moved - non- perman enr
Nail va rni s h - quick tem porary fi xing - cheap, eas ily rem oved - soft
- surface g rinding - d issolves in oils
Eukitt ® - good qualit y - easy ro use - slow se rring
soft fix ing meciium - no ha rden er - remain s sofr
- e mbed ding on ly - g ood op tical cla ri ty - un sta bl e ove r rime
- eas il y re moved - ca n de rach from slide
334
a _ _ _ _ ___ b
Figure VIII.C .3 - Preparation of the embedding resin under a fume extraction hood . a) Example of mixture according to the weight
(e.g. epoxy resin). The catalyst (green arrow) is added to the resin (red arrow) in a polypropylene becher directly posed on a bal-
ance. The mixture have to be stirred thereafter. b) Example of direct mixture in a number of drops (e.g. polyester resin). The
drops of catalyst (green arrow) are added to the resin (red arrow) in a polypropylene becher according to the resin volume. The
mixture have to be stirred thereafter (photos© lfremer O. Dugornay).
Table VIII.C.2 provides a simple calcularion of rhe quanriries of resin

and caralysr ro be mixed , depending on rheir rario. Polyesrer resins
are dissolved in sryrene and require rhe addirion of borh an acce lera-
ror and a caralysr to cure. As there is a risk of explosion if rhe undi-
lured acceleraror and caralysr are mixed rogerher, polyester resins are
supplied pre-accelerared for safery reasons . Even so, an excessive
amounr of catalysr added ro rhe pre-accelerared polyester resin still
leaves a risk of explosion.
Table VIII.C.2 - Simple dosage of polyester resin (in volume and/or weight)
and catalyst (in drops) depending on their ratio. The greater the quantity of
catalyst the higher the speed of setting. Users should not exceed a maximum
of 2% catalyst to avoid the risk of explosion.
Resin Resin Number of drops of catalyst
volume (ml) weight (g) 1% mixture 2% mjxture
910 - 100 20 40
45.5 - 50 10 20
233 - 25 5 10
lI S - 12.5 2.5 5
91 - 10 2 4
The media and its catalyst are mixed carefully in order ro prevenr bub-
bles forming in the resin mix, as these would make ir difficult to pre-
pare and /or observe the cs. The mixture is rhen lefr ro rest for few
minures to allow the biggesr bubbles to escape. The hardening time
of the media varies from a few seconds (Super Glue®, UV denral glue)
335
co a few hours (epoxy, polyester). We recommend the use of a dry oven

to complete the polymerisation process after the resin has set [0 the gel
stage. For example, the polymerisation of polyester is better after a
minimum of 24 h in a dry oven (around 30°C).
The resin can be coloured by the addition of a pigmenr, generally
black, or it can be purchased pre-coloured. All the resins and catalysts,
often known as "hardeners", are available from specialiSt suppliers.
Mounring kits conraining small quanrities of pre-coloured resin and
catalyst are also available from some suppliers.
2.2.2. Embedding individual structures for sectioning

Individual CS are embedded before secrioning, grinding and/or polish-
ing. They are then prepared for further processing by embedding in
rransparenr media. The cs are cleaned and dried at room temperarure
or in an oven before embedding.
The use of individual moulds is necessary (fig. VIII.C4). Moulds are
usually made of silicon elasromer, and are dimensioned according ro
the size of the cs. They are often individually labelled (with a reference
number or symbol impressed inro the base of each cell in the mould).
Silicon elasromer is a flexible material that facilitates the removal of
the castS when the resin has set.
First, a base layer of the medium (e.g. polyester resin) is pi petted inro
the cells of the mould and allowed [0 set. After sufficienr time for
hardening (e.g. 4 ro 24 h for polyester), a CS is placed in each cell (fig.
VIII.C4a) and a covering layer of embedding medium is poured over
it (fig. VlII.C4b and 4c), making sure that it completely covers the
CS. At this step, it is importanr ro rurn over the cs in its mould ro
eliminate air bubbles that are often trapped, and also [0 re-position it
in case it has floated our of position. Re-positioning should take inro
accounr the next steps of preparation (e.g. the secrioning plane). After
approximately 20 minures (the time will vary with the resin medium
used and the age of the resin), the catalysed resin will begin [0 set,
after which it will be impossible [0 remove the cs. The [Otal time of
setting is from a few minures [0 a few hours, e.g. a minimum of 24 h
for polyester (chap. VIII.C2.2.1). The embedded strucrures are then
ready for secrioning and /or polishing in preparation for further analy-
sis (fig. VIIl.C4d).
2.2 .3. Embedding for incident-light observations

Calcified structures, mainly oroliths, are embedded in a clear medium,
usually high-transparency polyester resin, in cylindrical cavities on
black plastic slides. The black background of the slide provides better
conrrast for observations of annuli. This technique, evolved from
336
~~ ________________________ ~ b
c d
Figure VIII.C.4 - Example of embedding: otolith embedding in an elastomer mould. a) A layer of resin (yellow arrow) was run on
the bottom of each location: after polymerisation, it is the base of the support of the otolith (red arrow). Each otolith is deposit-
ed on the resin bottom . b) The otolith is well oriented and it is embedded in one second layer of resin which has been just pre-
pared (yellow arrow). c) It should be made sure that the liquid resin (yellow arrows) comes to fill the mould completely. Here the
resin did not reach yet the right edge. The otolith must be then turned over to drive out the captive air bubble s. d) Otolith (red
arrow) definitively embedded in the polymerized resin (yellow arrow!. The block of resin carries an identification number (20)
molded on the lower face (photos© Ifremer O. Dugornay).
methods first described by Parrish & Sharman (1959), Rain (1961)

and Watson (1965), and is used for incid ent light observarions of rh e
exrernal faces of large numbers of sagittae from pelagic species , us ually
Clupeid and Scombridae. le is also suitable for incident light observa-
tions on the surfaces of small pieces of CS and provides an efficient
long-rerm mer hod of conservation for repearecl observarions at a later
dare.
Grids of rwenty-five flar-bortOmed caviries, each 7 mm in diamerer
ancl approximately 1.2 mm deep, are machined into 65 mm x 60 mm
slides cur from 3 mm-rhick Perspex™ sheer. The grid of caviries is
offser to leave a la mm wide un-drilled strip at rhe tOp of each slide.
337
This allows each slide to be with a reference number. Slide

reference numbers can be with a hardened sreel stylus or an
electric tool fiered with a tungsten carbide tip. Reference
numbers may be made more legible yellow or white wax
crayon inro the text.
Each of otolirhs must be within ies cavity on
the slide, downwards and
small otoliths Sprat
a static electrical
of the otoliths more difficult. Static
the
of moliths in the cavity, that the

covered VIIIC.5b). At this stage, it is
any otoliths that have floated Out of
the spaces between
the layer of resin that completely
covers the all otoliths on the slide. A custom-sized mm mm
x 0.5 mm thick) mIcroscope cover is then floated over the
resin (fig. and left in the fume cabinet until set
(fig. VIII.C.Sd). Thin cover will deform above the cavities and
may crack as the thicker of resin contracts more on
The resin will set firm within a few but observations must not
sec and there no risk of styrene
and a num-
reslI1 are
now J!1 use. numbers are
and are now available commercially. Where machined
slides are used, the hard and inert and the
drill bit must be to ensure that the base of
each in the slide is smooth and level. If the base of the pits
and uneven, or if the resin does nor adhere co the
thin of partial vacuum may form between the base and the
hardened resin when the resin sets and cools. It will then be very
ficulr to observe the cs zones as the interfaces of these will
have the silvered effeer of a mirror.
2.2.4. Multiple embedding of otoliths for

Orolitbs must be embedded in a support medium
thin
fcom solid
aluminium blocks, constructed from flexible or assem-
bled from machined aluminium parts, are used for this purpose
338
L .":JI/~I
~ . t'o(i.I>~~r ·
• \ , Ij IJ" ) ,Lf~ ~l' l." ":/ :~l ~)
...
< "",.
"7>11 ., -4
...'\
~ , ~
" '. ,.
-,
•• ~,
...
,
.~
.' ,
""
,
'.
• •"
~
••
,
.. ~b-""
- ....
-.
(",
~
. • •,- .. ..
~~'-1fL
a b
J(' , 'l ", .~ ,- . > , rf!

I' "
,.
,~
- I! ~-;
" ". v ..
,
•• ~ .~
.- 4...
.. .. ••
.. ., •• ~
f_
. • I · ... <
c
~~ ..-1 \..10 (/
J) d
Figure VIII.C.5 . Embedding whole oto liths on a black plastic slide. a) Placing otoliths in the pre pared slide. b) Filling the cavities
with catalysed resin. cl Placing the cover slip on the slide. dl The comp leted slide (photos R. Rosell).
(fig. VULC.6a). The latter moulds are normally used where larg e
numbers of otolith s have to be processed, for example from dem ersal
species, where age information is required for stock assessments. These
moulds ca n be partially disassembled in order to faci litate the easy
removal of the resin blocks contai ning tbe embedded otoljths. One
variant of this process now uses an ejectio n lever that permits the rapid
removal of the res in blocks without the need to dismantle the m ould s
(Va n Beek et at. , 1997). Aluminium moulds typically use guidelines
339
Cut inro the sides of the moulds ro line up th e orolith cem res for Cut-
tin g or sectioning at predetermined loca ti o ns (fig . VIlLC.6a).
Monofi lamem nylon located in the g rooves of the mould (Bed ford,
198 3) or X/Y embedding tab les fitted wi th a modified video ca mera
(fig. VIlI.C.6b) and monitor (Va n Beek et al., 1997) are used to
minimise the effeers of parallax when otoliths are being positioned in
the mould s. In the latter case an indi caror line across the cem re of the
moniror gives the operaror a more com fortable working posi ti on when
placing the oroli th s in the mould.
The bas ic principle is the same for all a luminium moulds. The mould
is sprayed or painted with a mo uld release age nt before the first laye r
of catal ysed black polyes ter resin is pouted. The oroliths are placed on
t OP of th e first layer of res in wh e n this has se t ro th e ge l stage
(fi g. VIII.C.6c). A covering laye r of resin is poured over the oroliths
and allowed ro harden; (the moulds ca n be placed on a level tab le to
Figure VIII,C .6 . Otoliths embedding in series in aluminium

mould. al View of aluminium moulds. b) System of alignment
control in mould with a video (photos Bennett Ltd')' c) Line s of
otoliths (red arrow) are laid out on a bottom of polyester resin
beforehand colored in black and polymerized. The second
layer of black polyester re sin (yellow arrow) is run in the mould
(photo© Ifremer O. Dugornay). c
340
ensure that the layers of resin are of uniform The resin

blocks the embedded otoliths are removed from the
of resin has sec. It is very
imponanr to number and the fish num-
ber in the sample for the otoliths in each part of the mould.
This information will later be used to label the finished resin blocks
containing the embedded oroliths.
the first resi n can be allowed co set
before the ocoJiths are fixed in position with a small amount ofu""Hv;)ICU
resin that allowed to set before adding the of resin.
The former method is bur care must be caken to ensure chat
is added to resin. If too much cara-
the resin will not remain at the" stage
the ocolichs to be fixed co the surface. This method
turned ctctt.rtimJ upwards) before positioning

on the resin in order to prevent air bubbles from at
the concave surface of the otolith.
Markers of dry ro indicate the start of
each row of O(oli ths, are at the left hand side of each mould
on top of the first of resin when this has set to the
stage. One type of mould (Van Beek et al., 1997) has a bevelled
at the left side of each mould that this function.
the type of resin that is used
(Bedford, 1983) In the
process, a mixture of
resin is used for bmh the first and the resin , while a mix-
resin and "flex" resin forms the base for the mp resin
the type of resin that is Llsed m form the
omer layer on GRP boat hulls, for (he tOp ensures a
smooth cop surface on (he resin blocks. Powdered mle or a
similar can off che surface if" lay-up" resin is
used for 1985). The addition of small
amounts of "flex" resin
blocks are slighdy flexible can be bent slighdy), and there is
less risk of the oroliths shattered by the action of the high-speed
saw. If small moliths are to be embedded, chalk can also be
added co the resin mixture as this prevenrs the diamond blades
ming-up" with resin and the cutting
341
Table VIII.C.3 - Polyester resin mixing tables used for the Otolin process.
Weight of material (g)
Boctom laye r
Toral layer we ighr (g) 5 10 20 30 40 50 60 70 80 90 100
Lay-u p tesin 45 9 18 27 36 45 54 6') 72 81 90
Flex resin (5 %) 0.25 0.5 1.0 1.5 2.0 2.5 30 35 4. 0 45 50
Black res in pig ment (5%) 0.25 05 1.0 l.) 2.0 2.5 3.0 35 4.0 45 50
Hardene r (MEKP) (l %) 00) 0.1 0.2 03 0.4 0.5 0.6 0.7 0.8 0.9 1.0
Top layer
Total layer weighr (g) 5 10 20 30 40 50 60 70 80 90 100
Cas ring resin 45 9 18 27 36 45 54 6'.) 72 81 90
Flex resin (5 %) 0.25 05 1.0 1.) 20 2. 5 ')0 35 4.0 45 5.0
Bl ack resin pigment (5%) 0.25 05 10 l) 2.0 2.5 30 ).) 4.0 45 5.0
Hard ener (MEKP) (0.6%) 003 0.06 0.12 0 .18 0.24 0 30 0. 36 0.42 0.48 0.54 0.60
2.2.5. Impregnation of bony tissue

Many bones consi sr of vascularised bony ri ss ue, and when rhe osseous
rrabecules are rel arively rhin and surrounded by la rge caviries rhe
whole organ is brirrle. For rhis reaso n, ir is necessary ro sr rengrh en rhe
bony srrucrures ro avoid breakag e durin g sec rioning . Embedding
should rherefore follow careful impreg narion wirh polyesrer, in order ro
ensure rhar rhe polyesrer enrers all rhe bone caviries, where ir hardens
during po lymerisari on like rhe polyesrer rhar surrounds th e cs. We
now describe the polyester resin (srratyl) impregnation process.
The cs, i. e. a bone, is d ehydrared in successive alcohol baths of 70 % ,
95 % and 100% , using at leasr one bath of24 h for each co ncentrarion ,
and poss ibly two barhs of each for large pieces. It is then immersed in
twO successive bat hs of acerone for 24 ro 48 h a nd fin a lly in rhe
monom er polyester (wirhoU[ caralyst) for 24 h. Depending on rhe
polyester used, suppl em e nrary barhs may be required betwee n the
m onomer step and rh e e mbedding srep. For rh e embedding stage, the
cs is immersed in rhe prepolymeri sed resin (monomer + catalyst 1-
2 %) in a mould which already conra i ns a hardened base laye r. I t is
somerimes recomme nded ro place th e m ould wirh the resin and the CS
for a few minures under vacuum in order ro remove bubbles and
improve impregnarion. Whe n rhe polym e rised block has hardened
(for example after 1-2 days at room temperature and th en one week at
600C, for srraryl) it ca n be sectioned with a low-speed sectioning
machine (chap. VIII.C.2. 3 1)
342
2.3. Sectioning
structures is step for a number of further prepa-
ration techniques such as and of thin
bur it is also necessary for
For where the are opaque or are too thick
for all the seasonal increments to be clearly observed on the upper SUf-
observations on the thin sections the core in the trans-
verse more reliable observation. There is no pre-
CS, and tbe and materials used
in simple and/or thin sections are very on the
natUre of the application et aI., 1998).
2.3.1. <;At'i"f'lmno
'-'","""PrY> when CS is to ensure that the section

centre of CS, ere.) (Chap. IILC). Seccion-
or, more often,
One of the most used saws is
equipped with a diamond disk.
All saws of this type L1se liquid media as the fluid: we recom-
mend water for non-hydrophilic structures such as otoliths and
diluted alcohol (70 to 95() for hydrophi lic tissues such bones.
Because of the of alcohol used must be
checked regularly. The of a CS
take from one ro five minutes.
The CS is usually embedded sectioned:
it is easier to it and is essential in the case of small CS like
certain otoliths. The section using the saw micrometer,
must first be located on the
into account: a diamond disc is about 300 pm tbick. With the

is tben sectioned simply
VIILC.7b, c). Some
tbem
saw
a the desited
thickness from 150 to
500 pm, rprnplnt~ for increments in
the CS section.
C a..-L.-_ ___
Figure VIII.C.7 - Section of the hard parts: example of section of an otolith embedded in resin and of a whole dorsal spine for
obtaining a slice. a) Location of the levels of cut (red arrows) for obtaining the otolith slice including the nucleus. b) Cutting with
a rotative saw With slow speed (lsomet®, Buehler®). The resin block with the otolith (red arrow) is cut by a diamond disc (yellow
arrow) in rotation (large blue arrow). The disk is humidified permanently. c) Detail of the preceding figure. The diamond disc (yel-
low arrow) turns while the resin block is supported above. The level of cut is indicated by the red arrows. d) Section of a slice
in a whole dorsal spine. The water flow is visible at the interface spine (red arrow) - diamonds disk (yellow arrow) (photos© Ifremer
O. Dugornay).
2.3.2. Multiple sectioning (fig. VIII.C.8)

Although low-speed saws have been used ro cut multiple thin secrions
from oroliths embedded in polyester resin blocks (McCurdy, 1985),
high-speed saws are more efficient when there is a need ro process
latge numbers of oroliths. High-speed saws are usually adapted from
ptecJSJon milling machines (Bedford, 1983) or preClsJOn
cutting/grinding machines (Van Beek et al., 1997). Purpose-built
high-speed orolith saws are also available. The choice of high-speed
saw wiJJ depend both on the quanti ty of oroliths to be processed and
the available budget for equipment purchase. The need for further
processing after the orojitils have been cur or thin-secrioned may also
have ro be considered.
344
Mul(iple rransverse rhin secrions are rourinely prepared from rhe sagit-
tae of demersal species, in laborarories where (he resulrs of fish age
esrima(ions are used for srock assessmenr purposes. European fish-age
esrimarion laborarories rend ro favour variarions of rhe rechnique
described by Williams & Bedford (1974), rhar produce (hin scrips of
cured polyesrer resin conraining cransverse secrions of orolirhs
(fig. VIIl.C.8a-c). Elsewhere rransverse rhin sec(ions are rourinely pre-
pared from individual sagittae for srock assessmenr purposes using
variarions of rhe merhod described by Nichy (1977). The Orolin sys-
rem developed by Van Beek et af. (1997) was rhe firsr srage in rhe
developmenr of a procorype orolirh produ(rion line for rourine age
reading of demersal species.
A number of variarions of Bedford's merhod have evolved
(rab. Vlll.C.4). Some of rhese are nor rhoughr co affecr rhe readabiliry
of rhe ocolirh growrh zones; e.g. rhe number of (lining blades and rhe
cur(ing speed (blade r.p.m.). However variarions in ocolirh secrion
a b
c
Figure vUl.e.s . Section of otolith series with high speed diamond saw (yeUow arrow). a) The first section crosses the otoliths.
The section levels had been located as a preliminary on the black resin. b) The second section carries out the slice of the otoliths
(red arrow). The water flow (blue arrow) arrives directly on the diamond disk (yellow arrow). c) Result of the slice of otoliths series
(photos© Ifremer O. Dugornay). (d) View of the high speed diamond saw of the Otolin system (photo Bennett Ud).
345
rhickn ess will affecr rhe readabiliry of rh e orolirh secrions. All circu-
lar curring blades have a preferred minimum secrion rhickness and
rhis is relared ro rh e amouoc of mareriallosr on eirher s ide of rhe blade
every rime a cur is made, rh e kerf loss. Facrors affecring kerf loss for a
parricular blade are rh e rhickness of rhe blade irself and rhe grir size
of rh e diamond compound . Arremprs ro produce rhinner secrions may
resulr in rhe desrrucrion of rhe orolirh rhin secrion. Thi s is of parricu-
lar imporrance for saws firred wirh rwo blades, where rhinner blades
may be required ro produce secrions of rhe desired rhickness. Wirh all
rypes of saw, rhe secrion musr be rhick enough for rhe embed ding
m edia ro su pporr rhe orolirh secrion during rhe cu rring process bur
rhin enough ro provide rh e required d egree of rransparency for obser-
varion of rbe growrh rings.
Table VIII.C.4 - Thin-section preparations used for the age estimation of Mer/angus mer/angiu5 otoliths at
some European institutes (ICES, 1998l.
Secrions mounred on glass microscope slides
Research Secrion Colour of Unmounred Mounced wirh Fixed to slide
insriwre rhickness embedding secrions cover slip
resin
Uncoared Pwrec rive
coa(ing
RIVO-DLO 0.8 mm Black No No Yes No
Nerherlands
FRC 0.4-0.5 mm Black No No No Yes
1rel and
CHAS 0.6-0.7 mm Black No Yes No No
K
AESD 0. 3-0.4 mm Black No Yes No No
N.lreland
lfremer 0 . .3 mm Clear Yes No No 0
Fran ce
2.3.3. Cryotome sectioning (fig. VIII.C.9)

The use ofEhrlich's hemaroxylin ro stain bone secrions requires decal-
cified marerial (chap. VIII.C 2.6. 2) . Secr ioning is managed on frozen
sampl es wirh a cryorome, for example rbe Cryomar™ (a microrome
wirh borh rb e knife and rhe plarine refrigerared) manufacrured by
lei ca lrd. The decalcified bones are frozen eirher in dry ice or direcrly
on rh e sample-holder rhar is refrigerared wirh merhanol in rhe
Cryomar™ The froz en sample is rhen cur wirh rhe knife of rh e micro-
rome (fig . VIII.C9a) and rhe secr ions are caughr in a perri-dish filled
wirh disrillecl warer (fig. VIII.C9b). The sections are about 15-20 ~m
346
thickness, depending on the material. The sections can be preserved in

70% alcohol before staining (chap. VIII.C2.8.2).
a b
Figure VIII.C.9 - Spine seclton with a cryotome. a) A decalcified spine (red arrow) is embedded inside ice (white arrow). The blade
of the cryotome (yellow arrow) is moving from the back (pink arrow) and cut very thin slice of spine embedded in the ice (red
arrow on the blade). b) The thin sections (red arrow), still embedded in the ice (white arrow), are deposited at the surface of a
water bath (blue arrow). After defrosting, the sections float on the surface (red arrows) and can be recovered (photos© Ifremer
O. Dugornayi.
2.4. Mounting
2.4.1. Section mounting for subsequent stages of preparation (fig. VIII.C.IO)
When transverse thin slices of otoli ths (or other CS) are to be prepared,
the resulcing thick section including the core is attached to a glass
slide with thermoplastic glue (Crystal Bond™) before being gtound
and polished (fig. VIII.C10a, b). The block can subsequently be
turned over, fixed again to a slide with the polished face down, and
gtound and polished on the other side (fig. VIII.ClOc, d).
2.4.2. Mounting otoliths from small fish (fig. VIII.C.lla)

Small otoliths are often directly mounted in embedding media (chap.
VIII.C2.2.1). After extraction the otolith is sucked into a drop of the
same medium, and put directly in the medium on a cover slip. One
can use polyester resin which permits the otolith to be manipulated
for a while before it sets. The mounted otolith can be observed directly
within a few hours of setting, and conserved as it is for a long period.
The most important precaurion is to check the thickness of the
medium above the otolith, as the distance of the focal plane may be
very shorr under high magnification. It is often necessary to remove a
certain amount of embedding media before it sets: any excess resin can
be trimmed from the cover slips with a scalpel; otherwise it will be
necessary ro grind and/or polish it before observation.
347
Manual of fish sclerochronollJgy
a b
c d
Figure VIII.C.J 0 - Preparation mcunting: example of aHixing otolith section with thermoplastic glue and turning over on hotplate.
a) Affixing of the otolith slice on the thermoplastic glue (e.g. Crystal Bond™). The block of resin containing the slice (red arrow)
is brought (grey arrow) on the thermoplastic glue at a viscous state (yellow arrow). b) the slice is supported (grey arrow) on the
glue (yellow arrow). The pressure drives out the air bubble s 01 the glue captive under the preparation. In lact the slice (red arrow)
is paste on the glue (yellow arrows) on a slide itself paste on a microscopic slide (green arrows). c) View of the finished prepa-
ration outside of the hotplate . d) Tu rn over 01 the resin block. When the thermoplastic glue is sufficiently liquid at high tempera-
ture (yellow arrow) the slice is taken off, turn ed over (grey arrow), and redeposited on the glue on the slide (green ar row). The
finished preparation is withdrawn from the hotplate (photos© lIre mer O. Dugornay).
2.4.3. Slide mounting of multiple otolith sections

The black resin srri ps conraining rhe rransverse rhin sec rions rhroug h
rhe orolirh growrh cenrres (fig. VIIJ.C. 8c) are fixed ro cusrom-sized
g lass microscope s lides (76 mm x 50 mm) , covered wirh caralysed
clear casring resin and prorecred by glass cover slips. Each microscope
slide is eng raved wirh rhe idenrifi carion numbers of rhe orolirh sec-
rions co be mounred on ir.
A disposable plasric piperre is used co app ly narrow srrips of caralysed
clear casring res in ro rhe microscope slide and rh is is used co g lue rh e
s lices of rhin secrions co rhe slide. The resin is rh en allowed ro sec.
Caralysed clear casring resin is dropped onro rhe g lass slide in rhe
spaces berween rh e black resin scrips and on cop of rh e srrips, ro form
a complere laye r of resin on cop of rhe slide. Gende srirrin g/ mi xing is
348
essencial co prevenc bubbles from forming in che resin as rhese would

make it difficulc to observe che ann,,!i on che otolichs . After about 20
minuces che resin begins co set and a glass cover slip is gencly placed
over ic. The slide is carefully placed inside a fume cupboard or under
a fume extraction hood unci! che resin has complecely sec. Excess resin
can be trimmed from the microscope slides with a scalpel once the
resin has sec co che gel scage, and any remaining resin smears can be
removed by using a drop of ace cone and a piece of cissue paper.
Very chin sections of stained bone (e.g. spinal sections) are mounted
on miccoscope slides in a hydrophilic medium (e.g. Permounc®) (fig.
VlII.C.llb).
Figure VlIl.e.]] - Example of piece mounting. a) Embedding of

larvae otoliths in resin. The larva otoliths (red arrow) are
deposited using a dissecting needle (green arrow) in a resin
polyester drop (yellow arrows) deposited on a cover slip. The
otolith itself is transported in a drop of resin at the end of the
dissecting needle. The right picture is a magnification of the
process. Scale bar = ] mm. b) Embedding of a stained slice
of spine in resin. The spine slice (red arrow I is deposited in an
hydrophilic resin (yellow arrows) using a dissecting needle.
(photos© Ifremer O. Dugornay). b
2.5. Grinding and polishing (fig. VIII.C.12)

There is noc a greac difference becween che meanings of "grinding"
and "polishing". The laccer term is probably besc reserved for the final
scages of the grinding/polishing procedure. Calcified structures can be
349
directly ground, or ground after embedding or sectioning. Like sec-

t ioning, CS grinding is employed in order: (1) ro improve readability,
(2) to distinguish marks that are not visible on the whole CS, (3) as
a precursor ro many other preparation techniques (e.g. burning,
staining, SEM, impressed acerate, microradiography). As for sectioning,
the grinding process requires a careful check of the CS plane (trans-
verse, sagirral or frontal).
Grinding and polishing procedures are often carried out by hand using
wet abrasive papers, with or without abrasive pastes. Grinding L1ses wet
sandpapers with grit grades between 120 and 1200. Polishing uses
polishing cloths with different grades of alumina pastes (from 3 flm ro
113 pm) or diamond powder. In manual preparation, grinding and
polishing movements must be random, in order ro avoid systematic
disrorrion of the plane of preparation (fig. VIII.C.12).
a b
Figure VIII.C. 12 . Grinding and polishing of preparations. a) Grinding. The preparation, an otolith slice mounted on microscopIc
slide (red arrows) is ground on a wet sand paper (yellow arrowsl. The hand movement is irregular (grey double·arrow) to avoid
systematic scratches. b) Polishing. The preparation, an otolith slice mounted on microscopic slide (red arrows) is polished on a
polishing cloth (yellow arrows) with and alumina slurry (white powder) and water. The hand movement is irregular (grey double·
arrow) to avoid systematic scratches (photos© Ifremer O. Dugornayl.
Grinding and polishing machines may be used both for orolith serial
preparation and for specific applications in which a perfect surface
state (perfect flatness and relief-free) must be obtained, as for thin sec-
tions prepared for microchemistry analysis, particularly with WDS,
EDS or PIXE. Some grinding machines allow the amount of material ro
be removed to be specified (i.e. the final thickness of the sections). The
grinding medium is silicon carbide (SiC) powder. It is used:
• to grind glass slides in order to standardise thickness and obtain a
surface roughness that will enhance subsequent resin adherence, when
the oroliths are mounted on the slides;
• ro grind preparations in order to remove the maximal thickness of
resin covering embedded otoliths.
350
Aftet slides or are

sonic baths (MilliQ water is required for
tions are [hen
several steps:
water
microchemistry
of rhe plane [Q be
.. polishing, which removes the scratches and

prevlOus stages. It is carried out in
with diamond or sprays in the aqueous
3!-lm 1 ~Im - 0.25 tlm sizes). The
steps IS
on
diamond
During are checked under [he
in reflecced light, as this is the only way to check surface
ete.). To facilitate this Plexi-
holders may be used, as these are also more advama-
geous in terms of contamination risk.
2.6. Acid etching, decalcification

2.6.1. Otolith acid
The action of the acid on the otolith or its section superficially
Several diluted acids have
acid at concentrations of between

acetic acid at 1% (Richter &
diamine tetra-acetic acid (EDTA) at 5%
EDTA seems preferable because its attack is more
the surface of the otolith less &
1985). Times of attack acid are very on
and otolith size: a few minutes are to obtain good prepara-
evaluation tests must be made for each par-
ticular case.
After the must be rinsed in abundant clear water
for several minutes. It must then be dried before further
SEM).
2.6.2. Decalcification of bony tissue

To prepare frozen sections for
bones must be decalcified.
agent is lIsually nitric acid at a concentration
of 5- 10%, depending on the volume of the sample. The duration of

the decalcification stage also vari es, d epe ndin g both on sa mple
volume and on the degree of minerali sation of the bone. In any case,
the decalcification process must be kept strictly under control because
after the bone has been completely cleared of its mineral content the
collagenous matrix may rap idly become damag ed and thus no lo nger
stainable.
D ecalcification is stopped by washing in tap- water. Samples can then
be preserved in 70% alcohol for severa l days before process ing. The
nex t step in sectioning is al ways preceded by careful washing in run-
ning tap-water fo r 12- 24 h in order to remove a ll the nitri c acid,
which would other wise disturb (be staining process.
2.7. Heating and burning (fig. VIII .C.13)

In some cases (e.g. flatfish) the quality of the observations may be fur-
ther improved by heating the otolith until its protein denatures and
turns brown in co lour, rende ring the winte r (translucent) g rowth
w nes more visible. Burning lasts for a few seconds (0 a few minutes ,
a b
Figure VIII.C.J 3· Otolith burning and cracking . al The otolith is

put direclly in a fla me with forceps. bl The otolith proteins burn
at the end of a few moments. The time of burning is variable
according to the specie s and of the size of the otolith. The
otolith must acquire a brown color. cl The otolith flaring left the
flame and it is broken by the medium on a rigid support with
a spike or a blade of scalpel (photos© "remer O. Dugornayl. c
352
depending On the and the otolith size. After burning the

otolith is broken with a scalpel blade or other hard tool (fig.
VIILC.13c). A of methods have been tested at the
bur only Gadus morhua
otoliths are heated by baking in a radiant heat oven at 275°C for three
to six minutes & Sheehan, 1997). In some cases, broken
otoliths or oroliths that have been cut in half with a diamond bladed
saw, are burned after in order to improve annuitls readability.
The stability of the burned surfaces can be maintained by
the orolith in polyethylene vials fiJled with dear
2.8. Staining (fig. VIII.C.14)

2.8.1. Staining otoliths VIII.C.l4a-b)
is a method of which reveals fine chromophilic
increments, sometimes to those obtained after burning.
Developed for the first time Albrechtsen (1 the technique
'-V"~""~, after polishing and
it in contaCt with a histological stain. The stain used
was methyl violet (l a stain for cellular
whole range of histological srains may be used.
& McDermott (l who tested several stains, 1 % aniline blue
called light blue) and toluidine blue the best
colouring and cartilage, and the cell kernel. It is
not really known what is the target of these in the otolith,
which contains neither nor cellular kernels. It may be that
they are attraCted by
nents of the otolith. Gauldie (1990) act
physically while settling in the discontinuities underlined by aci
etching, rather than with In sum
mary, we recommend the use of toluidine blue for a few minutes, aftt
acid as this is a universal dye for otoliths.
Bouain & Siau (1988) another method of staining after firSt
the otolith, immersion for 12-14 h in fuchsin acid, then pas-
sage for several minutes in starch black (5%): the increments
appear dark blue on a pink It seems that the physical
principle of coloration is not excluded, Another method of
treatment consists of a bath of acid and
staio before
often
the time of attack acid is very variable, depending
and otolith size: a few minutes are sufficient to obtain
but trials should be carried out in each case.
the must be gently rinsed with clear water
for few second to preserve the
353
2.8.2. Staining bony tissue (fig. VIII.C.l4c)

The specific sta in for b o nes is hema[Qxylin , which stains th e
cemeocing lines because of their richness in proteoglycans. This
technique is thus very valuable for deteccing the AGl.
Before staining, the decalcified bone seccions are rehydra ted if neces-
sary, picked up with a paint brush, drained off and dropped in a bath
of hemacoxylin. After ten co twenty minutes (the time required will
vary depending on the m ate[ial and the quality of the hemacoxylin),
the secc ions are collecced and washed in tap-water till the stain turns
bluish. The sec tions are then delicately picked up using a paim brush
and mounred in a hydrophilic resin (e.g. Aquamount ®) (chap.
vmc 2.4.3 and fig . VIII.Cll b).
Ehrlich's hemacoxylin (Game[ & Jolles, 1969) is prepared usi ng the
following quantities:
- hemacoxylin 4g
- ethanol 95% 200ml
- distilled water 200 ml
- glycerol 200 ml
- potassium alum 6g
- acetic acid 20 ml
a b
Figure VIII.C .14 . Preparation stai ning. a) b) Otolith staining. A

transverse etched section of an embedded otolith (red arrow)
is stained with the depositIOn of a staining solution (toluidine
blue). Thereafter the preparation must be rinsed for several
minutes with distilled water. c) Spine decalcified slices stain-
ing_ The spine slices, flo ating on distilled water, are covered
with a staining solution (Ehrlich hematoxyli n). Thereafter the
preparation must be rin sed for several minutes with distilled
water (photos© Ifremer O. Dugornay). c
354
Preparation and obsel'\lation techniQues
First dissolve the hemaroxylin in

leave the mixture to macure in the laboratory in light and air-
for at least two weeks. Ehrlicb's well for several
years.
the older the the bener is [he
of Ehrlich's hematoxylin. For this reason, it is advisable to
Ehrlich's hemawxylin available in a though it
should be flltered in order to avoid or at least
lust before use.
2.9, Some special considerations for microchemistry

Microchemistry is the of tbe
For the most pan, the are the same as
for observation of increments. HCl\,,,'vc'r there is always a risk of con-
and some of
smoothness. In order to reduce

siderations in the choice of material and
ration should be borne in mind.
As a all should be
measure at discrete on the sur-

The CS are embedded in resin and sectioned
The resin should be seleered bearing
[WO considerations in mind:
• of the resin. The resin used should basically inert so that

it does not introduce any contamination duriog infiltration. Most
epoxy resins do not contain detectable levels of the elements usually
found in owlitlls. and
resins may contain
resins should also be avoided because can conrain conmminanrs
which can affect the measurement of otolith composition,
• of the resin. When laser
(lA-ICP-MS), the energy of the laser can be very destructive. If the sam-
is held loosely in the resin the energy can cause it to vibrate,
and fly out of the resin, resins such as Spurr or
Araldire are most suitable fot le is par-
ticularly to ensure the purity of tbe resJns,
because do infiltrate oraliehs so
The reagents and and polishing media must be selected so as
to reduce tbe risk of contamination. This may the use of dif-
ferent compounds for and for observation studies.
are an obvious source of metal
355
contamination. The use of diamond sptays or suspensions in aqueous

phase is therefore recommended. Less obvious sources of contamina-
tion include polishing suspension media and the lubricants used for
saws (MilliQ water is recommended).
Some analytical techniques (EDS, WDS, PIXE) tequire a perfeer final
polished surface and flatness that are difficult to achieve by hand
polishing. Automatic grinding and polishing (see chap. VIII.C.2.S)
are recommended. Frequent checks on the progtess of the preparation
(with respect to the plane to be reached and the surface state) should
be made using a refleered light microscope.
Final cleaning with MilliQ water in an ultrasonic bath has often been
suggested in preparation protocols.
It is also important that prepatation stotage containets are ftee of con-
taminants. Cettain analytical methods are very sensitive to moisture,
so prepatations should be srored in a desiccating cabinet until analy-
sis. Various decontamination processes have been suggested prior to
sample analysis:
• pre-ablation when preparations are analysed with LA-ICP-MS (Cam-
pana et af., 1995);
• 1 Ss rubbing with a tissue soaked in ultra-pure HN0 3 (Milton et af.,
1997). This procedure is not tecommended when the analytical tech-
nique is sensitive to surface roughness.
Bulk analyses are microchemistry measurements that are made from
dissolved samples of CS. The sample is usually dissolved in an acid,
and the tesult is introduced intO the analytical equipment in the form
of a liquid.
Handling always carries an increased risk of contamination of samples,
and extraerion, cleaning and drying of bulk sample preparation should
therefote ideally be done in clean conditions, in a class 100 laminat
flow fume cupboatd.
Care must be taken when weighing samples, because the results of
bulk analyses are usually expressed telative to the amount of material
analysed, and any uncertainty introduced into the procedute may
affect the reliability of the results.
The purity of the acid used for digestion is extremely important for
these measurements, because of sample contamination and also
because impurities in the acid used fot standatd and procedural blanks
will result in highet limits of deteerion (LOOs) and thus poot sensi-
tivity. Sub-boiling redistilled nitric acid diluted with ultra-pure water
is tecommended. A number of methods are used to dissolve CS sam-
ples for bulk analyses. Most use nitric acid because it teadily attacks
CaC0 3 . The sample can be dissolved at room temperature, bur the
digestion is not always complete. In the case of otoliths, room tem-
perature digestion may leave a tesidual "ghost" composed of the
356
otOlith that resists acid in nitric acid

will completely involves extra
handling that may result in contamination. A compromise
to use a microwave oven to heat the in acid in order to achieve
complete The matrix is an important consideration
in microchemistry studies because there is very little information on
what elements are associated with the (as to the
mineralised components). While it might appear to be to
a digestion that would leave tbe
untouched, in fact it is probably better to ensure
of the in order to avoid any introduced
the protein.
et a/. (2000) the result of a decontamination
the results of five procedures.
Ba, Li and that
among treatments.
357
D. Observation
B. Morales-Nin,j. Panfili
There are abour as many cs observarion rechniques as rhere are prepa-

rarion rechniques, ranging from simple ro very complicared. The
researcher should select from rhem according ro rheir suirabiliry for
rhe objecrives of his srudy. As is rhe case for prepararion , rhe choice of
observarion tool depends on rhe rime required and available bur also
on rhe experimenral cosrs of rhe srudy. For example srandardising illu-
minarion condirions is very imporranr in image analysis (Mosegaard et
et!', 1998): rhe accurare descriprion of i1luminarion condirions is of rhe
urmosr imporrance when rhe oprical ptoperries of cs are being com-
pared. Incremenrs can appear co vary depending on rhe lighr source
used.
In the following sections, we summarise some of the most frequenrly
used observarion techniques available for sclerochronological srudies.
1. light microscopy
1.1. low-power magnification (binocular)

Large skeletal elements such as deithret of Esocids can be viewed and
measured withour magnificarion. Age can be assessed with the naked
eye, even under field conditions and even up to 30 years (Casselman,
1974 ). Neverrheless incremental strucrures are rarely large enough to
be observed accurarely by the naked eye, and ir is usual ro view them
under low-power magnification. Ar rhis scale only seasonal and annual
increments ate visualised. This observarion merhod is rhe cheapesr
and most rapid, permitting large numbers of samples to be observed.
The usual range of magnificarion is from Ix to abour 100x.
1.1.1. Whole structures

Whole-CS observation is mosr suitable for relatively rh in rranslucenr
strllcrures such as scales, opercular bones, cleithra and cerrain otolirhs.
For fish wirh thick CS rhat are too dense for rhe innermosr increments
to be observed, sections are used (chap. VIII.C.2.3).
1.1.2. Clearing media

The use of clearing media is recommended in any rype of observarion
wirh or wirhout prepararion. A clearing medium is a liquid or
hardening compound which enhances rhe visualisation of rhe incte-
ments. lr has ofren rhe same or nearly rhe same oprical densiry as thar
of rhe CS, allowing the lighr to penetrare more easily in order to reveal
358
the internal structures, Several liquid clearing media exisr: the most
used is water, saline solutions (more followed by
mixtures of water-alcohol (various
glycerine-alcohol (30:70), immersion oil. Essential oils (clove, rose-
mary, pine, ete.) also offer very results, Other
media such as creosote, methyl are now less used
because many of them are health hazards. Care should be taken to
ensure that the increment structures are not damaged and/or rendered
unreadable action of the clearing media. After observa-
tion in viscous and/or the CS must be cleaned
sue with a solution of ether-alcohol For
for the choice of
small andlor thin water or
mended while for thicker refringent oroliths media are most
suitable (e,g, rosemary Most media clear CS to some extend,
1.1 ,3, Mounting media

media refer to media that fix the cs, to a
slide (Secor et at., 1992), Some media are also llsed to con-
serve cs and enhance the visibility of the increments,
These also act through their optical
mostly
with some media due to their relative opacity and

colour, their hardness andlor their poor after storage; such
media include Canada balsam and epoxy. Per-
manent media that cannot be removed limit the of
the CS if the observation res this: for ins ranee
fish otoli ths on black sheering with
polyester resin. We recommend non-hydrophilic CS prepa-
rations with glue that melts at low temperarures
60°C) or with and hydrophilic cs
thin sections) with Permount®.
11 .4, Ilumination
Two means may be ro illuminate the surface viewed.
is either directed through the cs from below (transmitted
or on to rhe surface from above (reflected Reflected
light is usually used ro observe seasonal while for
microstructural daily) otolith examination we use transmitted
some trials uansmitted and reflected
light should be carried out on seasonal increments bones) before
on a rourine method, The aspect of the seasonal increments
on the rype of i1luminarion VIILD.l):
359
under rransmi[(ed light uanslucem incremems are bright and opaque

increments are dark, while under reflecred lighr rranslucenr incre-
menrs are dark and opaque ones are brighr. The facr rhar one and rhe
same increment can be described as "brighr" or "dark", depending
upon rhe form of illuminarion, can lead ro considerable confusion and
for rhis reason rhe rerms "opaque" and "rranslucent" should always be
used (Casselman, 1974; Williams & Bedford, 1974; Casseiman,
1983)
1.1.4.1. Transmitted light (fig. VIII.D.la and Ib)

When using rransmitted lighr a rransparem container should be used
if rhe cs is ro be observed immersed in a clearing medium. Two
sources of lighr are also available and should be resred: direcr
(fig. VIII.D.la) or diascopic (fig. VIII.D.l b) rransmission. When
observing rhick CS secrions, rhe besr view is obrained when rhe base
of rhe cs is illuminared from rhe side by fibre-opric lighr sources, and
rhe surface shaded. Surface shading causes lighr ro srrike rhe lower
parr of rhe secrion and scatter upward ro rhe surface, rhereby enhancing
rhe comrast berween rhe rranslucenr and opaque increments. The
brighrness and contrasr of rhe CS surface can be conrrolled by varying
rhe posirion of rhe illuminaring fibre-opric sources (Esrep et ai.,
1995 ).
1.1.4.2. Reflected light (fig. VIII.D.lc)

The qualiry of observarions is improved by using a comainer wirh a
black background. The lighr source musr come from eirher rhe side or
rhe rop of rhe surface of rhe CS prepararion under examinarion.
Reflecred lighr requires rhe use of a clearing medium ro avoid surface
lighr disrorrion. The orienrarion of rhe lighr source is very imporram
and ir may have ro be moved in order ro obrain the besr contrast
berween growrh incremenrs. Ir is preferable ro use a cold lighr source
for incidenr lighr observarions ro minimise evaporation of the liquid
observing medium: fibre-opric bundles are used ro direcr light from a
high-inrensiry lamp.
1.1.4.3. Profile projector

The profile projecror is mainly used for scale-reading. In the early
1920s the profile projecror was developed ro projecr an enlarged
image of a scale on ground glass. This was copied in many laboraro-
ries and was available commercially (Carlander, 1987). This appararus
allows the manual measuremenr of disrances on rhe ground glass.
Nowadays computerised image analysis sysrems are employed In
high-tech research laborarories.
360
a - -
c
Figure VIII.D.l - From left to right, whole otolith of P/euronectes platessa, spine section of Pangassius hypophthalmus, and scale
of Oicentrarchus labrax, observed In direct transmitted light (a), diascopic transmitted light (b) and reflected light against a dark
bottom (c). Scale bar = 200 ~m (photos J. Panlili).
361
1.2. High-power magnification (compound microscopy)

High-power magnificarion using compound microscopy is mainly
llsed for microsrrucrural examinarion of orolirhs, for reading primary
incremenrs , and somerimes for observarions of rhe inrernal srru crure
of or her CS (e.g. bone consriruenrs). As in binocular microscopy, various
sources of lighr are used: alrhough rransmirred li g hr is mosr ofren
used, refleered , polarised, phase co nrrasr, and fluorescenr illumina-
rions also exisr. Ar rhis level rhe observer needs ro make a choice afrer
co mparison of differenr lighr sources. Microsrrucrural examinarions
are besr made wirh a compound microscope wirh rhe following mini-
mum fearures (Campana , 1992): binocular eyepieces, ar leasr one of
which can be focused, objecrive lenses wirh nominal magnifi carions of
20x, 40x and 100x (plus orhers such as 10x and 60x), a movable
specimen srage (or berrer a mororised srage moved by m eans of a joy-
srick), a subsrage condenser lens, an aperrure diaphragm, and a vari-
a ble inrens iry illuminaror wirh irs own focu s ing /cond enser le ns.
Poorer microscope charaererisrics induce bias when inrerprering rhe
micro incremenrs . Image visualisarion is facilirared by use of a video
camera link ro a videoscreen projecror.
l.2.l. Resolution
The limir of resolurion corresponds ro rhe smaJJesr disrance rhar sepa-
rares rwo adjacenr visible srrucrures . The resolurion of lighr
microscopy depends mainly on rh e hardware and parricularly on rhe
objecrive lens available. Lenses correcred for colour and spherical aber-
rarion, and wirh a low numerical aperrure are essenrial. Campana
(1992) provides an inreresring rabl e rhar illusrrares rbe limiring
charaererisrics of rhe main rypes of obj ecrive lens: rhe numerical aper-
rure of rhe objeerive ulrimarely conrrols borh rhe magnificarion and
rhe resolurion rhar can be obrained, and rhe larrer increases as rhe
numerica l aperrure falls. N everrheless , rhe resolurion limir of a per-
fecdy sec up compound microscope is lower rhan rhe rh eorericallimir
(around 0. 3 flm), and ofren limirs observarion of rhe smallesr srruc-
rures, i.e. incremenrs < 1 flm (Campana, 1987, 1992; Morales-Nin ,
1988). In such cases rhe use of a SEM is essenrial. On rhe orher hand ,
rhe possibiliries of mag nificarion are limired only by rhe hardware
available. The maximum objeerive used is normally 100x (eirher wirh
or wirhour oil) and, wirh rhe appropriare combinarion of eyepieces and
body rubes, rhe maximum useful m ag nificarion is 1000x-1250x
(Campana, 1992).
l.2.2. Immersion v. dry objectives

Low-power lenses are dry objecrives (up ro 60x) while immersion
lenses are used for hig h mag nificarions . In bi ology mosr users believe
362
that the maximum magnification can be obtained oil immer-

sion of 100x However 100x dry
are used in and these can be also utilised for sclerochrono-
purposes. One of their main is the of
state of the cs thin otolith slices) as it is
not necessary to clean it after the observation. When using immersion
oil the must be cleaned afterward an ether-alcohol
solucion (50:50). We thus absolutely recommend the use of dry objec-
tive lenses at all
1.2.3. illumination
from the and an optimised source of illu-
mination has the greatest JOfluence on image quality
1992). All steps when adjusting illumination in
are much the same, the light source,
the light on the of observation with the and
the aperture and field ad justed
aperrure diaphragm will balance contrast, of field and resolu-
tion. All these parameters should be checked at the of a
series of examinations bur the same parameters should be used
between series in order to standardise the process.
1.2.3. L Filters
Achromatic lenses tend to improve
is closed down, and when light of a colour is used.
The change in can be attributed to the fact that lenses
are not correcred for all or for the entire field
of view. Green used. A coloured filter over
the source can raise the limit of resolution 15-20%
pana, 1992). Polarising filters observation of and
are also recommended (chap. VIlI.D.L3).
1.2.3.2. Confocal
Con focal also named Laser
observations of confocal seccions (single-focal
around 0.5 within internal planes of thick
to examine cs, mainly morphology and microstructures of
in order ro evaluate growth and the choice of sec-
et al., 1995). to convemional
of LSM include simultaneous measure-
meors in 3D and improved comrast and resolution in certain
tions. LSM can operate in epi-illumination
transmission modes. Observations based on the
mode are dependent on otolith autOfluorescence. The
363
ill so used ro d eteer fluor esce nc marks be neath the oro lith surface.
Ho wever the confocal t echniq ue cannoc d elineate th e depositi on of
incremems imo o wliths, beca use it d epend s on the fluorescence of the
spec imens. The exami na tion o f orolith m ic roin c rem e nts requires
transmi ssion-mode LSM.
1. 2.3.3 . Trans mitted v. refle cted light

In compo und mi croscopy, there are two poss ible so urces of lig ht , the
m ost frequ entl y used being transmi t ted lig ht which allows obse rva-
tions ro be made throug ho ut a shall ow depth of fieJd , an d reflec ted
li g ht (a lso na med epi-refl ected) whi ch all o ws rh e s ur face to be
observed . The latter so urce of illuminat io n was origin ally d eve loped
fo r g eol og ical studies and requires speci aJ equi p ment o n a standard
mi croscope. Thi s allows the li g ht bea m w pass through the o bjective
lens, refle er off rh e surface, and retllrn throu g h the lens w g enera te the
image . It is parti cularl y useful fo r observing the surface after spec ial
preparation such as g rin d in g and /o r po li shing w chec k its qualit y.
Ano th er appli ca tion is comro l of the acid etc hing of CS surfaces for
SE M prepa rati on. Etching comrol ca n also be don e direc rl y un de r the
mi croscope (c hap. VIII .C.2 .6). le is possibl e w co mbine both sources
of li g ht, transmitted and refleered , when necessary.
1.3. Polarised light

P olari sed li g ht is obta ined by means o f twO po lari s in g filt ers and
t ra nsmitted light. The first filter is p laced betwee n the so urce o f light
and the pre parat io n, ilnd th e other between the prepam tion and the
eyepieces. The rel ative pos it ion of the tWO filters can be auju steJ and
offers the poss ibili cy of revealing differem states of po lil risilc ion of the
li g ht through the CS prepara rion . Thi s ma y reveal so me st ru c tures
which are no t norm ally vi sible. O rolith s or bo nes the mselves have
po lari sing cha raereri sti cs. O w lith s are refrin ge m w p olari sed li g h c
and t his p ro perty a llows them to be vi sualised even in very sm all indi-
viduals such larvae of a few mm in leng th (chap . V111B.3.2 ). Pobri sed
li g h c is necessary w view certain orol.ith incremems, pil rti cularly t he
microincremenrs nea r the owlich edge (Mosegaard et Cl!., 1998) . For
ultrastruct ural bone examinat ion, po lari sed light is used to reveal the
ori e ncat io n of ch e coll ag en fibres, for exa mpl e isotro pic fibr es in
woven-fi bred bone matri x, or anisotrop ic fibres in parallel -fibred bone
matri x (Francillon- Vieill oc et ClI., 1990 ) (chap. l1. C. 1.l. 2).
364
1.4. Ultraviolet light

Ultraviolet light (UV) is only used to reveal fluorescent marks incor-
porated into CS (chap. IVA.1.2.1). The equipment needed to produce
UV light is usually specially adapted for compound microscopes,
which unfortunately restricts its use to small preparations. The route
of the light beam is similar to that of the reflected light (chap.
VIII.D.l.l.4.2) arriving from above and passing chrough the objec-
cive. The name "epi-fluorescent light " is also given ro this type of illu-
mination. The user musr check the compatibility of the UV source and
the microscope objeccives. Depending on the fluorescent substance
which is ro be revealed, specific filters may be needed. Table IVA.l
summarises the characteristics of fluorescent dyes, their excitation
wave-lengths, their fluorescence wave-lengths , their excitation rays
and che filters required. In order to view both fluorescent marks and
CS increments, ic is possible to simultaneously use epi-fluorescence
and transmitted light (fig. IVA .2).
2. Electron microscopy
Electron microscopy uses electrons instead of phOtons and thus offers

much higher magnification and resolurion. Two principal types of
microscope are available: the scanning electron microscope (SEM) and
the transmission electron microscope (TEM). Most sclerochronological
studies use only SEM. For chis reason we offer only descriptions and
recommendations for the use of SE M, and the reader should refer to the
numerous specialised references available for the use ofTEM. SEM falls
into an intermediate position between light microscopy and TEM in
term of resolution and image information. It is also similar to reflected
compound microscopy (chap. VIII.D.1.2.3.3).
2.1. SEM
The SEM is a surface topographic examination tool which is widely
used in the study of otoliths. This instrument offers a better than 300-
fold improvement in the depth of field over the highest quality light
microscope, as is reflected in the superb 3D images it provides. In
addition, studies of beam-specimen interactions using the SEM can
provide useful information about the chemical composition of the sur-
face of the specimen as well as che crystallographic, magnetic, and
electrical characteristics of the specimen . For microchemical analysis
purposes the SEM is coupled to an EDS (Energy dispersive spectrometer)
(chap. VII.G.l).
365
Manual of fi sh sc leroc hronology
2.1.1. SEM characteristics

The elec rron beam used ro "illuminare" rh e specimen is accelerared by
a volrage of 1 ro 30 kV. Inceraerions be rwee n rhe beam and rh e speci-
men used ro ge nerare rhe image have rhree possible our comes:
- some primary elecrro ns, depending upo n rhe acceleraring volrage
employed , penerrare rhe solid ro deprhs as much as 10 rm . Their rra-
jec rories vary and rhey lose m os r of rh eir energ y as rh ey penerrare rh e
speCI men;
- some primary elec rrons inreracr wirh rh e uppermosr arom s of rhe
specimen so rhar rh ere is a change in momenrum (bur no exchange of
energy ) wirh rhe resulr rhar rhe elec rron is backscarrered rhroug h a
larg e ang le and is effecrively reflecred from rh e specimen. Such elas ri -
ca lly refleered pr imary elecrro ns are known as "back scatrered elec-
rrons";
- some primary elecrrons inceracr wirh rh e hosr a rom s wirh rh e resulr
rhar collisions cause a cascad e of "second ary elecr ro ns " ro form along
rhe penerrarion parh . Some of rhese eleerro ns rhar are loca red in rhe
ourermosr stre/it/m diffuse rowards rh e surface, losing energy as rhey do
so. However, if rh ey re rain enoug h en erg y rh ey escape, in a process
known as "secondary elecrron emi ssion ";
- spec imen-beam inreraerio ns, as well as produ cing backscatrered and
secondary eleer rons, also produce pho rons, specimen currenrs, Aug er
elecrrons and X-rays, which are charac rerisri c of rhe probed specimen .
While any signa l g enerared can be urilised in principle ro produce an
image, in praer ice ir is rhe low-e nerg y second ary el ec rron s released
from rh e sample rh ar are m os r frequenrl y used. U sing surface-emirred
elecrrons, rarher rh an rhose rhar pass rhoug h rh e specimen as in TEM,
surface images wirh some rhree-dimensional qual iry can be obrained.
The impression of rh e image of rhe 3D surface is rh e res ulr of rhe di s-
rriburi on of lig hr and d ark areas . Thi s disrriburion is largely du e ro
[he faer rh ar rhe incidenc beam ge nerares more collecrable seco ndary
elecrrons pe r unir area when ir srrikes a sharply curved edg e or sloping
surface rhan when ir hirs a flar surface. Biologi cal speci mens, being
heavily conroured, fac ilirare rhe induerion of such a differencial effe cr.
Where rhe surface is smoorh , rilring rhe sample ar an an g le ro rh e
p robe wi 11 enhance rhe desired vari ari on in coUeered secondary elec-
rrons. More subrle effecrs involving rh e manner in which srrucrures
lying above a primary surface eirher defleer or absorb rhe probe elec-
rrons a lso come in ro play in rhe process. In any case, rhe "shad ows"
seen are a rrue represe nrarion of rh e 3D cha raerer of rhe specimen sur-
face und er srud y.
366
Preparation and observa tion techniques
on the beam which is

around 15 kV for molith observation, the ratio between the low-
backscattered electrons wi II be
penetrate the surface
due to the low conductivity of
depend on the of the (1)

ulrrastmcture examination or morphology.
To reveal the ultrastructure of an otolith it is necessary to cut it
(chap, VIILC2,3), Due to the low
power of the SEM, the section must be made through the
otolith area, Once the observation has been reached and
checked the surface must be polished VIILC25), Reflected
showing rhe surface sample enables the of the prepara-
tion to be checked (chap, VIILD.l.l In order to eliminate any
attached material the otolith should then be cleaned in a
cleionised ultrasonic water bath for] 5-30 seconds, After the
seCtion must be etched to reveal the ultrastructure, The two most
reagents are dilute HC! 2,0-5,0) and EDTA
(chap, VIII The etching time (1 10 min)
on the room temperature and the studied, Tri-
als should therefore be carried our to determine the rime to
alone
before the next preparatory steps for SEM,
should be dried at 30°C for 6-8 hours and free of dust.
are then attached to a SEM stub with carborundum ther~
glue or double-sided tape, A thin line of colloidal silver
the smb to the surface of the in order to pre~
from building up observation, The section is then
with gold (100 for
carbon (1 O~ 50 for
3. Microradiography (F.J. Meunier)
In some cases the best technique for visualising growth marks on

bones is microradiography of ground sections (fig. VIII.D.2) (Caillet
et aI., 1983; Casselman, 1983; Gruber & Srout, 1983; Yudin & Cail-
liet, 1990; Boujard & Meunier, 1991; Francis & Mulligan, 1998). The
principle of the technique is the same as that of medical radiography,
but using an appararus adapted to thin calcified objects such as
ground sections (Boivin & Baud, 1984, and others). Sections (chap.
VIII.C.2.3.1) are carefully laid on high-resolution film (Kodak
S0643) (fig. VIII.D.2a) and then exposed in an X-ray apparatus which
delivers X-rays of 10-30 kV at 10-15 Marintek, depending on the
parameters of the apparatus. The films are developed in HRP devel-
oper, washed and fixed. Then they are observed with a binocular
microscope after mounting between glass slides (fig. VIII.D.2b).
368
a 0......;;_ __ _ _ _- - - '' - '
Figure VIII .D.2 - Microradiograph of fin ray slices (Hoplostemum lit/orale). a) In a dark room , the
fin ray slices (red arrow) are deposited on a photographic film (yellow arrow) before being sub-
jected to X-rays. bl Result of microradiography under diHerent magnifications (photos@ Ifremer
O. Dugornay).
369
E. Conservation of preparations
w.J. McCurdy
1. Conservation of calcified structure preparations
Cool dark srorage is rhe besr conservarion environmenr for many cs

prepararions. The chosen mer hod of conservarion should prorecr rhe
CS from dereriorarion and also from loss or mechanical damage. All
conserved CS should be properly referenced and a copy of rhe reference
dara should be kepr in a secure place. Table Vlll.E.l lisrs rhe princi-
pal conservarion merhods for calcified srrucrure prepararions.
Table. VIII.E.1. - Conservation methods for calcified structure preparations.

Calcified srruccure prepararion Conservarion Srorage conrainers
Unmounred scales Conse rve dry Microrube vial s, paper envelopes
or plasri c scale packers
Scales mounred Conserve dry Slide srorage nays and boxes
on microscope si ides
Cellulose acerare sca le Conserve dry Srorage boxes
im pressions
Unmounred small oro lirhs 1 . Conserve dry l. Microrube vi a ls or plasri c slides
2.2': 95% alcohol 2. Mierorube vial s
Unmounred large orolirhs Conserve dry Subdivided conrainers or paper envelopes
(w hole and broken)
Di rec dy embedded larval orolirhs Mounring media Microscope slide rrays and srorage boxes
Plasri c slides wirh many orol irhs Mounring media Cusrom si ide rrays and sro rage boxes
Mounred oroiirh rhin seceions Mounring media Microscope slide rrays and srorage boxes
Many orolirh rhin secr ions mounred Mounring media Cusrom slide rrays and srorage boxes
on cU$rom microscope slides
Unmounred bones Con se rve dry pcorecred Paper envelopes, individual conrainers
and orher skeleral parrs from insecrs and fungi or subdivided conrainers
Mounred rhin secr ions of bones Mounring media Mi croscope slide rrays and srorag e boxes
and orher skeleral parrs
Bones and orher skelera I parrs Embedding media Individual conrainers
prepared for secrioning or subdivided conra iners
2. Scales
2.1. Unmounted scales

Scales are usually conserved dry (chap. VII1.B .2). Preprinred paper
envelopes and plasric scale packers are ofren used where large numbers
of scal e samples are processed, e.g. for Norrh Arlanric Salmon, Safmo
safar and or her Salmooid species. Envelopes and packers conraining
370
scales should be scored in labelled boxes CO reduce the risk of loss or

damage. Referenced microtube vials can be used co score both dry
scales and otoliths. The vials should be scored in special suppOrt racks
(chap. VIII.B. 3.3.2).
2.2. Mounted scales and scale impressions

Preparation methods for scales mounted on microscope slides and cel-
lulose acetare impressions are desc ribed in (chap. VIILC.1.2) and
(chap. VIII.C.l.3). Prepared microscope slides should be conserved by
scorage on slide trays and cell ulose acerate impressions shou Id be
scored in suirable boxes in a cool place.
3.0toliths
3.1. Otolith collections

Sometimes pairs of ocoliths are collected, even when only one ocolirh
is needed for the analysis. The reasons for doing so vary, but ir is
almost rourine practice when sampling sagittae in adulr fishes. Some-
rimes one particular otOlirh is preferred for analysis e.g. Plaice, Pleu-
roneftes platmet, bur often rhe orolirh co be read is selected at random.
In such cases considerarion should be given co conserving the unused
ocoliths, as rhese are a valuable reposicory of sclerochronological infor-
marion. The combination of a shortage of ocolirhs from older speci-
mens in some habirats and advances in ocolirh microchemistry has
highlighred the value of these otolirhs. Exisring collections of ocoliths
need to be evaluared, caralogued and conserved, in a way rhar will
make borh the otoliths and rhe corresponding fish life hisrory infor-
marion, more accessible to other researchers.
3.2. Whole and broken otoliths

Many otoliths are concavo-convex in shape and some smaller oroliths
are thin and fragile. If they are stored in paper envelopes as previously
recommended (Williams & Bedford, 1974), rhere is a significant risk
of damage every rime rhe co1Jection is handled. Unmounted otolirhs
can be stored in labelled vials (chap. VIILB.3. 3.2) or in cusrom plas-
ric slides and trays with covers. Some aurhors recommend conserva-
tion in alcohol >9 5%, which can be useful for fragile and small
otoliths (chap. VIILB. 3.3.2 ). Large Gadoid otoliths are frequently
stored in Repli dishes®, clear plastic trays with twenty-five 2 cm
compartments and a lid that can be raped to the base.
When alcohol or water have been used as observation media, borh
whole and broken otolirhs are best wiped dry with sofr tissue paper
and conserved in their original storage vials (chap. VIII.B. 3.3 .2). If
clearing oils (chap. VIII.D.l.1.2) have been used to improve the
371
quality of the observation, the oil may be genrly wiped off before
returning the otOl iths to their ori g inal storage containers. Small
pelagic otOliths e.g. Sprat, SpraltllJ spmltUJ are often stored and
observed on custom moulded plastic slides . Any water or alcohol used
to observe these otoliths should be allowed to evaporate and the
otoliths allowed to dry our thoroughly, before the glass or plastic pro-
tective cover is secured to the slide. The slides may then be stoted in
a suitable tray or container.
3.3. Embedded otoliths and otolith thin sections

Individual otoliths, e.g . from larvae, can also be conserved by direct
mounting in media (chap. VIII.C2.4 .2). Otoliths or otolith thin sec-
tions mou nted on glass microscope slides and plastic slides containing
many pairs of embedded whole otoliths (chap. VIII.C2.2.3), may be
stored on plastic or metal trays. The trays containing these slides
should be srored in suitable containers with lids ro protect their con-
tents . It is very important to maintain the highest possible standards
of slide preparation, as the cover slips will break easily if they are not
completely supported by the embedding medium. Excess embedding
media may also cause slides ro stick together during storage. We rec-
ommend the use of custom cover slips with a thickness of 0.5 mm,
when using polyester resins to prepare plastic slides with many
embedded whole oroliths and custom microscope slides with many
otolith thin sections (chap. VIII.C2.3.2 and chap. VIII.C2.4.3).
Thinner cover slips may crack or shatter during handling and obser-
vation.
4. Other skeletal parts
Conservation is very simple and is usually done dry. Generally

speaking, bones are best conserved by returning them to their origi-
nal paper storage envelopes. Preca urions must be taken to prevent the
growth of fungi and attack by specialised insects (chap. VIlI .B.4.2).
For very fragile samples, ethanol between 70% and 90% can be used
as a fixative (Meunier, 1988), (chap. VIII.B.4.2). If alcohol is used the
calcified materials should be stored in suitable referenced vials. Tissue
sect ions mounted on microscope slides may be stored in standard slide
storage racks or boxes. Larger samples and embedded mate rial pre-
pared for sect ioning should be sto red in suitable referenced individual
or sub-divided containers.
372
Glossary
Glossary
H. H,
373
Glossary
From Francillon-Vieilloret at, 1990; Kallsh et al., 1995.
(0), terminology reserved for ololiths; (S), terminology reserved for scales: (Sk), ter-
reserved for skeleton.
(entre centre formed beyond the otolith

core that leads to a new and from which a new series
of growth increments appears to emanate, Formation of these struc-
tures is often associated wirh life transitions such as mecamof-
centres are often referred to as accessory
the term accessory because
these features are different do
nor contain primordial
has also been used. See
The closeness of a quantity estimation or com-

puted value) to its true value.
estimation: Th is term is
assigning ages to fish, The rerm
it refers to time-related processes and the alteration of the
structure, and function of an over time. The term
to determination" due to the uncer-
Up-rnfJUll' The cohort of fish of a VCcll-'JlU age-

group). The term is not synonymous with
Annu/m annuli): One of a series of concentric zones on a structure

that may be in terms In some cases, an annulus may
not be continuous or obviously concentric. The appearance of
these marks on the calcified structure and [he and
should be defined in terms of characteristics of the structure.
This term has traditionally been used ro year marks even
though the term is derived from (he Latin "anus", nor
from "annlls", which means year. For otoliths, the variations in
microstructure that make an ctnnlllus a distinctive of an otolith
are nor well understood. See II.A.9, ILA.lO and ILC.9 .
Anterostmm . see Antirostmm.
Antirostmm (0): Anrerior and dorsal projection of the

shorter than the rostmm. See II.A.4.
375
Asterisells (pi. asterisei) (0): One of the three orolith pairs found in the
membranous labyrinrh ofOsteichthyan fishes. It lies within the lagena
("flask"') of the pars inferior. In non-Ostariophysan fishes the asterisetts
is small and shaped like a flattened hemisphere or quarrer moon. In
the Ostariophysi rhe asterisClls is roughly circular and laterally com-
pressed and is considerably larger than the sagitta. See figure II.A.l.
Axis 0/ measurement: A line along which growth incremenrs are num-

bered and measured.
Band: See wne.
Bone remodelling (Sk): The process of reshaping of the bone tissue which
occurs inrernally or at the edge of the bone. It can affecr primary or
secondary bone and every type of tissue. Ir is due ro morphogenesis
during the early life, or ro physiological demand or mechanical con-
srrainrs.
Bone resorptioll (Sk): The acrion of the erosion of the bone surface by
osteoclasts or osteocytes at their own peri phery.
Calcification: The process of deposition of calcium carbonate crystals in

oroliths and calcium phosphate crystals in bones and scales.
Ccmalicttllis (pI. canalimli) (Sk): The thin space of the bone rissue
including the cyroplasmic extension of the oSteocysrs and osteoblasts.
See figure II.C.2.
Cementing line 0/ resorption (Sk): There are two kinds of cemenring lines:
resorption lines (reversal lines) that occur on irregular resorbed sur-
faces and resting lines (rest line) that occur on unresorbed surfaces.
Both are thin chromophilic lines that show a greater degree of miner-
alisation than the surrounding bone tissue. The resorption line sepa-
rates the secondary bone from the primary bone. The resting line is a
line of disconrinujty within the bone tissue which corresponds ro tem-
porary but complete cessation of growth. See figure II.C.8.
Check (0): A disconrinuity (e.g., a stress-induced mark) in an otOlith

zone, a pattern of opaque and translucenr zones, or mictOincremenrs.
Microsrrucrural checks (e.g. hatching checks) often appear as high-
conrrast microincremenrs with a deeply etched D-wne or an abrupt
change in the microstrucrural growth pattern. If the term is used, it
requires precise definition. See figure II.A.ll.
CirCttlw (pi. cirmli) (S): A concentric crest on the external face of elas-
moid scales caused by tissue elevation of the superficial layer of the
scale.
Coh&rt: A group of fish of a similar age that were spawned during the
same time inrerval. Used with both age-group, year-class, and day-class.
376
Glossary
bone . A kind of bone architecture in which the tissue

volume is greater than that of the vascular cavities. Compact bone can
be avascular.
Core The area or areas surrounding one or more and

bounded by the first D-zone. See !I.A.5. Some fish
Salmonids) possess multiple and cores.
Corroboration: A measure of the or of an age

estimation method. For example, if two different readers agree on the
number of zones present in a hard part, or if two different age estima-
tion structures are as having the same number of zones,
corroboration nor validation) has been accomplished. The term
"verification" has been used in a similar sense; bowever, tbe term "cor-
roboration" is nrl'tPFrF,rt as verification that the age estimates
were confirmed as true.
(spine) on the external face of tbe

in the on
D-zone (0): That of an otolith microincrement that appears

dark when viewed under transmitted and as a
when acid-etched and viewed with a
This component of a microincrement contains greater amount of
organic matrix and a lesser amount of calcium carbonate than the L-
zone. Referred ro as the discontinuous or matrix-rich zone in earlier
works on "D-zone" is the term. See L-zone
and IIAS.
An increment formed over a 24-hour In its

a increment consists of a D-zone and a I.-zone. The
term is synonymous with "daily growth increment" and "daily
The term "daily is and inaccurate and should nor be
used. The term "daily increment" is See increment and
HAS.
or hatched on a
nVHMnpr1 date
Whether this refers co the date of
must be specified.
See check,
Dottble zone (or or . Two that are close

relative to the size of rhe calcified structure and rbe distance berween
two which are considered as one annulus. As such a double zone
includes both a zone and an annuLus, This structure has also
been termed a
377
Excisllra major (0): The cleft separating rostrum and antirostrlJm.
Exrisura minor (0): The cleft separating postrostmm and pararostrttm.
Field: An area of a calcified structure, defined on a side or a section,

and delimited at least by the cenrre and the edges (e.g., anrerior, pos-
terior, dorsal, venrral fields). It has also been termed as "region".
Focus (pi. fori) (5): The cenrral part and the centre of origin of the scale.
Growth mark (or ring or zone): See mark or zone.
Growth pattern: The notion of the relative growth of incremenrs dur-

ing a period of the life of the calcified structure (e.g. annuli or daily
increments).
Hatch date (0): The date on which a fish has hatched, typically ascer-
tained by counring daily incremenrs from a presumed hatching check
(see check) to the otolith edge.
Haversian system (Sk): See secondary osteon.
Hyaline zone: A zone that allows the passage of greater quanrities of

light than an opaque zone. The term should be avoided; the preferred
term is "translucenr zone". See translucenr zone.
Increment: A reference to the region between similar zones on a struc-

ture used for age estimation. The term refers to a structure, but it may
be qualified to refer to portions of the hard part formed over a specified
time inrerval (e.g . sub-daily, daily, annual). Depending on the portion
of the hard part being considered, the dimensions, chemistry, and
period of formation may vary widely. A primary incremenr consists of
a D -zone and an L-zone, whereas an annual incremenr comprises an
opaque zone and a translucenr zone. There may also be seco ndary
structures such as sub-daily increments and false and double zones
within annual incremenrs.
Initium (Sk): The cenrre of origin of growth of the fin ray.
L-zone (0): That portion of a microincremenr that appears light when

viewed under transmitted light, and as an elevated region when
acid-etched and viewed with a scanning electton microscope. The
component of a mictoincrement that contains a lesser amount of
organic matrix and a greater amount of calcium carbonate than the D-
zone . Referred to as "i ncremenral zone" in earl ier works on dail y i ncre-
ments: "L-zone" is the preferred term . See D-zone and figure Il.A.:5.
378
Glossary
Lapillus (pt. (0): One of the three otolith found in the

membranous labyrinth of Osteichthyan fishes. The most dorsal of rhe
it lies within the utriatl11S pouch") of the pars
In most this otolith is like an oblate and it is
smaller than the See ItA.I.
Lobes (0): The minor rounded protrusions of the the dor-

sal and the ventral edge.
Macrrxentric scale (or r~0P111'ra.tPLl See scale.
A1acroincrement: Increments that are more than 50 Ilm in

width; the "macro" serves to indicate that the denoted is
of size and that it can be seen with a binocular micro-
scope. Often used to describe seasonal increments. See increment.
V'/"ITn.rUll increment: The the last identifiable mark at the

margin of a structure used for age estimation. this
increment is usually pv,..,rpo<p'r! in relative terms; that
or of the last annual or daily increment.
Alark. A an hisromorphological mark of

similar structure or optical density laid down during the of
hard parcs. See zone .
ivledttlar . The first vascular in the median position in

the long for II1 rays of catfishes. See II.C.6.
Microcentrtc scale (5): A scale present since the early life of the individual,
which has not been resorbed.
Aficroincrement An increments that is less than 50 pm in width,

typically from one to 20 Ilm; the "micro" serves to indicate that
the object denoted is of small size and that it can only be
observed Often used to
describe otolith increments. See increment.
interruption (0). A in
the deposition of an organic zone. It may be localized
concentric feature. See check
M inera/isctfion: The biological process of the of crystalline

or mineral material in or on an
zone (Ill' or A number of closed zones,

the size of the calcified struCture and the distance of the
are ~F,'~"'~" as one annulus. See also false zone.
379
Nucleus, Kernel (0): Collective terms originally used to indicate the pri-
mordia and core of the otolith. These collective terms are ambiguous and
should not be used in descriptions of mictostructure. The preferred
terms are "primordium" and "core" (see definitions). When viewed
mactoscopically, the term "nucleus" has been used to refer to the region
around the core, although the precise extent of this area is generally ill
defined. If the term is used in describing macrostructure, it requires
precise definition.
Opaque zone: A zone that restricts the passage of light in comparison

with a trans lucent zone. The term is a relative one because a zone is
determined to be opaque on the basis of the appearance of adjacent
zones in the otolith (see translucent zone). In untreated otoliths under
transmitted light , the opaque zone appears dark and the translucent
zone appears bright. Under reflected light the opaque zone appears
bright and the translucent zone appears dark. An absolute value for
the optical density of such a zone is not implied. See translucent zone.
Optical focus plane (0): A plane at a certain depth in the 3D otolith

structure where microincrements can be distinguished when viewed
under a light microscope. The orienrations of the radial growth direc-
tions at an optical focu s plane are perpendicular to rhe direction of
observation.
Ossification (Sk): All the processes involved in bone formation.
Osteoblast (Sk) : A specific bone cell which synthesizes the bone matrix
and is located on the inner (endost) or on the outer (periost) surfaces of
the bone tissue .
Osteoclast (Sk): A specific bone cell which is involved in bone resorp-

tion, generally multinucleated in higher Vertebrates; they may also be
mononucleated in fish .
Osteocyte (Sk): A specific type of bone cell embedded in the bone tiss ue
and carrying out the trophic needs of the bone. It is an osteoblast
incorporated in tissue.
Osteogenesis (Sk): The process of bone tissue formation by the specialized

cells (osteoblasts).
Otolithometry (0): Age estimation from marks recorded in the otoliths

ofTeleost fish.
Pararostrttm (0): The posterior and dorsal projection of the sttgitta.

Generally shorter than the postrostrum (used in connection with Clu-
peid orolith morphology).
380
Glossary
Postrostrum . The posterior and ventral of the

Generally than the pararostrum in connection with Ciu-
peid otolith morphology).
Precision: The closeness of measurements of the same quan-

tity. For a measurement that is free of bias,
implies accuracy but the two terms are not
Primary bone The bony tissue which is deposited where antecedent

bone does not exist.
Pr,m,7rv osteon A vascular canal surrounded by concentric bone

lamellae which are deposited centripetally and which does not
on
Primordial grcmule The primary or initial components of the pri-

mordium. There may be one or more primordial granules in each
mordium. In the may be whereas
the rest of the /mm(Jrcilum
Primordium structure of an
it consists or fibrillar material one or
more optically dense nuclei from 0.5 pm to 1.0 pm in diameter. In the
stages of otolith if several are present, they
generally fuse to form the otolith core.
Pseudo-lamellar bone tissue A bone tis-

sue which is of a matrix with
from one to another bone layer.
Radius (pI. . A radially oriented groove,

from the scale focus, and to the absence of an
(external) of the scale. See ILB.l.
IIp~"d,n'"
Special terms used in the jargon of sclerochro-
of a calcified structure consists of
its growth pa[(erns. A reader is a person who tries to
marks recorded in a given calcified strucrure.
macrocentric scale) . A scale which has been

rapidly after the removal of the
scale.
Kf"rtlri?t1,Gtl:The loss of the original material of a calcified structure

a physiological process removed from its original place).
Rest line line of growth See cementing line.
Ring: See increment and zone.
381
Rostmm (0): Anterior and ventral projection of rhe sagitta. Generally

longer rhan rhe antirostrtlTn.
Sagitta (pI. sagittae) (0): One of rhe rhree orolirh pairs found in rhe
membranous labyrinth ofOsreichrhyan fishes. Ir lies wirhin rhesacCtl-
Ius ("Iirrle sack") of rbe pars inferior. Ir is usually compressed larerally
and is elliprical in shape; however, rhe shape of rhe sagitta varies con-
siderably among species. In non-Osrariophysan fishes, rhe sagitta is
much larger rhan rhe asteriscus and lapillm. The sagitta is rhe o[Olirh
used mosr freguenrly in orolirh srudies. See figure II.A.l.
Scalimetry (S): Age esrimarion using marks recorded in rhe scales of

Teleosr fish.
Sclerochronology: The merhod of esrimaring age and rhe durarion of life

hisrory events (or remporally-based events), from marks recorded and
conserved in calcified srrucrures.
Secondr:try bone (Sk): Bony rissue which is deposired in an area where rhe
primary bone has been resorbed (bone of subsrirurion).
Secondary osteon (or Haversiart system) (Sk): An erosional caviry iniriared

from a vascular canal and secondarily filled wirh concentric bone
lamellae deposired centriperally.
Secondary stmctllre: A rerm used for all macroscopic zonarions rbar do

nor appear ro conform ro rhe opaque and rranslucent zones of an cmntt-
The main examples are false and splir or double ringslzones.
IllS.
Skeletochronology (Sk): Age esrimarion using marks recorded in rbe

skeleral srructures ofTeleosr fish.
Spongy bone (Sk): A rype of bone archirecrure in which rhe rissue volume
is very vascular. The volume of rbe vascular caviries is much larger
rhan rhar of rhe rissue.
Stctined ring (or line): A cbromophilic/srainable ring or zone wirh a

variable inrensiry.
Sub-daily increments (0): An increment formed over a period of less

rhan 24 hours. See incremenr.
Sltfem amstictlS (usually shorrened ro Slllet/S) (0): A groove along rhe

medial surface of rhe sagitta. A rhickened porrion of rhe orolirhic
membrane lies wirhin rhe sulcus aCllstims. The sulClls acusticttS is ofren
referred co in o[Olirb srudies because of rhe clariry of rhe increments
near rhe sulcus in rransverse secrions of sagitta.e. See figure ILA.4.
382
Glossary
'tt/),errJ~ttnler'trv mark (or ring or

estimation or retained as an anrutltts, This mark is
Transitton zone (0): A in otolith structure between two

similar or dissimilar In some cases, a transition zone is reco-
due (0 its lack of structure or or it may be
as a change in the form (e,g. width or contrast) of the
increments. Transition zones are often formed in otoliths me ca-
morphosis from larval to stages or habitat
such as the movement from a
a marine to freshwater habitat. If the term is used, it requires
definition.
Translucent zone: A zone that aJJows the passage of

than an opaque zone, The term is a relative one because a zone is
determined to be translucent on the basis of the appearance of adjacent
zones in the structure (see opaque An absolute value for the
optical of such a zone is not implied. In untreated calcified
structures under transmined light, the uanslucent zone appears
and the opaque zone appears dark. Under reflected light the
translucent zone appears dark and the opaque zone appears
The term has been bur "translucent" is
Ultrastructure: The structure of a rissue observed at levels of

(particularly with electron microscopy).
Validation: The process the accuracy of an age estimation

method, The concept of validation is one of and should nor be
considered in absolute terms. If the method involves
then part of the validarion process involves
of the zones counted. Validation of an age estima-
indicates that the method is sound and based on fact.
The process of that is true. Indi-

vidual age estimates can be verified if a validated age estimation
method has been employed. Verification implies the of some-
thing, such as a that can be determined in absolute terms
to be either true or false. See corroboration,
Vertebral The circular and central parr of a vertebra,
or hatched in a
Whether this term is used to refer to the
of similar structure or density.

with , "band" and "mark". Where the use of this term
should be illustrated.
383
384
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448
Manual 01 lish sclerochronology
.o.",rnn\lm". and chemical ~\I.nhnl ..
20 2-dimensional view
3D 3-dlmensional view
A age
AAS speC[ romct ry
ADC
AES aromlc emission spectrometry
ACL arrested line
AGR absolute
AI aluminium
ALK
AMD diStance
AMS acceleraror mass specuomerry
ANOVA of variance
APE average percent error
ASA srock (+ see SPA, VPA)
Ba barium
BCF back calculatlon formula
BPH hVf)f)f'h'-<1< (in back-calculation)
C carbon
Ca calc!um
CAAGE and growth estimarion
calclUm carbonate
CCD device
CClR Comite Consultatlf International des Radio-communications
Cd cadmium
Ce cef!um
Cl chlorine
carbon dioxide
CRM certified
calcified ,trucrure
Cu copper
cv coefficienr or variation
converrer
DCT discrete cosine rransform
OGR dady grow rh rare
EDS energy spectromerry
EFAN European Fish Ageing
Fe iron
fast Fouriet rransfotm
449
GRE growrh rare eHecr

H hydroge n
HR-J CP-MS high resolurion-inducrively coupled plasma-mass specrromerry
ICES [nrernarional Council for rhe Explorarion of rhe Sea
ICP -AES inducr ively coupl ed plasma-aromic emission specrromerry
ICP MS inducrively coupl ed plasma-mass specrromerry
ID-I CP-M S isorope dilurion-inducrively coupled plasma-mass specrromerry
IRMS isorope rario mass specrromerry
)P EG joint phorographic experrs group
K porassium
LAC "Ligne J'arrer de croissance" (French)
LA-rCP-M S Laser ab larion -i nducrively coupled plasma-mass specrro merry
Li lirhium
LOD limir of derecr ion
l"IANOVA mulrivariare analYS IS of varia nce
Mg magnesium
Mn manga nese
MSVPA mulri spec ies virtual pop ul ar ion analysis
N nir roge n
Na sodium
Ni ni ckel
o ox)'ge n
OES oprical em ission spec rrom erry
OMC orolirh mi croc hemi srry
P phosphorus
PA percentage agreemem
PAS period ic ac id Schiff
Pb lead
PCA principl e co mponem analysis
PDB Peedee Belemnire
PJXE panicle-induced X-ray em ission
Ra radium
RAM rand om access memory
Rb rubidium
RGR relarive growr h rare
RMD relarive marginal di sca nce
Rn radon
S sulphur
SEM sca nning elecrron microsco pe
SGR specific growrh rare
Si silicon
SIMS secondary ion mass spec rromerry
SL srandard lengrh
SLA P srandard lighr Anrarcric precipirarion
450
Srn samarium
SMOW standard meRn ocean
SPH (in back-calculation)

Sr stronrium
Sr/Ca strontium/calcium
SRM standard reference materials
total allowable catch
Te thm
TEM
Th thorium
T1FF file fotmat
TIMS rhermal ionisation mass specrromcrry
Tl thallium
U uranium
VGBf
VPA
VPDIl vienna-Peedee belemnite
VSMOW vienna srandard mean ocean WJter
WDS spcctrometry
WIP water-msoluble
WSP warer-soluble
Zn zinc
451
Author addresses
Abdesslam Benzinou
EN1B, laborawire RESO, Technopole Bresr-Iroi se , Sire de la Poince
du Diable, BP 30815, 29608 Bresr Cedex, France
Tel : + 33 2 98 05 6692 - Fax: + 33 29805 6689
E-mail: benzinou @e oib.fr
Arild Folkvord
Universiry of Bergen, Deparrmenc of Fisheries and Marine Biology
Bergen High Technology Cencer, 5020 Bergen , Norway
Te!. : +475 5584456 - Fax: +47 5558445 0
E-mail: arild.folkvord@ifm.uib.no
Audrey J. G effen
----------------------------------------
School of Biologi cal Sciences, U niversiry of Liverpool, Porr Erin
Marine Laborawry, Porr Erin, Isle of Man lM9 6JA GB, UK
Te!.: +44 162483 10 16 - Fax: +44 162483 1001
E-mail: geffen@liv.ac.llk
Willie McCurdy
Deparrmenc of Agriclllrure and Rural DeveJopmenc for Norrhern
Ireland, Aquaric Sysrems Group,
18a Newforge Lane, Belfasr BT9 5PX, Norrhern Ireland, UK
Tel: +44289025551 3 - Fax: +442890 382244
E-mail: willie.mccurdy @dardni.gov.uk
BenOlr MesniJ
Ifremer, cenrre de Nances, laborawire MAERHA
BP 21105,44311 Nances Ced ex 3, Fran ce
Tel. : +3 3 240374009 - Fax: +33240 374075
E-mail: Benoir.Mesnil @i fremeLfr
Fran~ois J. Meunier
MNHN, FR CNRS 145 1, laborawire d'I chryologie generale
er appliquee, 43 rue Cuvier, 752 3 1 Paris Cedex 05, France
Te!. : +3 3 1 407 93761 - Fax: +33 140793771
or,
Universire Deni s Dideror-Paris 7, UMR CNRS 8570,2 place Jussi eu ,
75251 Paris Cedex 05, France
E-maiJ : meunier@mnhn.fr
452
Beatri z Morales-Nin
GOI -IMEDEA (UIB/CSIC)
Miguel Marques 21, 07190 Esporles, Spain
Tel: +349716 11721 - Fax: +34971611 761
E-mail: ieab mn @c\us Luib.es
Henrik Mosegaard
Danish Insriruce for Fisheries Research, Charlorrenlllnd Casrle,
2920 Charlorre nlllnd, D en mark
Tel : +45 3396346 1 - Fax: +45 3396 3333
E-mail : hm @dfu.min.dk
)acq lles Panfili

!R D, centre de recherche halieurique medirerraneenne er rropicale,
BP 171, 34203 Sere Cedex, France
E-mail: panfili@ird.fr
Helene de Ponrual
Ifremer, cenrre de Bresr, Drvlrh /LASAA ,
BP 70, 29280 Plouzane, France
TeI: +33298224 692 - Fax: +3329822 4653
E-mail: ponrual@ifremer.fr
Peter). Wrighr
Fi sheries Research Servi ces Marine Laboratory,
PO Box 101 , Victoria Road , Aberdeen , ABll 9DB, Scorland, UK
TeL: +4 4 1224295436 - Fax.: +44 1224 295511
E-mail : wrighrp@marlab.ac.uk
453
Index
Page
AAS 287,290 Back-calcularion - body proponional
Abrasive paper 350 hypothesis 160
Absolure marginal distance 129, 131 Back-calcularion - scale proponional
Accessory grow th centre 42,10 1,176 hyporhesis 160
Accrerion rate 52, 122,17 3 Backscacrered electron 280, 366
Accuracy 141,236,301 Balric 173,177,281
Acellular bone 66, 83 Basal plate 58
Acerare impression 332 Bigger is bener mechanism 167,171
Achromatic lenses 363 Biological interaCti ons 187
Acid acrack rime 351 Biomineralisation 53,2 51,255
Acid erc hing .351 , 47 Birch date 96,1 37,174
Acrylic glue 359 Birch dare distriburions 174
AES 287,290 Birth mark 82 , 95
Age at firsr maturiry 99, 175 Black plasric si ides 336,339, 359
Age class 96, 135 Blood plasma 53,99,255
Age gro up 96,135,180 Bomb chronomerer 273
Age-Iengrh key 135, 190,1 91 Bone 65
Age-structured model 180,181 Bone hisrology 68
AI 254, 296 Bone morphogenesis 82
Alcohol 317,327,359 Bone remodelling 68,76 , 108
Alizarin 117,120 Bony riss ue 66,316,342
Allomerry 149 Bootstrap 165, 175 , 279
Alumina pasre 350 Breeding 75,86,125
AMS 276, 291 Breeding mark 82
Anadromous 103,260,279 Bulk analysis 277,290,356
Annual increment 43,93,132 Bulk recllniques 286,287,290
Anosteocyric bone 66 Burned surfaces 353
Aquac ulture 126 Burning 352
Aquamount® 354 14C 273
Aragonire 35, 5.) ,252 UCl 12 C 247
Archaeology 88 Ca 55,99,252
Arresred growt h line 71,80 CaCOo 251,252,254
I1JteriJctlJ (p I. ClJteriJci) 31,42 , 92 Calcire 53, 252, 265
Automarion 201 Calcium 36, 52, 254
Avascular bon e 73,83 Calcium carbona re 35,44,25 1
Average fdrer 216 Calcium me tabolism 86, 177
Average percent error 139 Calcium phospha re 70
Avoidance of gear 178 Canad a balsam 359
Axis of measurement 108,129 Cartalmdm (pI. canaliculi) 69
Ba 70,254,257 Carbonare 56, 70, 264
Back-calcularion 154 Cartilage 27,258,353
Back-calcularion - bio log ical intercepr Caralyse 334
merhod 158
454
CCD 203 Cubic 175

Cd 302 d isrri burioos
Ce 279
Ce 11 tamnae 80
71
32
Chronometer 276 increment 92
Circular vascular canal 74 Data quaky 293
eircttltlS 59 Decalcification 351
Citrace 70 Decontamination
Cl 17
71,352
Dendrochronology 19, 1
Denticules 27
Demine 27
Dermis
Climatic variations Detection limits 299
252,268 Diadromous 260
Coefficient of variation 39 Diamond blade
Cohort , 181 Diamond
Cold season 80 Diamond
285 Diet
fibri Is 364 176
Com pact bone 46
conservation seleCtion 176
Contamination Dissolution
increments) Distal
Convolution 211,214 Disuibmion coefficient 258
Coral 274 Diurnal variation 257
Core Dominance hierarchies 172
Dorsal fin 92
season
Creosote D-zone
Cross-seccional 176 Ecotoxicology 256
Cross-secrional data 147,175 of CS
EDS 287,
Ehrlich's
,25 257 Elasmoblast 59,62
Elasmoid scale 58
70 Elememal 252,286
89,105,133 Elemental
95, 1 230 Elememal 255
105,
Ctenoid scale 58 Embryo 325
Cu 302 Embryonic 174
Enamel 27,28 5 Grinding and poli shing machines 3 50

End oge nou s fac cor 105, 264 Growch axis 34, 108 , 133
Endo lymph 3 1, 54,255 G rowch incremem 93,1 05, 129
Endosc 66 G rowch mark 62,7 9 ,9 1
Environ me mal hi scory 149, 260, 28 1 Growch mo rcalicy hypochesi s 16 7
Ep idermis 60 Growch paccern 9 1,1 4 0, 222
Epi ge neci c fac ror 79 Growch race effec c 15 1
Epoxy cesin 334,3 55 Growch race mechani sm 168
Erosi on 63 ,68, 32 3 G rowch rhychm s 64,8 7
Essenti al oils 359 G rowch trajectOries 148,15 1,1 63
Ecching 351 H 55, 247,257
Echer-alcohol 359, 363 Habicac 101 ,252 ,27 9
Echyl ene di amine cecraacecic acid Half-life 248, 27 1
(EDTA) 351 ,3 6 7 Handling 3 26
Eukicc® 334, 359 Hacch check , hacching check 41, 9 5
Exogenous fac ror 105, 264 H acch-d ace di scribuci o ns 174
Ex cernal bias 95 H acching 39, 174
Excernal laye r 60 H ea ring 35
Ex trinsic fi brils 68 H emaroxylin 346 , 35 1,35 4
Fe 254, 258 Hig h-pass filcer 2 14
Fi n ray 20,26 ,328 Hisrophysiology 65
Fine gr inding 35 1 H omeos casis 35, 284
Firsc macucicy 99 , 175,1 77 H owship's lacuna 68
ficn ess 17 1, 174 HR-JCP-MS 290
Flac bon e 77 Hydroxyapacice 66, 70 , 284
Flo-cex xv 359 Hypercalcemia 86
Fluorid e 70 , 284 H yperm inerali sed 58, 68 , 72
Focal p lane 105,3 47, 363 Hyperosrosi s 77
Food resources 82 H ypervasc ulari sed bo ne 7 5 , 83
Foraminifera 264 IC P-AES 287 , 290
Forceps 31 8, 326, 333 lCP-MS 287 ,2 89, 297
fossi l 74, 264, 280 ID-JCP - MS 290
From of mineralisacion 70 Illuminacion 206, 359
Frontal (plane) 34 Image 3D 22 8 , 366
Fu chsin ac id 353 Image acqui siri o n 206
G AG 269 Image ana lysis sysrem 20 2, 23 0
Gan o id sca le 58 Image anamorphosis 211
Gan oine 58 Image brig hcness resoluci o n 20 4
Gau ss ian fllcer 21 6, 220 Image comrasc 2 12,23 5
Ge nec ic faeror 79 Image d eblur ring 22 0
G lyceri ne 294 , 3 23, 35 9 Image fo rmar 208 ,239
G M reg ress ion (see al so Ricker Image imeg rar io n 20 8
reg ression ) 16 1 Im age m osa ic 208
Gom percz g rowch funcci on 15 4 Im age profi le 2 13 ,224
Gonad al d evelopmem 177 Image qualicy 201, 235
G radi ent filcer 22 0 Image spacial resoluc io n 204, 209
456
Immersing medium l'v1aclIla 31, 55,

Immersion oil 362
Incineration 71 Mandible 83
Increment width pattern 167,170 Mandl's 61
Incremental lines 62 Margin width 1 1)2
Individual growth 154, 163 Marginal increment 132
Inner ear 1, 53 zone 129,208
Inotropic 71 1 279
Instrument drift 300 Mass fraccionation 247
Intercali bration 91 Mass spectromerry 289
Interference 299 Mathematical morphology 220
Internal bias 95 Maximum likelihood 135, 279
105,1 Median filter 211
criteria 95, 1 201 Medullar
Intrinsic fibrils 68 Mesocosm
Ionic conteO[ Metabolic
IRMS 298 Metabolic control of otolith accretion
Isometric rate 173
saw Metabolic 75
70 Metabolic rate 268
290 Metabolism 267
247
K 251, 301 Methyl benzoate
test 175 Mg 70, 302
31,35,317 245
1,35 Microdrill 291
LA-rCP-MS 301 Microincrement 108,1 362
Lamellar bone 69, 79
31 Microscope slide 1, 370
220 Microstructure 137,1 173
Laser Microtube vial 327,370
Lateral li ne 318 260
151 MilliQ water 351,356
Li 357 Mineralisation 61,70
Life history events 41, 1 Minor element 277
Life-time fitness 175 Mixed-fisheries model 186
358 Mn 258
60 Modal 135
Limi tS of detection (lOD) 298 Monthly 85
78 Mould
100,273 331, 359
vascular canal 74 N 252, 290
Long-lived fish 93, 271 Na 251,254,301
fj Iter 216 Ni 258
L-zone 96 spline-based
Macrostructure 1 314 175
457
Nursery ground 189, 260, 278 Preservation in alcohol 317,329

0 252,257,290 Primary bone 66, 76, 79
Objeccivity 236 Primaryeleccron 289, 366
Observation 33 1,358 Primary incremenr 36,48
OES 287 ,290 Primary vascular canal 74
Oil - clearing 333,371 Pri1lZordimn (pI. primordia) 38
Oil - immersion 333, 359, 363 Producriviry 238
Onion model 105 Prorein 35,251,352
Onrogenetic 103, 189,268 Proteoglycan 66, 280, 354
Onrogeneric transirion 176 Protexx® 35 9
Opercular 78, 328, 358 Proximal 34, 56
Opercular bones 285,333, 358 Ra 252,271,292
Oprical densiry 101,232,358 Radial vascular canal 74
Orienration plane 34,317,319 Radioaccive decay 248
Osteoblasr 66, 74, 76 Radiocarbon 273
Osreoclast 66, 74, 76 RadioisotOpe 248,252, 271
Osteocyre 66,7 5,80 Rad iometric agei ng 250, 272, 291
Oric sac 31, 35 Radionuclide 271,284,291
OtOconia 35 Radius (pI. radii) 58
Otolin 35,251 Rank correlation 173
OtOlith 31 Rare of mineral 53,71
Ova 270 Rb 252
P 252, 254 Reference material
Paleoclimat 88, 280 (SRM, CRM) 286,297,302
Paleotemperarure 248, 280 Regenerared scale 62,318
Para Ilel - fi bre bone 69,80 Relative marginal distance 129
Pb 254,271,302 Reliability 138,356
PCA 278 Repeatabiliry 95, 138
PDB 247,298 Repeared-measures 154, 171
PectOral fi n 26,285,318 Repeated sampling 169,175
Percenrage ag reemeor 138 Reproducrion 86,270
Periost 66,76,83 Reproductive riming 174
Permounr® 349,359 Resin 334
Phoroperiod 50, 123 Resin embedding 334
Physiological relay 87 Res in impregnarion 34 2
PIXE 289,301,350 Resin puriry 355
Planes of extraCtion 317 Resin viscosiry 355
Plexiglass® specimen holder 351 Resolurion power 133, 362
Poikilorhermic 81,84 Resorprion _36, 74, 95
Polarised lighr 69, 323, 364 Resring line 71,76,83
Pollu rion 280 Reversal lioe 72
Pol yester resi 0 334,355 Ridge 36,58,63
Polymeri sarion 336 Ring 38,41,214
Population discri mi narion 276 Rourine 189, 240, 345
Precision 141,2 36,301 S 247,252,256
Pre-recruit survival J 67 Sacot//IS 31,35,5_3
458
31
Salinity 73
Sr 254,261,301
Scale Sr/Ca 288
Scale selection 176
(in 160 261, 291
Scleroblast 59, 66 Stage duration mechanism 168
Seasonal 75, 353
Seasonal increment 92 Standard axis 108
Seasonal merabolic Standardisation 108,
Seasonal variation Stare a study 91, 307
Seasonal zone Starvation 52, 1 ,256
Statoli rh 209
Stock assessment 234
Stock assessment model 180
centre 41, 101 Stock discrimination 235, 276
Stock 299
74 317,
343 Stratum 58
plane 105, Stratyl
Section thickness 345 Srromium 279
Selection of CS 92 Strontium-calcium ratio
SEM (Sr/Ca) 263, 288
Setdemem 335,
Sexual 126
fibres 68 S1Jic1JS ams/jellS

Si layer
demod ulation Sllpra-occipi tal
SIMS Surface
Size at emergence 171 Surface
167 Surface 350, 356
Size-rank 173 Survival pattern 171
Size-selective morcality 171 Swimbladder 35
Skeletal bone 65 test 140
Skin 116 Synchronizer 82
SLAP 247 114,127,280
Srn 279 TC fibres 60
SMOW 247, ,298 Teetb 27,
Source of error 293
211,214 Temperature
event
Tb
16 (glue,
27 Time scale
459
TlMS
TI 252
Toluidine blue
examination
Trace element 290
Transverse 34
79
55
U
Ulrrasonic bath
Utrim/lis 31,35
Validation 114,273
Vascular bone 73
Vascular canal 7 76
Vascular 67
Vaterire
Verification
VPDB
VSMOW
WDS 287,
Weber
Wet
Whirlockite
WIP
Woven-fibre bone
WSP
Xylenolorange 117
Year class 192
Yolk sac 42
Yolk-sac stage 171, l76
Zn 259, 302
taxa and common names
Acanrhurids 51 Cod 121,171

84 Cod 99, 177
285 Cod larvae 171
70 Cod Pacific 138
273 Cod juveniles 171
Alosa 278 Cod setded 1 1
A/osa spp, 260 Coelacanrh 63
Amiidae 58 Coelacanthidae 58
41,101,178 C%ssoma macropommlt 131
26 59,279
1, , 119 39,
269 Coumarou 86
260 278
Anguilliformes 320
273
Anostomidae 85
mirmlt't 39
85
67,81,85 Dicentrarchm labrax
270 Dipnoi 58
83
Balistes 84 77
Balistidae 92 Eel 119,1
45 41
Bichir 64 guttattts 263
Bream 125
BrctJooYtia /Jatmnl1s 261,285 Esox it,cius 285
Brevoortia tyranntIJ 173 Flatfish 101
Brown trout 123, 172, 1 Freshwater fish 85,103,255
Callichthyidae 83 Ftmdulm heteroditllS 285
177 Gadidae 77,321
77 Gadoids 107
Carassim auratus 50 Gadus morhua J, 51,99
82 Gasterostetts aculeatlfs 124
Catfish 46
Characiformes G ireila efevata 256, 263
319 Haemulidae 7.3
1 Haenm/on
41,101 :Hake 106
Clupeid 337 Halichoeres
461
Manual of fish sclerochron ology
H ererodonri fo rmes 27 Pagrm clUmtlls 275,280,285

Himndichthys af/inls 273 PagritS major 263
HuplosterntlrlZ littorale 40,74, 83 Paftgassius hypophthalmlls 109,36 1
H oplostethlls atlantlws 100,177, 207 Pe lagic 34,46, 92
H orse m ac kerel 137 Perch 26, 66
Hygophl1171 46 Percifo rmes 67,83
lsriop horids 92 Phori chthyidae 33,3 7,47
KatsIIUJOn/JS pel?!1nis 72 ,84 P laglosc/on sq1la1llosissillZlts 79
Lat/meria 74 Plaice 44 ,101 ,134
Lepolllis macrochi r 48,49 Platichthys stel/cIl!!s 48
LejJorinllS /riderici 60,85,86 Plettronectes platessa 41,46, 48
Leptocepha lus 260, 269 Pod os temonacae 86
Lesser sand ed 101,178 Poeci/ia ret/wlclta 270
Lethri liltS lIebltioSIJJ 67, 69, 83 Pogon/as cromis 276
Loligo pedei 209 Pol/achillJ pol/ClchillS 219
LO/lhim 84 Pollachilts virem 42 ,224
Llitjafllls sebae 302 Pollock 219
LlltJanllS sfi/!. 273 PolY/lterm bichir 72
l'vIaertlrorllts novaezelcmdiae 273 ,277 Pomacentms coelestis 42
iHal/otlls viiloslls 177 Pomc/dasys hasta 73
Megaleehls thoracata 40 Porich/hys notatltS 49
MdanogrammllS etegle/in/Is 41, 15 1 POLlting 226
Merlangllls merlcmglls 49,98,191 R ep tiles 75
i'vler/lleeiLtS cajJemis 46 Salmo setlm' 39,48,50
M er/!lccim mer//iccl IIJ 41 Sallllo trtlllcl 123,172,177
f\'lieropogollim Itrldltlettlls 266 Salmon 24, 62, 63
M ierost017tIIS paci/ims 101 Salmo n Atlamic 172,37 0
Monkfis h 84 Saumo n chi nook 177
iV! orone sa xati/Is 260, 263 Saumon masu 17 3
Mligil cephalllJ 74 Salmo nid 42,63 ,87
Myctophids 48 Setl/la salpa 140
M yle/ts rhomboidalis 78 , 85, 86 Salvelirtlts alpinllS 259,276,285
N on-Ostariophysean 31 9,323 Sarcoprerygii 70
OncorhYllchm gorbllSchet 41 Sarotherodon melcmotheron 115 ,119
On{()rhynchlls keta 41, 280 Sciaenidae 79
Om orhYllehlls kistltrh 125,279 SeiCleno/ls oullatllS 25 6, 263
Oncorhynchlls mClStI 17 3 SromberOJllorus cauCllla 281,285
Oncorhynchlls nerka 41, 48, 159 Scombridae 32 3
Onwr/Jynchm tshmuytseha 48, 177 Seo/lhthalmll.f IJlaximm 42,256
Orange roughy 177,207 Sebasles entomelcls 51
Oreochromis f/ i!otlctlJ 4l,85, 110 Sebastes Jordani 42
Osrariophysean 32, 34, 35 Sebastes ru{f.tS 238
Osreic hrhyan 23, 26 Sebastes spp. 100, 27 3
462
Se/af boo!}s 1
Serrasalmidae 85, 131
Shark 27,1 285
Si! uriformes 40,67,85
84
So/ea so/ea 224
138
338, 372
338,372
Squid 209
SttZOlledion ! u"'flIW'YFa 119
Sturgeon 84,87,284
Swordfish 92,320
Teieosrs 25,31,35
Temperate
Tent/ailfst:! fofi
Thunnidae
ThltfllUIS
Thmml/J 280
Thmm/fJ 285
ndotica 50
Trachuftts mediterranem
67
Tuna
lUmbar/a
Viper
191,207
Wrasse 66
463
Published by XLC, Le Re[ccq Kerhuon , Fran <.."<..'
Primed in Fran<"T a( C ;dhgril phy, Renoes
L..:ga l depos H: VJ CJuarrt r, 2002
Cove r designer: Adanrid e, I3rts r, France

Manual of Fish Sclerochronology
Sclerochronology, the study of calcified structure s to re-
construct the past history of living organisms, is central to
fi sh biology and fi sheries management. This manual aim s
to provide an overview of the current theoretical and prac-
tical aspects of sclerochronological studies. By providing
information on the nature of calcified structure s (otoliths,
scale s, skeleton), their uses in fi sh re search and method s
for preparation and examination, the manual constitutes
a comprehen sive guide for researcher s, technicians and
students either new to the field or interested in expanding
their range of expertise. The enclo sed multimedia version
(DVD) is supplemented by videos illustrating the main tech-
nical proc edures with an alternative naviga tion mode
ba sed on decision trees.
Keywords: otolith, scale, skeleton, age, growth, image

analysis, microchemistry.
Manuel de sclerochronologie
des poissons
La sc /ero chronologie , discipline qui etudie le s pieces
calcifiee s pour re construire I'histoire individuelle des or-
ganismes vivants, est essentielle pour la connaissance
de la biologie des poissons et la gestion des peches_Ce IRD Editions
213, rue La Fayette
manuel presente une synthese actualisee sur les aspects 75480 Paris Cedex 10, France
theoriques et pratiques des etudes de sclerochronolo- Tel. +33 (0)] 4803 76 06
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ca/Cifiees (otolithe , ecaille, squelette), leur utilisation
dan s le s re cherches en ichtyologie et les methodes de Diffusion
preparation et d'ob servation, ce manuel constitue un IRQ
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de sequences videos et beneficie d'un mode de naviga-
tion alternatif base sur des arbres d'aide a la decision. ISBN 2-7099-1490-5
II~ ~I ~~~ ~II~ 11111~ ~11111

Mots-c/es: otolithe, ecaille, squelette, age, cro;s-
sance, analyse d';mage, m;croch;m;e_
9
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Tel. +33 (0)29822 4013
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ISBN 2-844 33-067·3
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Manual of fish

In homage to H erve Troadec ,

righrs No be reproduced, scored (0 r<.'cric:val or (rans~

mmed, in any form or mf"chanical, photocopying,

© Edirions Ifremer, 2002

Chapter 11 - Types of calcified structures

1.1. Constituents of bony tissue 66

r ' .... ~."~ IV Validation and verification methods

A. Direct validation 114

1.2.1. Fluorescent 116

Ch:~nh~r V - Some uses of individual data

A. Growth and 146

B. Ecological applications 167

Chapter VI - Computer-assisted age estimation

1.2.2.3. Spectral filtering: the Fourier transform 216

Chapter VII - Otolith microchemistry

H. de Poncual, A.J. Geffen

2. Metabolism and ontogenic events 267

Chapter VIII - Preparation and observation techniques

A_Aid to decision trees 308

3.3. Otolith and conservation

1.1.4. Illumination 359

1. Conservation of calcified structure preparations 370

some consider that

In Europe, the Union financially the concerred

The breadth and depth of this coorribmion will make it

The rev iewers

John M. Casselman Er/end Moksness

42 videos which illustrates the main technical procedures, interac-

the term is derived from the Greek

Calcified structures have the potential to grow the life of

The assessment of individual age and through CS

What does each CS record? ----~

As permanent CS constituce more or less

whose idemity is sometimes anything bur obvious. Moreover, infor-

What are the methods employed to reveal the information required?

Data provided by cs an alysis result from an acquisition process in

Are the data obtained accurate and precise?

Are the chemical patterns recorded informative

The idea that CS possess chemical tags or "chemo-prints" was recog-

which is particularly difficult to assess, as well as those of interpreta-

How does this work?

Despite a rather long hisrory and development, sclerochronology is far

The literature on fish cs is enormous: for instance the ASFA (Aquatic

Estimates of the age of individual living organisms are needed in

the various stages of Kunrzman (1824), later challenged the

periphery of the scale

salmon. these pioneer

Although in its early stages at the of the 20th

The first works on sclerochronology for Chondrichthyans are much

Calcified structures have different

The inner ear, which is found in all functions both

mechanoreception . The lumen of the entire system is filled with

Figure lI.A.l Asteriscus

used in the reconstruction and

Ocoliths are formed extracellularly from the crystallisation of the

Otoliths exhibit a range of incremental structures that are often

1 ocoliths do not appear [0 be subject co mineral

2.1. Primary increments

Sub-daily increments and discontinuities in the increment record may

somatic growth. Experiments have shown that otoliths continue to

2.l.2. First ring

be formed on the day of hatching, and may be properly termed a hatch