Environmental biology of fishes, tập 95, số 4, 2012

Environ Biol Fish (2012) 95:415–418
DOI 10.1007/s10641-012-0076-4

Chemical signatures of otoliths and application in fisheries
Yongwen Gao & David L. G. Noakes

Received: 23 June 2012 / Accepted: 20 July 2012 / Published online: 1 August 2012
# Springer Science+Business Media B.V. 2012

Keywords Stable isotopes . Trace
elements . Carbonate . Equilibrium conditions

The very successful 141th American Fisheries Society
Annual Meeting was held in Seattle from September
4–8, 2011. During the meeting we organized an otolith
symposium titled “Chemical signatures of otoliths and
application in fisheries”. Twelve oral and three poster
presenters from China, Japan, Norway, and United
States were presented in the session, and contributed
as manuscripts for publication in a special issue of
Environmental Biology of Fishes.
Why do we think this is important? First, the chemical signatures of otoliths have received increased
attention in recent years because otoliths contain a
great deal of information about the life history of the
fish and that information can be extracted from stable
isotope and trace elemental analyses. Second, although otolith chemistry has been investigated since
the 1960s (e.g., Lowenstam 1961; McIntire 1963;
Devereux 1967; Degens et al. 1969), very few symposia have concentrated specifically on the topic.
Y. Gao (*)
Makah Fisheries Management,

P.O. Box 115, Neah Bay, WA 98357, USA
e-mail:

Therefore, we think it is important and timely to
highlight our AFS otolith symposium and publish the
presentations.
Otoliths are mm-sized, laminated calcium carbonate structures (CaCO3) found in the inner ears of
teleost fish (Carlstrom 1963). They grow from a fluid
medium (endolymph) that mainly contains calcium
ions Ca2+ and bicarbonate HCO3- (Schwarcz et al.
1998). From the mineralogical perspective, carbonates
consist of 3 minerals: rhombohedral calcite and dolomite, and orthorhombic aragonite. Aragonite is unstable in nature and commonly inverts to its polymorph
calcite (Nesse 1986). During the precipitation and
physicochemical processes minor or trace elements
are incorporated into carbonate mineral phases by
substituting for Ca2+ in the crystal structure. The divalent metal cation M2+ in the calcite group occupies
the octahedral sites with 6-fold coordination, whereas
M2+ in the aragonite group occupies the triangular
sites with 9-fold coordination (Berry et al. 1983;
Zheng 1999). Thus trace elements with larger ionic
radii, such as Sr2+, Na+, and Ba2+, are preferentially
incorporated into orthorhombic aragonite, whereas
rhombohedral calcite is enriched in smaller ions, such
as Mg2+, Fe2+, Mn2+, Zn2+, and Cd2+. At normal or
low temperature conditions this substitution is governed by a distribution coefficient (df) of a special
trace element between liquid and solid phase that can
be expressed by:

Y. Gao
College of Fisheries, Huazhong Agricultural University,

Wuhan, Hubei 430070, China

ðm Me=m CaÞS ¼ df ðm Me=m CaÞL

D. L. G. Noakes
Department of Fisheries and Wildlife,
Oregon State University,
Corvallis, OR 97331, USA

at equilibrium (i.e., constant temperature and pressure; df01) and no concentration gradients between
the two phases (McIntire 1963; Brand and Veizer

ð1Þ


416

Environ Biol Fish (2012) 95:415–418

1980). In this equation, Me represent the trace
elements expressed in moles (m) in the solid (S)
and liquid (L) phases. At non-equilibrium conditions, however, the relationship will be changed to:
logðm MeI =m MeF Þ ¼ df logðm CaI =m CaF Þ

ð2Þ

where I and F are the initial and final concentrations of trace elements and Ca in solution (Gordon
et al. 1959; Veizer 1983). When df>1, the Me will
be enriched in the carbonate solid phase relative to
the Me/Ca of the liquid phase; when df<1, the Me

will be depleted in the carbonate solid phase.
The above statement is the essential theory for
carbonate element geochemistry, which is also applied to fish otoliths. However, the most challenging issue in elemental studies is how to interpret
the trace elemental data obtained from otoliths.
Because many factors, such as ion size, valence,
coordinate number, electronic potential, temperature,
crystal structure, and so forth, affect the elemental
partitioning coefficient or df (McIntire 1963), it is
often hard to determine which factor is distinctive
or dominant in the bio-geochemical process. For
example, the Sr/Ca ratios of otoliths are useful
tracers for freshwater and estuary settings, but less useful in the marine environment because the Sr2+ distribution is homogeneous in the world’s oceans. Many
fishery biologists have realized this weakness and turn
their attention to stable isotopes in otolith studies.
In stable isotopic analysis, we measure carbon and
oxygen isotope ratios (13C/12C or δ13C; 18O/16O or
δ18O) of otoliths by analyzing the CO2 molecule from
aragonite:
CaCO3 ) CO2À
3 ! CO2

ð3Þ

where CO2 is the gas purified from the reaction between aragonite powder and 100 % phosphoric acid.
The theory and practice of using otoliths in stable
isotope analyses are rooted in Urey’s hypothesis
(1947) and the subsequent experiments that calcium
carbonates are precipitated in oxygen isotopic equilibrium with the surrounding waters in which the
organism (including fishes) lived, and thus they
retain the isotopic records of the life history of

the animal (e.g., Epstein et al. 1953; Friedman
and O’Neil 1977; Grossman and Ku 1986; Kalish
1991). Therefore, stable oxygen isotope ratios
(18O/16O) extracted from otoliths can provide unique

information about habitat alteration, water temperature,
and decadal-scale ecosystem changes that an individual
fish encountered (Devereux 1967; Nelson et al. 1989;
Gao 2002; Gao and Beamish 2003).
A major concern in isotopic application is how well
or how accurately we can use the oxygen isotopic
temperature scale to estimate the environmental conditions. From the famous isotopic temperature equation for biogenic carbonate (Epstein et al. 1953):
Tð CÞ ¼ 16:5 À 4:3ð d S À d W Þ þ 0:14ð d S À d W Þ2
ð4Þ
we see the oxygen isotopic temperature scale was presented as a function of the difference between oxygen
isotope ratios in carbonate sample and in seawater:
where δS is the δ18O value of solid carbonate (e.g.,
shells, corals, otoliths), and δW is the δ18O value of the
seawater in which the carbonate precipitated. We can
measure δS from otoliths, but have no direct means to
measure δW to which the fish was exposed. Furthermore, if there were changes in δW these would lead to
“apparent” changes in isotopic temperatures even
though temperature had remained constant (Gao
2008). Such changes in δW are in fact occurring in many
estuary settings, as a result of changes in salinity. Due to
the fact that salinity is not an independent variable in the
process of oxygen isotopic fractionation and the δWsalinity relationship varies from area to area (Craig and
Gordon 1965), using salinity to estimate δW generally
encounters many challenging problems.
Fortunately, Ghosh et al. (2006a; 2006b) reported a

new paleothermometer based on the ordering of 13C
and 18O into bonds with each other in the carbonate
mineral lattice. The new thermometer constrains temperature without needing to know the δW from which
the carbonate precipitated, or anything else besides
carbonate. By measuring the δ18O, δ13C, and abundance of Δ47 isotopologues (mostly 13C18O16O) from
the carbonate and phosphoric acid produced CO2, we
can calculate the enrichment of Δ47 isotopologues and
temperature (T) from the equation:
T2 ¼ 59200=ðΔ47 þ 0:02Þ

ð5Þ

where Δ47 is in permil (‰) and T in Kelvin.
Stable carbon isotope ratios (13C/12C), in contrast,
are not precipitated in equilibrium conditions with the


Environ Biol Fish (2012) 95:415–418

417

ambient waters and the basic principle is that: “you are
what you eat, plus a few permil” (DeNiro and Epstein
1976; 1978). Schwarcz et al. (1998) proposed that the
carbon isotopic composition of cod otoliths was in
isotopic equilibrium with the dissolved inorganic carbon (DIC) of endolymph, and used the following
equation:

d 13 Coto ¼ M d d þ ð1 À M Þ d sw þ 2

ð6Þ

to estimate the M-values in cod otoliths (M00.12~
0.43, depending on cod life stages): where M is the
fraction of metabolic carbon in the blood HCO3-; δd
and δsw are the diet changes for cod and inorganic
carbon from seawater, respectively. Solomon et al.
(2006) manipulated the δ13C of food and ambient
DIC in a factorial design with juvenile rainbow trout,
and experimentally determined that 80 % more δ13C
of otoliths were derived from DIC.
Even though there are significant differences between stable isotopes and trace elements in theory and
analyses (e.g., McIntire 1963; Friedman and O’Neil
1977; Wefer and Berger 1991), the two geochemical
tools have often been used together. Lowenstam
(1961) combined 18 O/ 16 O ratios and SrCO 3 and
MgCO3 contents in both brachiopod shells and seawater, and concluded that temperature affects 18O/16O,
and Sr/Ca and Mg/Ca ratios. In their experiments on
inorganically precipitated aragonite, Kinsman (1969)
and Kinsman and Holland (1969) reported an inverse
relationship between Sr contents and ambient temperature. These results encouraged some authors to use
both stable isotopes and trace elements for the same
otolith (e.g., Gauldie et al. 1995). Nevertheless, Gao et
al. (2005) found that stable isotopic data are more
sensitive and powerful than trace element concentrations in Pacific cod (Gadus macrocephalus) otoliths.
Since fisheries biologists generally combine the two
methods as “Microchemistry”, we use “Chemical signatures” of otoliths in this special issue.
This special issue includes manuscripts from both
stable isotope and trace element analyses in otoliths,
covering many interesting applications in fisheries

science and management such as experimental and
field oxygen isotopic temperature scales (Geffen; in
this issue, similarly hereinafter), identification of marine and anadromous fish stocks (Gao; Dou et al.), fish
migration and habitat use (Dou et al.; Chang et al.),
and otolith marking techniques (Hobbs et al.). In

particular, a couple of manuscripts from China represent the first study on chemical signatures of otoliths
in application to special Chinese fishes (Shen and
Gao; Liu et al.). By publishing this issue, we hope to
attract more fishery biologists and managers to use the
isotopic and elemental tools of otoliths for fisheries
issues and applications. Although ageing and experimental studies are still popular, we believe novel and
chemical investigations on multi-species of fish and
multi-tracers of elements should be the direction in
future fisheries science and management.

References
Berry LG, Mason B, Dietrich RV (1983) Mineralogy: concepts,
descriptions and determinations. WH Freeman & Co, San
Francisco
Brand U, Veizer J (1980) Chemical diagenesis of a multicomponent carbonate system-1: trace elements. J Sediment
Petrol 50:1219–1236
Carlstrom D (1963) A crystallographic study of vertebrate otoliths. Biol Bull 124:441–463
Craig H, Gordon LI (1965) Deuterium and oxygen-18 variations
in the ocean and the marine atmosphere. In: Tongiorgi E
(ed) Stable isotopes in oceanographic studies and paleotemperatures. Consiglio Nazionale delle Ricerche, Laboratorio di Geologia Nucleare, Pisa, pp 9–130
Degens ET, Deuser WG, Haedrick RL (1969) Molecular structure and composition of fish otoliths. Mar Biol 2:105–113
DeNiro MJ, Epstein S (1976) You are what you eat (plus a few
permil): the carbon isotope cycle in food chains. Geol Soc
Am, Abstracts with Programs 8:834–835

DeNiro MJ, Epstein S (1978) Influence of diet on the distribution of carbon isotopes in animals. Geochim Cosmochim
Acta 42:495–506
Devereux I (1967) Temperature measurements from oxygen
isotope ratios of fish otoliths. Science 155:1684–1685
Epstein S, Buchsbaum R, Lowenstam HA, Urey HC (1953)
Revised carbonate-water isotopic temperature scale. Geol
Soc Am Bull 64:1315–1326
Friedman I, O’Neil JR (1977) Compilation of stable isotope
fractionation factors of geochemical interest. In: Fleischer
M (ed) Data of geochemistry. US Geol. Surv. Prof. Paper,
440-KK
Gao YW (2002) Regime shift signatures from stable oxygen
isotopic records of otoliths of Atlantic cod (Gadus
morhua). Isotopes Environ Health Stud 38:251–263
Gao YW (2008) Decadal-scale stable oxygen isotopic variations
in seawater and implications for otolith thermometer studies. Am Fish Soc Symp 49:1545–1556
Gao YW, Bargmann GG, Brand U, Noakes DLG (2005) Stable
isotopic and trace elemental compositions of otoliths and
the stock structure of Pacific cod (Gadus macrocephalus).
Environ Biol Fish 74:335–348
Gao YW, Beamish RJ (2003) Stable isotope variations in otoliths of Pacific halibut (Hippoglossus stenolepis) and


418
indications of the possible 1990 regime shift. Fish Res
60:393–404
Ghosh P, Adkins J, Affek H, Balta B, Guo W, Schauble EA,
Schrag D, Eiler JM (2006a) 13C-18O bonds in carbonate
minerals: a new kind of paleothermometer. Geochim Cosmochim Acta 70:1439–1456
Ghosh P, Garzione CN, Eiler JM (2006b) Rapid uplift of the

Altiplano revealed through 13 C-18O bonds in paleosol carbonates. Science 311:511–515
Gordon L, Salutsky ML, Willard HH (1959) Precipitation from
homogeneous solution. John Wiley, New York
Gauldie RW, West IF, Coote G, Merrett N (1995) Strontium
variation and the oxygen isotope water temperature record
in the otoliths of the deep water fishes Trachyrincus murrayi and Coryphaenoides mediterraneus. Comp Biochem
Physiol 110A:271–280
Grossman EL, Ku TL (1986) Oxygen and carbon isotope fractionation in biogenic aragonite: temperature effects. Chem
Geol 59:59–74
Kalish JM (1991) Oxygen and carbon stable isotopes in the
otoliths of wild and laboratory-reared Australian salmon
(Arripis trutta). Mar Biol 110:37–47
Kinsman DJJ (1969) Interpretation of Sr2+ concentration in carbonate minerals and rocks. J Sediment Petrol 39:486–508
Kinsman DJJ, Holland HD (1969) The co-precipitation of cations with CaCO3-IV: the co-precipitation of Sr2+ with
aragonite between 16°C and 96°C. Geochim Cosmochim
Acta 33:1–17
Lowenstam HA (1961) Mineralogy, 18O/16O ratios, and strontium
and magnesium contents of recent and fossil brachiopods and
their bearing on the history of the oceans. J Geol 69:241–260

Environ Biol Fish (2012) 95:415–418
McIntire WL (1963) Trace element partition coefficients: a
review of theory applications to geology. Geochim Cosmochim Acta 27:1209–1264
Nelson CS, Northcote TG, Hendy CH (1989) Potential use of
oxygen and carbon isotopic composition of otoliths to
identify migratory and non-migratory stocks of the New
Zealand common smelt: a pilot study. NZ J Mar & Freshw
Res 23:337–344
Nesse WD (1986) Introduction to optical mineralogy. Oxford
University Press, New York

Schwarcz HP, Gao Y, Campana S, Browne D, Knyf M,
Brand U (1998) Stable carbon isotope variations in
otoliths of cod (Gadus morhua). Can J Fish Aquat
Sci 55:1798–1806
Solomon CT, Weber PK, Cech JJ, Ingram BL, Conrad ME,
Machavaram MV, Pogodina AR, Franklin RL (2006) Experimental determination of the sources of otolith carbon
and associated isotopic fractionation. Can J Fish Aquat Sci
63:79–89
Urey HC (1947) The thermodynamic properties of isotopic
substances. J Chem Soc 1947:562–581
Veizer J (1983) Chemical diagenesis of carbonates: theory and
application of trace element techniques. In: Arthur MA,
Anderson TF, Kaplan IR, Veizer J, Land LS (eds) Stable
Isotopes in sedimentary geology. SEPM Short Course 10.
pp 3.1–3.100
Wefer G, Berger WH (1991) Isotope paleontology: growth and
composition of extant calcareous species. Mar Geol
100:207–248
Zheng YF (1999) Oxygen isotope fractionation in carbonate and
sulfate minerals. Geochem J 33:109–126


Environ Biol Fish (2012) 95:419–430
DOI 10.1007/s10641-012-0033-2

Otolith oxygen and carbon stable isotopes in wild and laboratoryreared plaice (Pleuronectes platessa)
Audrey J. Geffen

Received: 26 September 2011 / Accepted: 27 April 2012 / Published online: 26 May 2012
# Springer Science+Business Media B.V. 2012

Abstract The relationship between water temperature,
growth rate, and otolith isotopic ratios was measured for
juvenile plaice (Pleuronectes platessa) reared at two
temperatures (11 and 17°C) and two feeding regimes
(1 and 3 prey items·ml−1). The otolith isotope ratios in
individual fish ranged from −2 to −4 for carbon isotope
ratios (δ13C) and from 0.2 to 1.9 for oxygen isotope
ratios (δ18O). The otolith oxygen isotope ratios were
significantly affected by water temperature, but not by
feeding level, and there were no significant synergistic
effects. The fractionation of oxygen isotopes during
otolith growth was independent of individual growth
rate. Carbon isotope ratios were not significantly affected by food ration or water temperature, but were related
to fish growth rate. The carbon isotope ratios were
negatively correlated with fish length in the colder water
treatments, and tended to increase with fish length in the
warm water treatments. The laboratory-determined relationship between otolith oxygen isotope ratio and water
temperature was applied to individuals of five species
(plaice, cod, whiting, haddock, gurnard) collected in a
single trawl sample. The otolith derived temperatures
A. J. Geffen (*)
Department of Biology, University of Bergen,
PO Box 7803, Bergen 5020, Norway
e-mail:
A. J. Geffen
Institute of Marine Research,
PO Box 1870, Nordnes,
5817 Bergen, Norway

often overestimated measured water temperatures. The
difference between real and estimated water temperatures varied between species, and the closest fit was for
field-caught plaice.
Keywords δ13C . δ18O . Otolith isotope fractionation .
Irish sea . Flatfish . Temperature proxy

Introduction
In recent years ecologists have become increasingly
interested in techniques which may be used to investigate the effects of environmental change (particularly
temperature) on the growth rates of marine organisms.
The temperature regime experienced throughout the life
of an individual impacts directly on patterns of reproduction and growth. In the study of fish biology, knowledge of the temperatures experienced by individuals can
be used to track migration patterns, and to construct
predictive models of growth and production. Such
information can be of enormous economic value, where
understanding fish migration and growth is of high
priority in the management of commercial fish stocks.
Obtaining reliable environmental temperature data
for large numbers of mobile marine animals such as
fish is difficult. However, otoliths grow continuously
throughout the lifetime of a fish, and it is possible to
generate a lifelong record of temperature change specific to that individual, based on the oxygen isotope
ratios (Weidman and Millner 2000; Gao et al. 2005;


420

Weidel et al. 2007). The use of otolith isotope measurements for temperature estimation depends on the
assumption that the fractionation of the oxygen isotopes during the precipitation of biogenic carbonate is
governed by predictable quantum energy equilibrium

conditions determined solely by the physicochemical
properties. Otolith δ18O increases inversely with temperature, but there are important processes that may
affect oxygen isotope ratios, and thus the temperature
estimates, from biogenic carbonate. The first factor is
the possibility of “vital effects” which encompass both
species-specific differences in the temperature–isotope
relationship, and physiological processes that can potentially affect the preferential precipitation of isotopes. Departures from equilibrium are commonly
attributed to biochemical and metabolic factors resulting in modification of the isotopic signatures preserved in calcium carbonate. Variations in growth
rate, for example, may cause changes in precipitation
kinetics and incorporation of respiratory carbon dioxide in addition to the marine dissolved inorganic carbon reservoir normally utilized during calcification.
Under these circumstances the isotopic record preserved by aragonite is no longer simply a function of
temperature variation and will reflect physiological
factors, which may be organism specific. As a consequence it is critical to test the assumption of equilibrium isotope fractionation, and especially to be able to
test independently the relative influences of individual
metabolism, or growth rate, and temperature.
Many studies of the isotopic composition of fish
otoliths focused on the use of these ratios in fossil
otoliths to predict paleotemperatures (Patterson et al.
1993; Smith and Patterson 1994; Picard et al. 1998).
The use of isotopic ratios to investigate aspects of the
biology of extant fish populations has primarily been
based on surveys of field collected samples, often from a
wide range of habitats (Devereux 1967; Iacumin et al.
1992; Meyer-Rochow et al. 1992). These studies relied
on the use of temperature–isotope relationships derived
from invertebrate studies (Epstein and Mayeda 1953;
Grossman and Ku 1986). Only one study seriously
analysed the accuracy of the resulting temperature estimates (Weidman and Millner 2000) and in this case
overestimates of temperature were common. Phylogenetic, age and life history stage, or metabolic differences
between species, populations, and individuals may affect

the stable isotope ratios (Kalish 1992; Newman et al.
2000; Gao et al. 2005). Investigations of several groups

Environ Biol Fish (2012) 95:419–430

of organisms such as bivalves (Dare and Deith 1991;
Mitchell et al. 1994); coccolithophores (Paull and Balch
1994); bryozoans (Crowley and Taylor 2000); and fish
(Rooker et al. 2008) have shown that oxygen isotope
ratios do not always reflect equilibrium fractionation. As
a consequence it is critical to test the assumption of
equilibrium isotope fractionation, and especially to be
able to test independently the relative influences of individual metabolism (or growth rate) and temperature.
Validation experiments allow for control or monitoring of important variables that can introduce added
variability, such as individual growth rate or the isotopic ratio of the water in the experimental tanks. Most
experimental studies on fish have found a good relationship between temperature and oxygen isotope ratios (Thorrold et al. 1997; Hoie et al. 2004; Godiksen
et al. 2010). Carbon isotope ratios are often assumed
to be more influenced by growth and metabolism than
by ambient temperature (Kalish 1991a, b; Wurster et
al. 2005; Weidel et al. 2007; Shiao et al. 2009), and
this has been confirmed experimentally in several
species (Thorrold et al. 1997; Solomon et al. 2006;
Tohse and Mugiya 2008).
Thorrold et al. (1997) used an experimental
approach to determine that oxygen isotope ratios were
a reliable indicator of temperature in Atlantic croaker
Micropogonias undulatus, and were relatively independent of individual growth rate. Similar experiments
have shown equilibrium precipitation of oxygen isotopes in the otoliths of cod Gadus morhua (Radtke et
al. 1996; Hoie et al. 2004), and Arctic charr Salvelinus
alpinus (L.) (Godiksen et al. 2010), also with no

evidence for growth rate effects. However, the fractionation equations differ markedly between studies,
and it is difficult to reconcile the differences in part
because the temperature ranges in these studies do not
overlap.
In view of the interest in temperature effects on marine fish, it is crucial to broaden the experimental approach to a wider range of species. Plaice (Pleuronectes
platessa L.) is an important commercial species in the
northeast Atlantic, with a relatively restricted thermal
range. Juvenile plaice inhabit shallow sandy nursery
grounds and are subject to high and variable temperatures during their first year of life (Nash et al. 2007).
Annual variation in growth rate and the subsequent
variation in recruitment and productivity are often linked
to temperature regimes (Geffen et al. 2011), but direct
evidence linking the temperature experienced as


Environ Biol Fish (2012) 95:419–430

juveniles to later growth is lacking. Temperature estimates from the juvenile portion of plaice otoliths could
prove useful, as shown for Arctic charr by Godiksen et
al. (2011).
Temperature dependent isotope fractionation has
not been tested yet in any flatfish species. Some variability may be expected in this group because of the
higher organic content often found in their otoliths
(Richter and McDermott 1990). Therefore, experimental validation of the use of oxygen isotope ratios
is advisable for application of this technique to field
studies. The present study examined the effects of
water temperature and growth rate on the δ18O and
δ13C values of juvenile plaice otoliths. The fractionation of oxygen isotopes during calcification should be
independent of metabolic processes for the reliable use
of isotope ratios as a measure of the sea water temperature experienced by individual fish. Two different

temperatures and two different feeding rates produced
a group of fast- and slow-growing animals at each
temperature. In this way, the effect of growth rate for
each individual was measured independently from the
effect of temperature to quantify the extent to which
metabolic processes can alter the relationship between
isotopic composition and temperature. In addition, the
data provided information on the level of accuracy and
precision to be expected for estimates of seawater
temperature calculated from oxygen isotope measurements for this species.
The experimentally determined relationship between otolith oxygen isotope ratio and temperature
was then applied to a sample of local field-caught fish.
The accuracy of the temperatures estimated from the
otolith isotope ratios was evaluated by comparing the
deviation between estimated and measured temperatures. The precision of the temperature estimates was
evaluated by comparing the variability within and
between fish species.

Materials and methods
Laboratory experiments
Pleuronectes platessa eggs were obtained from a captive broodstock and incubated at 10°C from May–
October 1996. After hatching, 500 larvae were stocked
into eight cylindrical 200l black plastic tanks. There
were four experimental treatments, each replicated in

421

two tanks. Two water temperatures (11 and 17°C)
were tested, with two feeding regimes (1 and 3 prey
items·ml−1) at each temperature. The temperatures

were controlled by a continuous flow of heated or
cooled seawater from thermostatically regulated header tanks. The larvae were fed with newly hatched brine
shrimp (Artemia salina) nauplii. The two different
temperatures and two different feeding rates produced
a group of fast- and slow-growing animals at each
temperature.
Water temperatures were monitored with continuous
temperature data loggers (Orion TinyTalk mkII, RS
Components) submerged in each tank. Water samples
were taken at intervals for determining salinity, oxygen
and carbon isotope ratios (Table 1). The warm tanks (17°
C) were emptied after 4 months and the cold tanks (11°C)
after 6 months. At the termination of the experiment the
largest plaice were 70 mm and the smallest 40 mm.
Field sampling
The oxygen isotope ratio–temperature relationship
established in the laboratory experiment on plaice
was tested on a sample of field caught fish collected
locally. Individuals of five fish species were collected
from a single bottom trawl sample in the eastern Irish
Sea, on the east coast of the Isle of Man in November
1995. Bottom water samples were taken at the end of
the trawl tow. The fish were measured, weighed, and
the otoliths removed and stored for 1 month in dry
envelopes. After cleaning in bleach to remove any
surface material, the most recently deposited otolith
layer was removed with a dentist’s drill, and the powder simultaneously collected on poly-carbonate filter
paper with a fine vacuum nozzle attached to the drill.
The otolith sample from each individual fish was
analysed separately, and it was anticipated that the

temperature estimates obtained would show broad
agreement both within and between species. The water
temperature experienced by the fish was estimated for
each individual by using the measured otolith oxygen
isotope ratio, and the measured water oxygen isotope
ratio. Four different equations were used; the relationship established in the plaice laboratory experiment,
Thorrold et al.’s warm water fish relationship (1997),
Hoie et al.’s cod relationship (2004), and Godriksen et
al.’s charr relationship (2010). The deviation between
the estimated temperature and the average water temperature for that depth (Table 4) was calculated for


422

Environ Biol Fish (2012) 95:419–430

Table 1 Salinity (psu) and oxygen isotopic ratios of water samples taken at intervals during the juvenile plaice experiment. Replicate
tanks of each treatment are shown
Treatment

Cool
low food 1

Cool
low food 2

Cool
high food 1

Cool

high food 2

Warm
low food 1

Warm
low food 2

Warm
high food 1

Warm
high food 2

34.58

34.58

34.59

34.59

34.68

34.13

34.58

34.73

0.2

0.12

0.19

0.14

0.27

0.41

0.17

0.23

34.58

34.59

34.61

34.61

34.65

34.65

34.68

34.68

.16

0.17

0.25

0.19

0.18

0.15

0.14

0.15

34.60

34.61

34.62

34.61

34.69

34.70

34.71

34.73

.23

0.18

0.17

0.22

0.25

0.15

0.25

0.26

34.62

34.69

34.81

34.78

0.10

0.20

0.25

0.23

Sample date
22/05/96
Salinity
δ18O
01/06/96
Salinity
δ18O
11/06/96
Salinity
δ18O
22/07/96
Salinity
δ18O
25/07/96
Salinity

34.66

34.66

34.66

34.66

34.77

34.78

34.78

.11

0.16

0.21

0.15

0.08

0.24

0.23

34.73

34.63

34.69

34.64

0.21

0.23

0.19

0.13

δ O
18

16/10/96
Salinity
δ18O

each individual, each species, and each relationship.
The accuracy and precision of the different temperature estimates was evaluated using ANOVA, quantifying the contribution of each factor (species, individual,
temperature relationship) to the deviation from the
independently measured temperature.
Isotope analysis
The isotopic composition (δ18O and δ13C) of the otoliths and water samples was analysed by mass spectrometry. Water samples and freshwater standards (calibrated
against VSMOW and SLAP) were measured following
a procedure similar to that described by Epstein and
Mayeda (1953) in which 2.5 ml of water were equilibrated with 100 μmols CO2 for 48 h at 25°C in a
shaking constant temperature bath. Following equilibration the CO2 was separated cryogenically and analysed
using an automated dual-inlet, triple collector stable
isotope ratio mass spectrometer (VG SIRA 12).
Prior to analysis, all otoliths were pre-treated to
remove organic contaminants by thermal decomposition under vacuum at a temperature of 180°C for 1 h

(Patterson et al. 1993). Individual otoliths (0.46–
1.05 mg) were then reacted under vacuum with anhydrous phosphoric acid at 25°C. The resultant CO2 was

separated cryogenically and analysed using an automated dual-inlet, triple collector stable isotope ratio
mass spectrometer (VG SIRA 12). Sample gas mass
ratios are measured against a reference gas calibrated
with respect to the NBS 19 international standard via a
secondary laboratory calcite standard prepared under
identical conditions to those used for unknowns.
Isotope ratios were corrected for 17O effects following
(Craig 1957) and equilibrium oxygen isotope fractionation between CO2 and water using a fractionation factor
(α) of 1.0412, and between aragonite and CO2 generated
by reaction with phosphoric acid using a fractionation
factor (α) 1.01034 (Friedman and O’Neill 1977). The
resultant data are reported in conventional delta (δ) notation in per mil (‰) relative to the VSMOW (water
samples) and VPDB (otolith samples):
ð1Þ
where R is the

18

O/16O or

13

C/12C isotope ratio in the


Environ Biol Fish (2012) 95:419–430

423

sample or standard. Replicate analysis of internal water

standards gave an analytical precision (σn−l) of 0.07‰.
For otoliths, the typical analytical precision (σn−1) for
replicate analysis of standard materials is 0.05‰ and
0.07‰ for carbonate carbon and oxygen isotope ratios
respectively.

Results
Final fish lengths differed significantly between the
cold and warm tanks of the low feeding regime treatment (MSE 7.83, p<0.05), and between the low and
high feeding regime tanks in the warm treatment
(MSE 8.76, p<0.05). Thus, the independent contributions of growth rate and temperature to otolith isotope
ratios were testable for fast and slow growing fish at
the same temperature (11°C), and for fish with the
same growth rate achieved at different temperatures
(11 and 17°C). The otoliths of the plaice juveniles
ranged in size from 0.4 to 1 mg (Table 2), and provided sufficient material for the analysis of individual
otoliths using the small sample protocol.
There were no significant differences in values
between left and right otoliths (paired t-test, df016,
t18O 00.79, t13C 0−0.79, p>0.05). The slopes of the

regression of the isotopic ratios between left and
right otoliths were not significantly different from
1 (Fig. 1), nor were there any consistent trends in
the residuals which might reflect a consistent bias
due to otolith or fish size.
The otolith isotope ratios in individual fish ranged
from −2 to −4 for carbon (δ13C) and from 0.2 to 1.9
for oxygen (δ18O) (Fig. 2). The otolith δ18O oxygen
was significantly affected by water temperature, but

not by feeding level, and there were no significant
synergistic effects between feeding level and temperature (Table 3). The variation between the two sagittal
otoliths of individual fish was not significant, nor was
the variation between fish within each tank or treatment. The fractionation of oxygen isotopes between
seawater and aragonite during otolith growth was independent of individual growth rate (Fig. 3).
Carbon isotope ratios in the otoliths were not significantly affected by food ration or water temperature.
The carbon isotope ratios declined significantly with
fish length in the colder water treatments, and tended
to increase with fish length in the warm water treatment (Fig. 4).
There was a significant negative relationship between the oxygen isotopic ratios in the plaice otoliths
and the measured water temperatures in the tanks. A

Table 2 Final fish size, otolith weight, water temperature and isotope ratios for the two temperature and two feeding treatments
(replicates combined) in the juvenile plaice experiment
Temperature →↓

Warm

Cool

Feeding level
High Food

Low food

44.25±2.06

48.25±6.85

Fish length (mm ± 1s.d.)

nd

0.63±0.005

Otolith weight (mg ± 1s.d.)

−2.98±0.16

−3.01±0.53

Otolith isotope ratio (δ13C ± 1s.d.)

0.76±0.11

1.71±0.14

Otolith isotope ratio (δ18O ± 1s.d.)

16.20±0.55

11.19±0.67

Estimated water temperature (°C ± 1s.d.)

17.40

11.16

Measured water temperature (°C, modal value)

7

11

N

53.60±6.87

43.78±10.79

Fish length (mm ± 1s.d.)

0.74±0.17

0.56±0.11

Otolith weight (mg ± 1s.d.)

−2.86±0.40

−3.05±0.44

Otolith isotope ratio (δ13C ± 1s.d.)

0.55±0.14

1.73±0.04

Otolith isotope ratio (δ18O ± 1s.d.)

17.88±0.80

11.24±0.21

Estimated water temperature (°C ± 1s.d.)

17.52

11.20

Measured water temperature (°C, modal value)

24

14

N

N number of fish used for otolith isotope analysis


424

Environ Biol Fish (2012) 95:419–430

(a)

0.8

There are numerous published equations relating water temperature and oxygen isotope ratios in
carbonates, including at least three experimentally
determined relationships for cod (Radtke et al.
1996; Gao et al. 2001; Hoie et al. 2004). The
temperature dependent relationship determined in
this study on plaice otoliths and the resulting
estimated water temperatures were compared with
previously published fractionation equations and
temperature estimation relationships:

0.4

1000
þ 15:99T
À 18 ln a ¼18 À24:25
Á
d Oc À d Ow ¼ 3:72 À 0:19T C

2

δ18O left otolith

1.6

1.2

ð3Þ

(This study)
0

0

0.4

0.8

1.2

1.6

2

δ18O right otolith

1000 ln a ¼ À41:14 þ 20:43T

ð4Þ

(Godiksen et al. 2010)
ln a ¼ À32:54
þ 18:56T
À1000
Á
d 18 Oc À d 18 Ow ¼ 4:64 À 0:21T C

(b)

ð5Þ

(Thorrold et al. 1997)

ln a ¼ À27:09
þ 16:75T
À1000
Á
d 18 Oc À d 18 Ow ¼ 3:90 À 0:20T C

ð6Þ

(Hoie et al. 2004)
Warm, Low Food
Warm, High Food
Cool, Low Food
Cool, High Food

2

Fig. 1 a Oxygen (δ18O) and b carbon (δ13C) isotopic ratios of
right and left otoliths of juvenile plaice

linear isotopic fractionation equation fit the data from
the juvenile plaice otoliths:
1000 ln a ¼ 0:059 ðÆ0:002ÞT þ 1:623ðÆ0:079Þ
r2 ¼ 0:94; F1;31 ¼ 537:4; p < 0:001

ð2Þ

otolith δ18O

1.6

1.2

0.8

0.4

0
-4

-3.6

-3.2

-2.8

-2.4

-2

otolith δ13C

where a ¼ d18 Ootolith þ 1000= d18 Owater þ 1000, and
T01000/TK. δ18Owater in SMOW was converted to
PDB following PDB ¼ ðSMOW Ã 0:97002Þ À 29:98

Fig. 2 Otolith carbon (δ13C) and oxygen (δ18O) isotope ratios
of individual juvenile plaice reared from hatching in two temperature and feeding regimes


Environ Biol Fish (2012) 95:419–430

Table 3 Analysis of variance of
the effects of water temperature
and feeding level on the oxygen
and carbon isotope ratios in
juvenile plaice otoliths

425

Source

SS

df

MS

F

p

Temperature

1.71

1

1.71

57.63

0.005

Food

0.01

1

0.01

0.22

0.67

Temperature × food

0.02

1

0.02

0.60

0.50

Error

0.09

3

0.03

Temperature

0.13

1

0.13

2.53

0.21

Food

0.001

1

0.001

0.02

0.89

Temperature × food

0.001

1

0.001

0.013

0.92

Error

0.15

3

0.51

δ18O

δ13C

where T°C is the estimated temperature, δ18Oc is
the oxygen isotopic ratio of the otolith and δ18Ow is
the oxygen isotopic ratio of the seawater. These relationships differ primarily in elevation, but in some
case also in slope (Fig. 5).
The slopes of all the relationships between real and
estimated temperature were <1, indicating that the
deviation between the estimated temperature from
the juvenile plaice otoliths and the real water temperature varies with water temperature.

To test the application of the experimentally determined otolith isotope–temperature relationship, the
otolith δ18O of the most recently deposited otolith
material were measured in 10 haddock, eight cod, 14
Warm, Low Food
Warm, High Food
Cool, Low Food
Cool, High Food

-2

0

0.4

16

0.8

14

1.2

12

-2.4

otolith δ13C

18

otolith δ18O

Temperature ( o C) estimated from otolith δ18O

20

whiting, four plaice, and three gurnard collected together in the same trawl. The species were characterised by distinct carbon vs oxygen values (Fig. 6), and
there were significant species differences in otolith
δ18O (ANOVA, F4,30 014.79, p<0.001). Plaice, haddock, and cod δ18O values were all significantly different. Cod and whiting otolith δ18O did not differ
significantly, and gurnard δ18O differed only from those
of cod and haddock (TukeyHSD post hoc p<0.01).
The actual water temperatures at 36 m depth vary
seasonally from 14°C in the beginning of August to
11°C in December. The minimum bottom temperature
measured during previous years was 6.7°C in March
1994, and the maximum was 15.7 in August 1995. An

-2.8

-3.2

1.6

-3.6
10

Warm, Low Food
Warm, High Food
Cool, Low Food
Cool, High Food

2
20

30

40

50

60

70

Fish length (mm )

Fig. 3 Otolith oxygen (δ18O) isotope ratios, and temperatures
estimated from these ratios, in relation to fish length for individual juvenile plaice reared from hatching in two temperature
and feeding regimes

-4
20

30

40

50

60

70

Fish length (mm)

Fig. 4 Relationship between the lengths of individual juvenile
plaice and the carbon (δ13C) isotope ratios of their otoliths


426

Environ Biol Fish (2012) 95:419–430

(a)

Temperature (oC)
18

16

14

12

10

33.0

32.5

32.0

31.0

30.5

30.0
Warm, Low Food
Warm, High Food
Cool, Low Food
Cool, High Food

29.5

29.0
3.54

3.52

3.5

3.48

3.46

3.44

3.42

Temperature (1/TK*1000)

(b)
24
Warm, Low Food
Warm, High Food
Cool, Low Food
Cool, High Food

Estimated water temperature (oC)

22
20
18
16

Whiting
Cod
Gurnard
Haddock
Plaice

-0.5
14
12

-1.0

10

-1.5

8
6
4
10

12

14

16

18

Measured water temperature (oC)

Fig. 5 Oxygen isotope fractionation in relation to temperature
(a), and correspondence between real and estimated water temperatures (b) for juvenile plaice otoliths (data points and thick
black line). Fractionation equations fitted to the plaice data are
shown for comparison: Thorrold et al. (1997)—light grey
dashed line, Hoie et al. (2004)—thin black line, Godiksen et
al. (2010)—thick grey line

average value of 13.95±1.09°C was used to represent
the actual bottom temperature conditions for the fish
sampled in the field. There were significant species

Otolith δ13C

1000 ln α

31.5

differences in the temperature estimates, and deviations
from measured temperatures (Tables 4 and 5). The temperature estimates from cod, haddock and whiting otoliths were not significantly different, and these species
gave the highest overestimates of actual temperature.
The temperatures estimated from plaice otoliths were
significantly cooler than the gadoids, and the difference
between actual and estimated temperature was significantly smaller. The temperatures estimated from the
gurnard otoliths were not significantly different from
either the plaice estimates or the estimates from the
gadoid fish.
The temperatures estimated by the oxygen isotope
ratios varied between individuals, species, and with
calculation method (Fig. 7a). Estimated temperatures
were generally warmer than the water temperatures
measured in situ (Fig. 7b). There was no significant
interaction between the effects of species and equation, confirming that the differences in estimated temperatures were purely the result of scaling due to the
different experimentally derived relationships. The
equation determined for plaice in this study (Eq. 4)
and Høie et al.’s equation (Eq. 6) gave the closest
temperature estimates, and the smallest overestimates
of measured water temperatures for the local area. The

-2.0

-2.5

-3.0

-3.5
-0.5

0

0.5

1

1.5

2

Otolith δ18O

Fig. 6 Otolith carbon (δ13C) and oxygen (δ18O) isotope ratios
of otoliths of fish from five different species collected together
in the same trawl in October, in the eastern Irish Sea. Average
values (± 1sd) of plaice otoliths from the laboratory experiment
temperature treatments are shown for comparison


Environ Biol Fish (2012) 95:419–430

427

Table 4 Water temperatures measured at 36 m depth at the Resa
station during Aug–Oct 1994 and 1995. The average temperature was used to evaluate the accuracy of the oxygen isotope
ratio estimates in field caught fish, reflecting the amount of
material removed from the surface of the otolith

Date

Temperature (°C)

4-August-1994

14.3

11-August-1994

14.7

15-September-1994

13.9

27-September-1994

13.3

4-October-1994

13.0

1-November-1994

12.2

10-August-1995

15.7

13-October-1995

14.5

Average

12.4

average temperature overestimates were 3.9 and 2.7°C,
respectively, significantly less than the other calculation
methods. Thorrold et al.’s (1997) equation (Eq. 6) overestimated temperatures for all species, by 6.9°C on
average, and Godiksen et al.’s (2010) equation (Eq. 5)
underestimated temperatures by 2.8°C on average
(Fig. 7b).

Discussion
Isotopic analysis of fish otoliths has enormous potential to reveal information about the past and present
environments. Stable isotope ratios in fish otoliths
provide valuable information for ecological studies
of individuals and populations (Kennedy et al. 1997;
Godiksen et al. 2011). Migrations (Northcote et al. 1992;
Rooker et al. 2008), population structure (Stephenson et
al. 2001; Gao et al. 2005; Shiao et al. 2009), and climate
change (Patterson et al. 1993; Gao and Beamish 2003),
have all been studied using the results of stable isotope
analysis in fish otoliths.

Table 5 Analysis of variance of

the effects of species and calculation method on temperature estimates from field caught fish, based
on difference between estimated
and average measured summer
temperatures at site (Table 4)

Source
Species
Calculation method
Interaction
Error

Temperature is probably one of the most important
environmental factors in aquatic ecosystem studies.
Temperature influences both abiotic and biotic processes, and thus is often a significant variable in ecological relationships. For this reason there has been so
much interest in being able to reconstruct the temperature record experienced by individuals (Godiksen et
al. 2011). The composition of calcium carbonate is
sensitive to temperature, and to temperature changes.
Thus there is the potential to recover the temperature
record from the calcium carbonate structures formed
by both fish and invertebrates. Stable isotope ratios
(oxygen and carbon) and trace elements compositions
have both been used in this way, but isotopic ratios
have only recently been subjected to the same experimental manipulation to investigate whether metabolic
processes may influence the values measured (Kalish
1991b; Thorrold et al. 1997; Tohse and Mugiya 2008).
The results from this experiment indicate that ambient water temperatures primarily determine the oxygen
isotope ratios in juvenile plaice otoliths, independently of
individual growth rate. This validates the assumption of
equilibrium precipitation of calcium carbonate, although
the form of the relationship to temperature is probably

not linear (Hoie et al. 2004; Godiksen et al. 2010). The
size, or growth rate, of the individual fish does not affect
the isotopic fractionation of oxygen. Plaice otoliths exhibit differential growth rates in the beginning during
metamorphosis from a bilaterally symmetrical larva to an
asymmetrically flattened juvenile, and the difference can
continue into adult life (Sogard 1991; Jearld et al. 1993;
Lychakov et al. 2008). Therefore some differences between right and left otoliths might be expected if the
otolith growth influenced the isotopic ratios. However,
the level of variability measured between individual
otoliths, and between individual fish, is not significant
compared to the variation induced by water temperatures. Variations in otolith δ13C are clearly related to fish
growth and metabolism, and the response of otolith
carbon isotope ratios to environmental conditions

SS

df

185.64

F

p<

4

13.05

.001

1893.74

3

177.59

.001

2.02

12

0.05

483.40

136

1


428

Fig. 7 Comparison of temperature estimates (a), and deviations
from measured temperature (13.96±1.09°C, shown as horizontal grey band) (b) in field-caught fish of different species, using
different relationships: Thrld Thorrold et al. (1997), Gdksn Godiksen et al. 2010, Hoie Høie et al. (2004), PlaL Plaice laboratory,
this study. Error bars01 sd

reduces their utility as a measure of temperature. However, as pointed out by Thorrold et al. (1997), they may
serve to indicate metabolic processes or variations in the

growth responses of individual fish.
In addition to determining that otolith oxygen isotopic ratios depend on water temperature, it is also
critical to determine how accurately the measured ratios
can be used to predict the temperature experienced by

Environ Biol Fish (2012) 95:419–430

individual fish. Of the relationships tested, (Hoie et al.
2004) was closest to the laboratory plaice relationship
and produced accurate estimates of the real water temperature measured by data loggers in the experimental
tank. The temperature estimates based on the fractionation relationships determined by Thorrold et al. (1997)
overestimated the water temperatures, and that of
Godiksen et al. (2010) underestimated water temperatures compared to the actual data. The slope of the
plaice fractionation equation was lower, but similar to
that determined for cod (Hoie et al. 2004). The slopes
determined for Atlantic croaker (Thorrold et al. 1997),
and Arctic charr (Godiksen et al. 2010) were both considerable higher. These relationships were determined
based on experimental work at higher (Thorrold et al.
1997) or lower (Godiksen et al. 2010) temperatures than
those experienced by the plaice in this study, and therefore the predictions are outside the range of the original
data. The experimental relationships determined by
Hoie et al. (2004) most closely fit the data for the
relationship between measured and estimated water
temperature for plaice. This result reinforces the importance of increasing the scope of experimental studies to
cover a wider range of species and temperatures.
The δ18O values measured in the recently deposited
otolith material in field caught fish mostly fell within the
range of those reported for 24 different species sampled
from the Adriatic by Iacumin et al. (1992). However the
otolith δ13C in the Irish Sea fish was generally lower,

and the otolith δ18O among the gadoids sampled in the
Irish Sea was considerably lower than those reported by
Iacumin et al. (1992). The isotope–temperature relationships developed for cod (Hoie et al. 2004) and for plaice
(this study) were similar, and produced reasonable temperature estimates for the wild-caught plaice, but overestimates of temperature in the other species.
A reliable relationship exists between the oxygen
isotopic composition of otolith aragonite and environmental conditions experienced by individual fish. There
are many applications where relative changes in temperature are valuable, and thus the issue of the accuracy
of the temperature estimates is not relevant. However,
where accurate temperature estimates are required, more
experimental data is needed to better define the exact
form of the relationship between otolith oxygen isotopic
ratios and actual water temperatures. Then, it should be
possible to extend the application of isotopic studies to
important questions such as models of fish population
growth and oceanic processes and productivity.


Environ Biol Fish (2012) 95:419–430
Acknowledgments The author thanks S Crowley and J Marshall (University of Liverpool) for their cooperation and advice
throughout the study. D Hornby assisted in the laboratory rearing. Otolith and water samples were analysed by SF Crowley in
the Department of Earth Sciences, University of Liverpool. This
study was performed at the Port Erin Marine Laboratory, funded
by the Ministry of Agriculture, Fisheries and Food Chief Scientists Group and the University of Liverpool Research Development Fund.

References
Craig H (1957) Isotopic standards for carbon and oxygen and
correction factors for mass spectrometric analysis of carbon
dioxide. Geochim Cosmochim Acta 12:133–149
Crowley SF, Taylor PD (2000) Stable isotope composition of
modern bryozoan skeletal carbonate from the Otago Shelf,

New Zealand. N Z J Mar Freshw Res 34:331–351
Dare PJ, Deith MR (1991) Problems with reconstructing sea
water temperature records from stable oxygen isotopic
profiles in shells of the scallop Pecten maximus. In: Barret
J, Dao JC, Lubet P (eds) Fisheries, biology and aquaculture
of Pectinids: 8th International Pectinid Workshop, Ifremer,
Plouzane, France, pp 258
Devereux I (1967) Temperature measurements from oxygen
isotope ratios of fish otoliths. Science 155:1684–1685
Epstein S, Mayeda T (1953) Variations in 18O contents of
waters from natural sources. Geochim Cosmochim
Acta 4:213–224
Friedman I, O’Neill JR (1977) Compiliation of stable isotope
fractionation factors of geochemical interest. Data of geochemistry, 6 edn. United States Geological Survey Professional Paper
Gao YW, Beamish RJ (2003) Stable isotope variations in
otoliths of Pacific halibut (Hippoglossus stenolepis)
and indications of the possible 1990 regime shift. Fish
Res 60:393–404
Gao YW, Schwarcz HP, Brand U, Moksness E (2001) Seasonal
stable isotope records of otoliths from ocean-pen
reared and wild cod, Gadus morhua. Environ Biol Fish
61:445–453
Gao YW, Svec RA, Joner SH, Bryant BL (2005) The life
history and stock structure of groundfish from stable
isotopic analysis of otoliths. Geochim Cosmochim Acta
69:A551
Geffen AJ, Nash RDM, Dau K, Harwood AJP (2011) Subcohort dynamics of O-group plaice, Pleuronectes platessa
L., in the Northern Irish Sea: settlement, growth and mortality. J Exp Mar Biol Ecol 400:108–119
Godiksen J, Svenning M-A, Dempson J, Marttila M, Storm-Suke
A, Power M (2010) Development of a species-specific

fractionation equation for Arctic charr (Salvelinus alpinus (L.)): an experimental approach. Hydrobiology
650:67–77
Godiksen JA, Svenning MA, Sinnatamby RN, Dempson JB,
Borgstrom R, Power M (2011) Stable isotope-based determinations of the average temperatures experienced by
young-of-the-year Svalbard Arctic charr (Salvelinus alpinus (L.)). Polar Biol 34:591–596

429
Grossman EL, Ku T (1986) Oxygen and carbon isotope fractionation in biogenic aragonite: temperature effects. Chem
Geol 59:59–74
Hoie H, Otterlei E, Folkvord A (2004) Temperaturedependent factionation of stable oxygen isotopes in
otoliths of juvenile cod (Gadus morhua L.). ICES J
Mar Sci 61:243–251
Iacumin I, Bianucci G, Longinelli A (1992) Oxygen and carbon
isotopic composition of fish otoliths. Mar Biol 113:537–542
Jearld AJ, Sass SL, Davis MF (1993) Early growth, behavior,
and otolith development of the winter flounder Pleuronectes americanus. Fish Bull 91:65–75
Kalish JM (1991a) 13C and 18O isotopic disequilibria in fish otoliths: metabolic and kinetic effects. Mar Ecol Prog Ser 75:2–3
Kalish JM (1991b) Oxygen and carbon stable isotopes in the
otoliths of wild and laboratory-reared Australian salmon
(Arripis trutta). Mar Biol 110:37–47
Kalish J (1992) Application of otolith oxygen and carbon
isotope data to the study of fish biology. In: Thresher
RE, Mills DJ, Proctor CH, Ianelli JN (eds) International Symposium on Skeletal Microanalysis of Marine
Fish Stocks,, Hobart, Tasmania (Australia), 1992.
CSIRO, pp 150–153
Kennedy BP, Folt CL, Blum JD, Chamberlain CP (1997) Natural isotope markers in salmon. Nature 387:766
Lychakov DV, Rebane YT, Lombarte A, Demestre M, Fuiman
LA (2008) Saccular otolith mass asymmetry in adult flatfishes. J Fish Biol 72:2579–2594
Meyer-Rochow VB, Cook I, Hendy CH (1992) How to obtain clues
from the otoliths of an adult fish about the aquatic environment

it has been in as a larva. Comp Biochem Physiol A 103:333–
335
Mitchell L, Fallick AE, Curry GB (1994) Stable carbon and
oxygen-isotope compositions of mollusk shells from Britain and New Zealand. Palaeogeogr Palaeoclimatol Palaeoecol 111:207–216
Nash RDM, Geffen AJ, Burrows MT, Gibson RN (2007) Dynamics of shallow-water juvenile flatfish nursery grounds:
application of the ‘self-thinning rule’. Mar Ecol Prog Ser
344:231–244
Newman SJ, Steckis RA, Edmonds JS, Lloyd J (2000) Stock
structure of the goldband snapper Pristipomoides multidens (Pisces: Lutjanidae) from the waters of northern and
western Australia by stable isotope ratio analysis of sagittal
otolith carbonate. Mar Ecol Prog Ser 198:239–247
Northcote TG, Hendy CH, Nelson CS, Boubee JAT (1992) Tests
for migratory history of the New Zealand common smelt
Retropinna retropinna (Richardson) using otolith isotopic
composition. Ecol Freshw Fish 1:61–72
Patterson WP, Smith GR, Lohmann KC (eds) (1993) Continental
paleothermometry and seasonality using the isotopic composition of aragonitic otoliths of freshwater fishes. Climate
change in continental isotopic records. Geophys Monogr Ser
78
Paull CK, Balch WM (1994) Oxygen isotopic disequilibrium in
coccolith carbonate from phytoplankton blooms. Deep-Sea
Res I Oceanogr Res Pap 41:223–228
Picard S, Garcia JP, Lecuyer C, Sheppard SMF, Cappetta H,
Emig CC (1998) δ18O values of coexisting brachiopods
and fish: temperature differences and estimates of paleowater depths. Geology 26:975–978


430
Radtke RL, Showers W, Moksness E, Lenz P (1996) Environmental information stored in otoliths: insights from stable
isotopes. Mar Biol 127:161–170

Richter H, McDermott JG (1990) The staining of fish otoliths
for age determination. J Fish Biol 36:773–779
Rooker JR, Secor DH, DeMetrio G, Kaufman AJ, Ríos AB,
Ticina V (2008) Evidence of trans-Atlantic movement and
natal homing of bluefin tuna from stable isotopes in otoliths. Mar Ecol Prog Ser 368:231–239
Shiao JC, Yui TF, Hoie H, Ninnemann U, Chang SK (2009)
Otolith O and C stable isotope compositions of southern
bluefin tuna Thunnus maccoyii (Pisces: Scombridae) as
possible environmental and physiological indicators. Zool
Stud 48:71–82
Smith GR, Patterson WP (1994) Mio-Pliocene seasonality on
the Snake River plain: comparison of faunal and oxygen
isotopic evidence. Palaeogeogr Palaeoclimatol Palaeoecol
107:3–4
Sogard SM (1991) Interpretation of otolith microstructure in
juvenile winter flounder (Pseudopleuronectes americanus):
ontogenetic development, daily increment validation, and
somatic growth relationships. Can J Fish Aquat Sci
48:1862–1871
Solomon CT, Weber PK, Cech JJ, Ingram BL, Conrad ME,
Machavaram MV, Pogodina AR, Franklin RL (2006) Experimental determination of the sources of otolith carbon

Environ Biol Fish (2012) 95:419–430
and associated isotopic fractionation. Can J Fish Aquat Sci
63:79–89
Stephenson PC, Edmonds JS, Moran MJ, Caputi N (2001)
Analysis of stable isotope ratios to investigate stock structure of red emperor and Rankin cod in northern Western
Australia. J Fish Biol 58:126–144
Thorrold SR, Campana SE, Jones CM, Swart PK (1997)
Factors determining δ13C and δ18O fractionation in

aragonitic otoliths of marine fish. Geochim Cosmochim
Acta 61:2909–2919
Tohse H, Mugiya Y (2008) Sources of otolith carbonate: experimental determination of carbon incorporation rates from
water and metabolic CO2, and their diel variations. Aquat
Biol 1:259–268
Weidel BC, Ushikubo T, Carpenter SR, Kita NT, Cole JJ,
Kitchell JF, Pace ML, Valley JW (2007) Diary of a bluegill
(Lepomis macrochirus): daily δ13C and δ18O records in
otoliths by ion microprobe. Can J Fish Aquat Sci
64:1641–1645
Weidman CR, Millner R (2000) High-resolution stable isotope
records from North Atlantic cod. Fish Res 46:327–342
Wurster CM, Patterson WP, Stewart DJ, Bowlby JN, Stewart TJ
(2005) Thermal histories, stress, and metabolic rates of
chinook salmon (Oncorhynchus tshawytscha) in Lake
Ontario: evidence from intra-otolith stable isotope analyses. Can J Fish Aquat Sci 62:700–713


Environ Biol Fish (2012) 95:431–443
DOI 10.1007/s10641-012-0032-3

Elemental signature in otolith nuclei for stock discrimination
of anadromous tapertail anchovy (Coilia nasus) using laser
ablation ICPMS
Shuo-Zeng Dou & Yosuke Amano & Xin Yu &
Liang Cao & Kotaro Shirai & Tsuguo Otake &
Katsumi Tsukamoto

Received: 19 September 2011 / Accepted: 20 April 2012 / Published online: 5 May 2012
# Springer Science+Business Media B.V. 2012

Abstract The elemental signature in otolith nuclei was
determined using laser ablation inductively coupled
plasma mass spectrometry (LA-ICPMS) for stock discrimination of adult anadromous tapertail anchovy,
Coilia nasus, in five Chinese estuaries. Five elements
(Na, Mg, K, Sr, and Ba) were well detected in the otolith
nuclei of the adult fish. Results showed that the elemental composition in the otolith nuclei varied substantially
among the estuaries. Age and fish length data showed
no significant influences on the elemental concentration
ratios across the sample sites. The Sr:Ca and Ba:Ca
ratios were inter-site distinct and could be used as natal
tags for discriminating among stocks. Discriminant
function analyses (DFA) showed that these ratios can
be used in discriminating the Liaohe River estuary (LD,
92.3 %), the Yangtze River estuary (CJ, 86.7 %), and the
Yellow River estuary (HH, 76.9 %) samples with high
S.-Z. Dou (*) : X. Yu : L. Cao
Key Laboratory of Marine Ecology and
Environmental Sciences, Institute of Oceanology,
Chinese Academy of Sciences,
Qingdao 266071, People’s Republic of China
e-mail:
Y. Amano : K. Shirai : T. Otake : K. Tsukamoto
Atmosphere and Ocean Research Institute,
The University of Tokyo,
5-1-5 Kashiwanoha,
Kashiwa-shi, Chiba 277-8564, Japan
X. Yu
Graduate School, Chinese Academy of Sciences,
Beijing 100049, People’s Republic of China

classification accuracy, followed by the Haihe River
estuary (BH, 58.3 %) and the Daguhe River estuary
(JZ, 46.2 %) samples. An overall classification accuracy
rate of 72.7 % from the discriminant functions indicated
that elemental fingerprinting appeared to have the potential to discriminate between tapertail anchovy stocks
in these estuaries.
Keywords Otolith chemistry . Elemental
fingerprinting . Population discrimination .
Anadromous fish . Chinese estuaries

Introduction
In the past two decades, the study of otolith chemistry
and application of elemental fingerprinting have been
proven to be a useful natural marker in reconstructing
the migratory history of individual fish and discriminating among populations (Edmonds et al. 1991;
Thresher et al. 1994; Campana 1999; Kennedy et al.
2000; Gillanders 2002). Several papers have critically
evaluated the recent advances in this field (Campana
1999; Thresher 1999; Campana and Thorrold 2001;
Elsdon and Gillanders 2003; Elsdon et al. 2008).
Otolith elemental fingerprinting is largely based on
the assumptions that the environment (e.g., water
chemistry) influences the incorporation of elements
into the otolith and that once these elements were
deposited, they are not altered or reabsorbed due to
the metabolic inertness of the otolith. Therefore, trace


432

elements in otoliths can be regarded as natural tags of
environmental history, albeit variations in ambient water
(e.g., temperature, salinity, and chemical composition)
and variability in physiology among fish individuals
may considerably influence the otolith microchemistry
(Kalish 1989; Campana 1999; Elsdon and Gillanders
2003). More importantly, elemental fingerprinting is
becoming more useful when it is combined with other
methods, such as otolith morphometrics and genetic
techniques (Longmore et al. 2010; Smith and Campana
2010; Clarke et al. 2011).
Laser ablation inductively coupled plasma mass
spectrometry (LA-ICPMS) is often used to examine
otolith chemical composition using a focused beam
because it combines the beam capacities of a highenergy laser with the rapid and accurate analytical
capacities of ICPMS (Campana 1999; Thorrold and
Shuttleworth 2000; Gillanders and Joyce 2005;
Stransky et al. 2005). Because the otolith nucleus
roughly corresponds to the fish spawning and hatching, the chemistry of otolith nuclei detected with LAICPMS can be used as a natural marker of the spawning and natal home for both marine and anadromous
fish (Campana et al. 1994; Thorrold et al. 1998;
Brophy et al. 2003; Ashford et al. 2006). In contrast
to marine fish species, one advantage for elemental
fingerprinting in anadromous fish is that their natal
rivers can be characterized by a unique chemical composition, suggesting that trace element uptake onto the
otolith nucleus of anadromous fish may show distinct
inter-site variation. Therefore, elemental fingerprinting
in otolith nuclei of anadromous fish could serve as an
effective delineator for discriminating among anadromous stocks (Thorrold et al. 1998).
Tapertail anchovy, Coilia nasus, is an anadromous

clupeid fish that inhabits the major estuaries along the
Chinese coast. It was once an important commercial
fishery species in China. Since the 1980s, however,
the wild populations have drastically decreased or
disappeared in most estuaries (Yuan and Qin 1984;
Luo and Shen 1994). Aside from anadromous stocks,
landlocked stocks that reside in freshwater over the
entire life were also reported in some affiliated lakes of
the Yangtze River, such as the Taihu lake (Cheng et al.
2005; Yang et al. 2006; Liu et al. 2008). Anadromous
adults start to migrate upstream at about 2 year old
from March to April (over 7°C SST and 17–30‰
salinity in the estuarine waters), during which sexual
maturity is rapidly developed. The matured fish

Environ Biol Fish (2012) 95:431–443

usually spawn in still waters at 15 to 27.5°C from
May to September, depending on spawning sites and
timing. After spawning, the fish stay nearby for a
certain period of time and then move downstream into
the local estuaries to grow before the end of winter
(Cai et al. 1980; Yuan and Qin 1984; Yuen 1987; Chen
1991; Liu et al. 2008). Although it is generally known
that larvae drift into the estuarine areas and reside
there until reaching sexual maturity for upstream
spawning migration, the early life history, particularly
the downstream larval transportation (e.g., freshwater
duration and larval drift process) is poorly understood.
To date, the tapertail anchovy populations in the

Chinese waters, which is essential for fishery management, are not well documented. Because the Chinese
rivers usually have distinct chemical properties (Hu et
al. 1982; Liu 1985; Chen 2006; Wang et al. 2009), it is
possible that the site-dependent elemental signatures
from the otolith nuclei will show the natal sources of
the tapertail anchovy. This provides the primary premise for elemental fingerprinting in discriminating
among stocks. In the present study, we investigated
the elemental composition from the otolith nuclei of
adult tapertail anchovy collected from five Chinese
estuaries to test if elemental fingerprinting can act as
effective natural tags for discriminating among the
stocks. To achieve this goal, otolith elemental concentrations were probed and analyzed using LA-ICPMS
to look at their inter-site differences. Intra-estuary data
of different age groups in each sample site was analyzed to explore if the otolith elemental signatures of
the fish born in a specific river may undergo dramatic
inter-annual variation.

Materials and methods
Sample collection
Samples used in this study were from one otolith
collection of the Chinese Academy of Sciences, which
was conducted during the fishery research surveys
using otter trawl gear along the Chinese coastal waters
in the 1980s. This collection included samples from
the Liaohe River estuary (LD), the Haihe River estuary (BH), the Yellow River estuary (HH), the Daguhe
River estuary (JZ), and the Yangtze River estuary (CJ),
respectively (Table 1; Fig. 1). Immediately after capture, the fish were labeled and frozen for subsequent


Environ Biol Fish (2012) 95:431–443

433

Table 1 Summary of tapertail anchovy samples collected in the five sample sites (codes as in Fig. 1) for chemistry analysis using LAICPMS (BL, body length; BW, body weight; W, otolith weight; Dmax, major axis diameter; Dmin, minor axis diameter)
Sampling site

Sampling date

n

Fish samples
BL (mm)

Otolith
BW (g)

Age (yr)

W (mg)

Dmax (μm)

Dmin (μm)

LD

1983

13

279–402

65–222

2–4

12.3–20.4

4012–4965

3740–4535

BH

1983

12

282–404

72–220

2–4

13.0–21.5

4206–5087

3980–4626

HH

1983

13

270–387

65–218

2–4

11.7–19.8

4131–5012

3858–4492

JZ

1983

13

287–385

76–198

2–4

13.1–19.6

4089–4526

3890–4417

CJ

1985

15

262–372

59–204

2–4

11.3–19.2

3970–4673

3755–4318

biological analysis, including otolith removal. In the
laboratory, fish length, weight, sex, maturity status,
and age (estimated according to annual rings in scales
or the empirical age-length relation) were determined
and recorded. Sagittal otoliths were removed from
each fish, cleaned of adhering tissue in distilled water,

and stored dry in sealed glass vials until further
examination.
The otolith sampling program of the present study
was designed to include fish of known stocks. As
previously described, adult tapertail anchovy tend to
return to the natal rivers to spawn and then migrate
downstream to reside in the local estuarine areas to
feed. To minimize the potential influence of stock
mixing, otoliths were collected from the adult fish that
had spawned (based on the sexual maturity) and resided in waters near the specific river mouths (according to sampling site). Samples are thus believed to be
reasonable representations of discrete stocks. To examine the inter-annual variation in elemental composition of the otolith nuclei, fish samples of 2–4 year
old (4–5 individuals representing each age cohort, i.e.,
born in different years) within a geographical stock
were chosen for elemental analyses. A total of 66
otoliths were collected and analyzed for otolith nuclei
using LA-ICPMS (Table 1).
Otolith preparation
Sagittal otoliths were sonicated in distilled water for
10 min to clean the otolith surface, and then were
oven-dried overnight at 35°C. Samples were measured
for the longest and shortest axes to the nearest 1 μm
under a microscope (Nikon, SMZ-1000) and were
weighed to the nearest 0.1 mg using an electrical
balance (Sartorius BS-124 S). Each otolith was

embedded in epoxy resin (Struers, Epofix), mounted
on a glass slide, and then cut and ground to expose the
core on the sagittal plane using a grinding machine
that was equipped with diamond cup wheels
(Discoplan-TS; 70 μm, Struers; 13 μm, Sanwa

Diamond, Tokyo). Because concentric rays from the
core to the edge of the otolith can be clearly visible
under microscopy, the otolith nucleus can be identified
(Fig. 2). After grinding, otolith sections with 400–
600 μm thickness were polished with suspension (OPS, Struers) on an automated polishing wheel (Struers,
Rotopol-35) to get finer and more exposed surfaces. To
avoid possible contamination, otolith samples were
cleaned in sonicated Milli-Q super water (Millipore
S.A.S.) in the present study. After decontamination,
sectioned otoliths were oven-dried at 35°C, and were
randomly numbered across sample sites and placed in
clean sealable plastic boxes until chemistry analyses.
Chemistry analysis
For LA-ICPMS analysis, concentrations of otolith elements were determined on a Merchantek UP-213 Nd:
YAG deep UV laser ablation system (New Wave
Research, Fremont, CA) coupled with an Agilent
7,500 s ICPMS (Agilent Technologies, Tokyo). The
lasers were operated at a wavelength of 213 nm with a
pulse rate of 10 Hz and an energy density of 0.3 Jcm−2.
In each analysis session, 11–12 otolith sections,
randomly numbered and selected from all sample
groups, plus a reference standard glass (NIST 612,
National Institute of Standard and Technology), were
mounted on a carrying stage and then fixed in the
sealed chamber where otolith material ablation took
place. Otolith analysis sequences were randomized so
that the order of analysis for any one sample group


434

Environ Biol Fish (2012) 95:431–443

Fig. 1 Map showing estuaries in which tapertail anchovy samples were collected. LD, the Liaohe River estuary; BH, the Haihe River
estuary; HH, the Yellow River estuary; JZ, the Daguhe River estuary; CJ, the Yangtze River estuary

was spread over the entire analysis sequence. When an
otolith is analyzed on an ICPMS, instrument drift
occurs due to the buildup of Ca ion on the instrument,
as well as the changes in temperature, plasma, and

electronics, resulting in low detection limits of the
elements (Campana et al. 1994; Elsdon and
Gillanders 2002). To minimize the potential effects
of instrument drift on the elemental analysis, the


Environ Biol Fish (2012) 95:431–443

435

Fig. 2 Photographs showing the reflected-light
microstructures of an adult
tapertail anchovy otolith
section (a). SEM photographs (b, × 30; c, × 400)
showing the laser ablation
points on the otolith nucleus
for chemical analysis on
LA-ICPMS

concentric rays

nucleus area

a
c

b

check

standard was analyzed at the beginning, the middle
(after ablating the first 6 otoliths), and the end of each
analysis session. Ablation points in both the standard
and the otoliths were positioned via a computer
connected to the UP-213 using the default New Weave
Research laser ablation system software. In the present
study, the otolith nucleus region was defined as the
round area that spread at a radius of 190–230 μm from
the core, which was measured under microscope for
each otolith section. The nucleus region was morphologically marked with its distinct circumference, from
which concentric rays around the nucleus occurred outwards the edge (Fig. 2). To precisely locate the otolith

laser point
diameter: 40 µm
depth: 15 µm
distance from core: 100 µm

cores for laser ablating, we took photos of each otolith
section under microscope before chemical analysis so

that the unique otolith characteristics (e.g., the concentric rays from the core outwards to the edge, the outline
of the core area, or even the ‘accidental markers’ created
during otolith preparation) could be referred for identifying the nucleus in the laser ablation system. On each
otolith section, one point in the otolith core and four
other points that were evenly positioned at a distance of
100 μm from the core were laser-ablated and chemically
analyzed. The four ablations around the core ablation
were expected to fall safely within the otolith nucleus
area. After elemental analysis, otolith sections were


436

examined under microscope to confirm the laser ablation locations or by scanning electron microscopy
(SEM, Keyence VE-8800) to verify the validity of each
laser ablation (Fig. 2). In this process, invalid laser
ablation data, if any, could be excluded from statistical
analysis. The average values of elemental data of the
five ablations were assumed to accurately represent
the elemental composition of the otolith nucleus.
The ablation craters produced had a diameter of
appropriately 40 μm with a depth of about 15 μm,
and the dwell time was 15 s. The ablated material
was transported from the ablation chamber to the
ICPMS, via a settlement cell, by an argon (Ar) and
helium (He) gas stream. The ablation chamber was
purged for 90 s after each opening to remove any
background gas or sample particles that may have
contaminated future samplings. Data were collected
using the default Agilent 7,500 s ICPMS software.

The concentrations of each element were standardized to calcium by expressing concentrations of
elements as ratios to Ca.
To determine the actual limits of detection
(LODs), blank ablations that only consisted of
measuring Ar and He gases were conducted before
and after each analysis session for approximately
120 s. Preliminary analyses had identified five
elements (Na, Mg, K, Sr, and Ba), which were
consistently detectable in the otolith nuclei. Relative
standard deviations (% RSD), based on replicated
measurements of the calibration standard, were
calculated to reflect the level of precision achieved
for each element.
Data analysis
Since K and Na are usually not bound to the lattice
of the otolith but are weakly occluded in the interstitial spaces, they are easily leached during otolith
preparation (grinding, washing, and polishing etc.).
Furthermore, the concentration of these two elements in the otolith is under strong physiological
regulation. For these reasons, they are believed to
be unsuitable as stable elemental signatures in stock
identification studies (Thresher et al. 1994; Proctor
et al. 1995). Thus elemental data of K:Ca and Na:Ca
were excluded from the statistical analyses in the
present study.
The elemental concentration ratios (Mg:Ca, Ba:
Ca, and Sr:Ca) of the analytical data were first

Environ Biol Fish (2012) 95:431–443

examined for within-group normality (KolmogorovSmirnov’s test) and for homogeneity of variances

among groups (Levene’s test). When both assumptions
were met, one-way ANOVA was applied to examine the
univariate differences in concentration ratios of each
element among the sample sites to test their spatial
differences. One-way ANOVA was also applied to examine the univariate differences in concentration ratios
of each element among the age groups in each stock to
explore their inter-annual variation so that the possible
effects of inter-annual differences on spatial differences could be evaluated. Post hoc multiple comparisons using Bonferroni test were applied to
compare the means between groups, when significant differences were computed. Furthermore, to
assess the possible age effects on the intra-site
differences of the elemental concentration ratios,
two-way ANOVA was performed on the data of
each elemental concentration ratio across the sample sites. Analyses of covariance (ANCOVA), with
fish length as a covariate, were separately run on
the concentration ratios of each element among
sample sites to examine the possible fish length
effects on otolith elemental compositions among
sampling sites. To achieve this, all the interactions
between site and fish length in each element were
tested using the custom model. If no significant
interactions were observed in all the elemental
data, the full factorial model was then applied for
ANCOVA to examine the fish length effect on the
elemental concentration ratios.
To determine the ability of elemental signatures
to correctly classify the samples, discriminant
function analysis (DFA, Fisher’s coefficient) was
conducted to identify the elements that contributed
the most to the spatial differences of the chemical
signatures. Three elements (i.e., Mg, Ba, and Sr)

that are likely to be influenced by the environment
were used for DFA in the entering independent
together method. Pooled within-group matrices
were produced to examine the correlation and covariance among the concentration data of the three
elements. Combined-groups plotting of the first
two discriminant function axes against each other
and the predicted group membership (cross-validated classification) were generated to reflect the
accuracy of classifications.
Statistical analyses were performed on SPSS 17.0 for
windows (SPSS Inc.) at a significance level of P<0.05.


Environ Biol Fish (2012) 95:431–443

437

Results
The analytical accuracy for the NIST standard averaged across all samples was high for all the five
elements with %RSD ranging from 2.55 (Na) to 4.65
(Mg). LODs (mmol mol −1 ) for Na (0.127), Mg
(0.0017), K (0.107), Sr (0.0046), and Ba (0.0002)
were all well below the detected concentrations in
otoliths. Among the three elements tested (Mg, Ba,
and Sr), they all met both within-groups normality
(Kolmogorov-Smirnov’s test, P> 0.05 in all cases)
and homogeneity of variances among groups
(Levene’s test, P>0.05 in all cases), except for the
Mg:Ca and the Ba:Ca ratios in the JZ sample. The
Sr:Ca and Ba:Ca ratios showed significant inter-site
differences (ANOVA, P < 0.05 in both elements),

whereas Mg:Ca ratio did not significantly differ
among sample sites (ANOVA, P > 0.05; Table 2;
Fig. 3). The Ba:Ca ratios of the LD and HH samples
were significantly lower than those of other samples
(Bonferroni test, P<0.05 in all cases; Fig. 3), but did
not significantly differ between any other two samples
(Bonferroni test, P>0.05 in all cases; Fig. 3). The Sr:Ca
ratios were not significantly different between LD and
CJ samples, JZ and BH samples, and JZ and HH samples (Bonferroni test, P>0.05 in all cases; Fig. 3), but
significantly differed among other samples (Bonferroni
test, P<0.05 in all cases; Fig. 3).
The elemental concentration ratios were not significant among age groups in any specific sample site
(ANOVA, P>0.05; Table 3; Fig. 4), except for the
Mg:Ca ratio (P<0.05) in the BH sample. Age components did not significantly affect the three elemental
concentration ratios across the sample sites (ANOVA,
P>0.05 in all elements; Table 4). Similarly, neither
Table 2 Results of one-way
ANOVA running on the elemental concentration ratios in
the otolith nuclei of tapertail
anchovy collected in the five
sample sites

significant interactions between site and fish length nor
fish length significantly affected the three elemental concentration ratios across the sample sites (ANCOVA, P>
0.05 in all elements; Table 4).
Pooled within-group matrices generated low correlations between the concentration ratios of the three
elements included in DFA (−0.065 between Mg:Ca
and Ba:Ca; 0.096 between Mg:Ca and 0.179 between
Sr:Ca and Ba:Ca), indicating that the three elemental
concentration ratios were not closely correlated. The

main DFA results were summarized in Table 5 and
Fig. 5. The scores of the first canonical discriminant
function (F1, eigenvalue09.31) explained 95.7 % of
the variance and could easily discriminate the LD and
CJ samples from other samples. It could marginally
discriminate the HH and BH samples as well. F1 was
closely correlated (canonical correlation00.95) to the
three elemental concentration ratios with Sr:Ca ratio as
the greatest contributor to the function scores (Fisher’s
coefficient 01.022). The scores of function 2 (F2,
eigenvalue00.37) could effectively discriminate between the LD and CJ samples and marginally discriminate the HH and BH samples, but it explained only
3.8 % of the variance. Ba:Ca contributed the most to
F2 scores (Fisher’s coefficient00.942). Scores of both
F1 and F2 through F3 significantly differed among
sample sites (Wilks’ lambda test, P<0.005 in both
cases), whereas function 3 (F3) scores were not geographically different (Wilks’ lambda test, P00.214).
The Sr:Ca and Ba:Ca ratios were the two most powerful elemental signatures to geographically discriminate the five samples. DFA produced an overall
classification accuracy rate of 72.7 %, with the highest
rate for the LD sample (92.3 %), followed by the CJ
(86.7 %), HH (76.9 %), BH (58.3 %), and JZ (46.2 %)

Sum of
Squares

df

Mean
square

Between Groups

0.045

4

0.011

Within Groups

0.524

61

0.009

Total

0.569

65

Between Groups

0.004

4

0.001

Within Groups

0.012

61

0.000

Total

0.016

65

Elements ratio
(mmol mol−1)
Mg:Ca

Ba:Ca

Sr:Ca

Between Groups
Within Groups
Total

63.534

4

15.883

7.256

61

0.119

70.789

65

F

P

1.299

0.280

5.099

0.001

133.539

0.000


438

Environ Biol Fish (2012) 95:431–443

Mg:Ca (mmol/mol)

0.50

assigned to the most geographically separated LD
sample. Two of the three misclassified cases in the
HH sample were assigned to the geographically separated JZ sample. Between the two most geographically
adjacent BH and HH samples, one of the five misclassified BH individuals was assigned into the HH
sample; whereas one of the three misclassified individuals in the HH sample was assigned into the BH
sample (Table 6).

A

0.40
0.30
0.20
0.10
0.00

Ba:Ca (mmol/mol)

0.06
0.05

B

b

0.04
0.03

b

a

b

Discussion

a

0.02
0.01
0.00
LD

Sr:Ca (mmol/mol)

5.00

BH

C

4.00

b

HH

c

JZ

CJ

bc

3.00
2.00

a

a

1.00
0.00
LD

BH

HH

JZ

CJ

Fig. 3 Concentration ratios (mean ± S.D.) of the three elements

consistently measured above LODs in the otolith nuclei of
tapertail anchovy collected in the five sample sites (codes as in
Fig. 1). A, Mg:Ca ratio; B, Ba:Ca ratio; C, Sr:Ca ratio. Different
lower case letters indicate significant differences between
groups at P<0.05

samples (Table 6). The seven misclassified individuals
in the JZ sample were incorrectly assigned to the BH
(4) and the HH (3) samples, both of which are geographically separated from the JZ sample. Meanwhile,
the two misclassified cases in the CJ sample were

Table 3 Statistical significances
(one-way ANOVA, P<0.05)
showing the inter-annual variation (among age groups) of otolith elemental concentration
ratios within a specific sample
site (codes as in Fig. 1)

Previous studies have documented substantial intersite differences in otolith elemental fingerprints among
geographically separated stocks in a variety of fishes
(Edmonds et al. 1991; Campana et al. 1994;
Humphreys Jr et al. 2005; Stransky et al. 2005;
Longmore et al. 2010). Otolith elemental fingerprinting has proven successful in discriminating among
fish stocks with a classification success as high as over
90 % in a number of species, such as American shad,
Alosa sapidissima (Thorrold et al. 1998; Walther and
Thorrold 2009) and macrourid, Coryphaenoides
rupestris (Longmore et al. 2010). In the present study,
classification success was relatively high for the LD,
CJ, and HH samples, but it was only at a marginal
level for the BH and JZ samples. Stock mixing is

commonly assumed to be a potential factor that may
cause low classification success in stock discrimination by elemental fingerprinting. Tapertail anchovy
tends to migrates to the natal rivers to spawn and their
residence is highly localized in the river and the adjacent estuarine areas at all life stages (Chen 1991; Luo
and Shen 1994; Liu et al. 2008). Therefore, mixing
among fish from the five estuaries was assumed to be
minimal. This was supported by the finding that the
LD (the most northern stock) and the CJ (the most
southern stock) could be discriminated with high classification accuracy from other geographical stocks. It

Elements ratio
(mmol mol−1)

LD

BH

HH

JZ

CJ

Mg:Ca

0.817

0.009

0.296

0.989

0.875

Ba:Ca

0.669

0.068

0.851

0.928

0.170

Sr:Ca

0.425

0.375

0.345

0.447

0.916