morphometry and composition of aragonite and vaterite otoliths of

Morphometry and composition of aragonite and vaterite

otoliths of deformed laboratory reared juvenile herring

from two populations

J. TOMAS* AND A. J. GEFFEN

Port Erin Marine Laboratory, School of Biological Sciences, University of Liverpool,Port Erin, Isle of Man, IM9 5AP U.K.

(Received 24 March 2003, Accepted 19 August 2003)

Vaterite otoliths were sampled from two reared populations (Celtic and Clyde Seas) of juvenile

herring Clupea harengus. The crystallography, elemental composition and morphometry were

analysed and compared with those of normal aragonite otoliths. The incidence of vaterite

otoliths in the juveniles sampled (n¼ 601) ranged from 7�8% in the Clyde population to

13�9% in the Celtic Sea population, and was 5�5% in the small sample (n¼ 36) of wild adults

examined. In all but one case fish had only one vaterite otolith; the corresponding otolith of the

pair was completely aragonite. Although the majority of the juveniles sampled showed cranio-

facial deformities, there was no link between the skull or jaw malformation and the incidence of

vaterite otoliths. All vaterite otoliths had an aragonite inner area, and vaterite deposition began

sometime after the age of 90 days. The vaterite otoliths were larger and lighter than their

corresponding aragonite partners, and were less dense as a consequence of the vaterite crystal

structure. The vaterite areas of the otoliths were depleted in Sr, Na and K. Concentrations of

Mn were higher in the vaterite areas. The transition between the aragonite inner areas and the

vaterite areas was sharply delineated. Within a small spatial scale (20 mm3) in the vaterite areas,

however, there was co-precipitation of both vaterite and aragonite. The composition of the

aragonite cores in the vaterite otoliths was the same as in the cores of the normal aragonite

otoliths indicating that the composition of the aragonite cores did not seed the shift to vaterite.

Vaterite is less dense than aragonite, yet the concentrations of Ca analysed with wavelength-

dispersive spectrometry (WDS) were the same between the two polymorphs, indicating that Ca

concentrations measured with WDS are not a good indicator of hypermineralized zones with

high mineral density. The asymmetry in density and size of the otoliths may cause disruptions

of hearing and pressure sensitivity for individual fish with one vaterite otolith, however, the

presence of vaterite otoliths did not seem to affect the growth of these laboratory reared

juvenile herring. # 2003 The Fisheries Society of the British Isles

Key words: biomineralisation; fish hearing; ICPMS; otolith development; Raman spectrometry.

*Author to whom correspondence should be addressed at present address. Grupo de Oceanografıa

Interdisciplinar (GOI), Institut Mediterrani d’Estudis Avancats (CSIC/UIB), Miguel Marques 21, 07190

Esporles, Illes Balears, Spain. Tel.: þ 34 971 61 17 21; fax: þ 34 971 61 17 61; email: [email protected]

Journal of Fish Biology (2003) 63, 1383–1401

doi:10.1046/j.1095-8649.2003.00245.x,availableonlineathttp://www.blackwell-synergy.com

1383# 2003TheFisheries Society of theBritish Isles

INTRODUCTION

Otoliths are involved in the perception of sound and the maintenance ofpostural equilibrium in fishes. The perception of sound is a fundamentalsense, providing information from the environment (Popper & Fay, 1999) forpredator avoidance, food availability, and the location of other individuals formating (Zelick et al., 1999). Otoliths are composed of CaCO3 that normallyprecipitates as aragonite. In aberrant otoliths the CaCO3 precipitates as calciteor vaterite. These three polymorphs of CaCO3 differ in the geometry of thecrystal: calcite is trigonal, aragonite is orthorhombic and vaterite is hexagonal.The formation of calcite or vaterite otoliths has been reported in a number ofmarine and freshwater species from different environments (Strong et al., 1986;Gauldie, 1993; Bowen II et al., 1999). Otoliths may be completely or partiallycomposed of calcite or vaterite but the mechanisms governing the switchbetween polymorphs are unknown. The presence of vaterite otoliths may affectthe functioning of the inner ear and the growth and survival of the fish, andmay lead to compensations in otolith size or shape, but these aspects have yet tobe examined.Juvenile herring Clupea harengus L. with deformed heads were examined in

this study after evidence was found in the zebrafish Danio rerio (Hamilton) thatmutations affecting the development of jaws, gills and cranium resulted in themalformation of otoliths (Malicki et al., 1996; Whitfield et al., 1996). Thecrystallography of the malformed zebrafish otoliths was not reported in thesestudies, but it is possible that the macrostructural changes were accompanied bychanges at the molecular level. Several reports of vaterite otoliths in wild fishes(Morales-Nin, 1985; Strong et al., 1986; Gauldie, 1993; Brown & Severin, 1999)have commented on the aberrant appearance of these otoliths.Populations of juvenile herring originating from fish spawning in the Firth of

Clyde and the Celtic Sea were raised in the laboratory, and fish with jaw andcranial deformities were selected, and compared with normal juveniles, in orderto study the relationships between otolith morphometry, crystal form andotolith and fish growth. Normal and aberrant otoliths were compared toexplore the relationship between composition and otolith structure. The mor-phometry of aberrant otoliths was also analysed to provide information aboutthe relationship between otolith growth and otolith shape.

MATERIALS AND METHODS

SOURCE OF MATERIAL

One year-old juvenile herring were sampled from populations originating fromgametes collected from the Celtic Sea (January spawning) and the Clyde Sea (springspawning) herring populations. Eggs from several females were distributed on glassplates, and artificially fertilized with sperm from several males. After hatching, the larvaewere reared in flowing sea water in 2000 l circular black tanks. Temperatures were notcontrolled and followed seasonal fluctuations. The light regime was maintained withfluorescent lighting, controlled with time clocks to follow the seasonal changes in photo-period. Larvae were fed with rotifers and subsequently Artemia sp. and were weaned atc. day 30 after hatching onto formulated dry food.

1384 J . TOMAS AND A. J . GEFFEN

# 2003TheFisheries Society of the British Isles, Journal of FishBiology 2003, 63, 1383–1401

The juvenile fish sampled ranged from 3–14 months in age (Table I). Adult Celtic Seaherring were also sampled to assess the frequency of vaterite otoliths in the wild popula-tion. Every fish was measured (total length, LT), weighed and the otoliths removed.Otolith dissection was carried out using acid washed glass probes and distilled water toprevent otolith contamination. Once extracted, otoliths were double rinsed in distilledwater, air dried and stored in acid washed polypropylene vials.Preliminary observations had indicated that deformed individuals had a high incidence

of crystalline otoliths, presumed to be composed of vaterite and easily distinguishablefrom normal aragonite otoliths (Fig. 1). To test this, a random sample of 112 juvenilesfrom the Celtic Sea stock and 109 juveniles from the Clyde were collected in 1998 toprovide a control sample of normal fish to investigate the relationship between skulldeformities and the incidence of vaterite otoliths (Table I).

OTOLITH MORPHOMETRY

Otoliths were weighed using a Cahn G-2 electrobalance (precision� 0�001mg) and theotolith dimensions (mm) were measured using an image analysis system [Fig. 1(b)]. Theotolith length was the distance between the anterior and posterior edges of the otolith;the width was the longest distance between the dorsal and ventral edges of the otolith,perpendicular to the length of the otolith. The perimeter of the otolith was tracedautomatically using an edge-recognition sub-routine, and the total otolith area (mm2)calculated by the image analysis software. All of the vaterite otoliths contained a centralregion that appeared to be normal aragonite. This aragonite area varied in shape and sizebetween individuals [Fig. 1(b), (c), (d)]. The perimeter of the aragonite area of the vateriteotoliths was outlined manually, and the area calculated by the image analysis software[Fig. 1(b), (c), (d)].

CRYSTALLOGRAPHY

X-ray diffraction spectrometry (XRD) was used to identify the polymorph composi-tion of the normal and apparently vaterite otoliths. Six otolith samples (three vaterite andthree normal) were crushed and mixed for bulk crystallographic analysis. The spatialdistribution of the different CaCO3 polymorphs within vaterite otoliths was examined byRaman spectrometry, which provides information about the metal and carbonate bondswithin the crystal lattice. A laser was used to excite the sample surface and the Ramaneffect and the distortion of the crystal lattice suite was recorded as energy spectra whichwere characteristic for each polymorph of CaCO3. A vaterite otolith was mountedproximal side up on a glass microscope slide and analysed with a Renishaw Ramanimaging microscope using the 1064 nm NIR line for excitation. The laser power at thesurface of the samples was 3mW. The scattering volume, which is the area encompassedin a single analysis spot, was c. 20 mm3. The spectra were collected in the range100–1500 cm�1 with a spectral resolution of 2 cm�1. The data were collected 40 times withan exposure time of 10 s. Additional instrument details are given by Williams et al. (1994).

ELEMENTAL COMPOSITION OF NORMAL AND VATERITEOTOLITHS

Aragonite and vaterite differ in the structure and spacing of the crystal, and this canlead to differences in elemental composition. The trace element composition of wholeotoliths was analysed with solution based inductively coupled mass spectrometry(SB-ICPMS). Twenty-two otolith pairs (11 pairs from each the Celtic and Clyde Seastocks) were analysed to assess the extent of elemental variation within normal pairs.Another two pairs of otoliths, each with a vaterite and a normal otolith were thenanalysed to examine the differences in composition related to vaterite. Individual otolithswere dissolved in 100ml of concentrated nitric acid and diluted in 1% nitric acid to a finaldilution ratio of 1 : 5000. The dissolved samples were analysed with a PlasmaQuad 3 (VGElemental) ICPMS. The concentrations of Sr, Ba, Mn, Li, Pb, Ni, Co and Sc were

HERRING VATERITE OTOLITHS 1385

# 2003TheFisheries Society of theBritish Isles, Journal of FishBiology 2003, 63, 1383–1401

TABLEI.

Number

ofcasesofvaterite

otolithsin

norm

alherringandin

fish

displayingheaddeform

ities(C

elticandClydeSeas)

Number

offish

Number

offish

withvaterite

otoliths

Population,yearclass

Agerange(indays)

Norm

al

Deform

edNorm

al

Deform

ed(%

oftotal)

Celtic1997

390

061

013

21

Celtic1998

348–410

112

107

17

911

Inleftotolith:20,in

rightotolith:19

Clyde1997

317

029

02

7Clyde1998

93–351

109

183

13

10

8

Inleftotolith:9,in

rightotolith:14

Celtic1999

Wildadults

36

02

05



measured following the spike method. Elemental concentrations were calculated based onthe standards addition method. Any elements that failed to give a linear response at anydilution or failed to give a correct spike concentration were discarded from furtheranalysis. Due to interferences with the argon gas, Ca had to be measured with atomicabsorption spectrophotometry (AAS) instead of SB-ICPMS. Samples were analysed in aPerkin-Elmer 5000 atomic absorption spectrophotometer. The final dilution ratio for themeasurement of calcium was 1:50. Standards were SB-ICPMS and AAS grade fromAldrich�.One pair of otoliths, containing a normal and a vaterite otolith, from a fish from each

population (Celtic and Clyde Seas) was analysed by wavelength-dispersive spectrometry(WDS) to assess the spatial variation in composition (Ca, Sr, Na and K) betweenaragonite and vaterite areas of the otoliths. The composition of the inner aragonitearea of the primarily vaterite otoliths was also compared to the composition of theinner aragonite sector from the normal aragonite otoliths in order to test whether initialchemical differences in the inner area of an otolith could explain the shift from aragoniteto vaterite. Otoliths were individually placed on glass slides, embedded in epoxy resin andground and polished along the sagittal plane using diamond suspensions. Samples wereultrasonically cleaned between each polishing stage and before carbon coating. Sampleswere analysed with a Cameca Camebax Microbeam WDS microprobe fitted with fourspectrometers (Table II). Quantitative analysis of each element was achieved by discri-minating the count rates at the peak of the element’s characteristic X-rays from thecontinuous spectrum, before a PAP (Pouchou and Pichoir) correction. The electron beamwas used in raster mode, covering an area of 8�5� 8�5mm to avoid sample damage

(a)

(c)

(b)

(d)

FIG. 1. Aragonite and vaterite otoliths of reared juvenile herring (scale bar¼ 1mm) Note the similarity in

shape of these otoliths, regardless of the morph or extent of vaterite area. (a) Normal aragonite

otolith, (b) otolith with vaterite areas, showing the dimensions measured for morphometry analysis

and (c), (d) examples of the variation between individual fish in the extent of vaterite areas.



(Kalish, 1989, 1991). Otoliths were all analysed along four transects between the nucleusand the outer edge of the otolith in the posterior, ventral and dorsal sides. Concentrationsof the oxide form of each element were transformed into elemental composition usingthe oxide : element mass ratio. Final elemental concentration was expressed in parts permillion (ppm).

DATA ANALYSIS

Fish growth and mass–length relationships were analysed for each population sepa-rately using regression and ANCOVA techniques, after appropriate data transforma-tions. The incidence of vaterite otoliths was compared for each population separately.The w2 statistic was corrected with the Yates correction of continuity (Scherrer, 1984) tocompensate for n< 40. When n< 20 Fisher’s exact test was used instead. Otolith pairswere categorized as normal if both right and left otoliths were aragonite, and vaterite ifone or both otoliths contained vaterite. All comparisons between the individual otolithsof otolith pairs (normal aragonite v. normal aragonite; normal aragonite v. vaterite) weremade for all fish pooled, since the level of comparison was within individual fish andbecause the Celtic and Clyde Sea fish were not significantly different in the proportion offish with vaterite otoliths. The dimensions of left and right otoliths within each pair(normal v. normal, normal v. vaterite) were compared with the t-test for paired samplesto test the relationship between aberrant crystallization and otolith size.

RESULTS

CRYSTALLOGRAPHY

The XRD bulk crystallographic analysis revealed that the abnormal crystal-line otoliths were composed of vaterite and aragonite. Surface analysis of thespatial distribution of the different CaCO3 polymorphs within vaterite otoliths

TABLE II. Analytical conditions of the WDS. The limits of detection (LOD) of the WDSfor each element were calculated using the formula: LOD ¼ 3m�1 �

ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiRBT�1

B

q, where

m¼ counts s�1% element in the standard�1, RB¼ count rate on background (counts s�1),and TB¼ count time on background (s, half time of count time on peak). The precisionof measurement (relative S.D., %) of each element was calculated for each sample usingthe formula: Precision ¼ 100

ffiffiffiffiT

p ffiffiffiffiffiffiRP

p�

ffiffiffiffiffiffiffiRB

p� �� 1, where T¼ count time on peak (s),

RP¼ count rate on peak (counts s�1) and RB¼ count rate on background (counts s�1)

Electron beam operated in raster mode 8�5� 8�5mm

Gun potential: 15 keV

Beam current: 10 nA

Distance between spots: 25 mm

Element Counting times at peak (s) Spectrometer Crystal LOD (ppm)

Calcium 30 3 PET 338Strontium 90 2 TAP 239Sodium 90 1 TAP 112Potassium 60 3 PET 219



with Raman spectrometry indicated that both aragonite and vaterite poly-morphs were present in close proximity, within a volume of 20 mm3 (Fig. 2).

INCIDENCE OF VATERITE OTOLITHS

The juvenile herring with deformed heads or jaws were generally smallcompared to the rest of the population. These abnormalities were observed infish as young as 3 months old (Table I). At c. 1 year old, the normal fish weresignificantly longer (ANOVA, Celtic Sea: d.f.¼ 1 and 107, P< 0�001; Clyde Sea:d.f.¼ 1 and 90, P< 0�001) and heavier (Celtic Sea: d.f.¼ 1 and 107, P< 0�001;Clyde Sea: d.f.¼ 1 and 90, P< 0�001) than the deformed fish (Fig. 3). Initially itappeared that the fish with skull deformities also had a high incidence ofvaterite otoliths, but in fact most had normal aragonite otoliths. The proportionof fish with vaterite otoliths was the same in both populations (w2, P> 0�05).Taking as a reference the fish sampled in 1998 when both normal and deformedfish were sampled, there were no significant differences between healthy anddeformed fish in the number of cases with vaterite otoliths (w2, Celtic and ClydeSeas both P> 0�05). Vaterite deposition occurred with equal frequency in rightand left otoliths of both the Celtic and Clyde Seas fish (Fisher’s exact test, bothP> 0�05) showing that the phenomenon affected equally left and right otolithsin fish from both stocks. None of the youngest fish dissected (Clyde Sea, 93 daysold) had vaterite otoliths, suggesting that the change in otolith formationoccurred in fish >3 months old, well after metamorphosis and the developmentof schooling behaviour. The length–mass relationship of fish with vateriteotoliths did not differ significantly from fish with normal otoliths in Celtic

0

500

1000

1500

2000

2500

0 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500

Rel

ativ

e fr

eque

ncy

(va

teri

te a

nd

vate

rite

+ a

rago

nit

e)

0

5000

10 000

15 000

20 000

25 000

30 000

35 000

Rel

ativ

e fr

eque

ncy

(ar

agon

ite)

Vaterite with aragonite tracesVateriteAragonite

Raman shift (cm–1)

FIG. 2. Raman spectra from the vaterite otolith of herring analysed with the Raman spectrometer.

Despite displaying the characteristic peaks of the vaterite at 106, 266 and 301 cm�1, the Raman

spectra from a different location on the same otolith also showed the existence of two very clear

peaks at 152 and 204 cm�1 characteristic of the aragonite (!�). The scattering volume was 20mm3 and

indicates the spatial scale at which both aragonite and vaterite may co-precipitate.



(ANCOVA, d.f.¼ 1 and 168, P> 0�05) and Clyde fish (ANCOVA, d.f.¼ 1 and144, P> 0�05). Thus, among the deformed fish examined, there was no furthereffect of otolith crystallisation on fish condition. Among the adult herringsampled in the wild in the Celtic Sea, two adults out of 36 had vaterite intheir otoliths (Table I).

MORPHOMETRY OF ARAGONITE AND VATERITE OTOLITHS

Left and right otoliths of normal aragonite pairs differed significantly inlength only; left otoliths were longer than right otoliths (Table III). In vateritepairs, the vaterite otoliths were significantly larger in length, perimeter and area,and were also significantly lighter than their aragonite partners (Table III). Theextent of vaterite deposition was highly variable and ranged from 12 to 91% ofthe total otolith area (Figs 1 and 4). There was a significant relationship between

Fis

h m

ass

(g)

Celtic

05

10152025

Healthy Deformed Clyde

Healthy Deformed

LT

(m

m)

Celtic

0

40

80

120

160

Healthy Deformed Clyde

Healthy Deformed

(a)

(b)

FIG. 3. Differences in (a) mass and (b) total length of healthy and deformed reared juvenile herring from

the Celtic and Clyde populations (mean� S.D.).

TABLE III. Summary of the statistical differences between normal (both aragonite) andvaterite (one vaterite, one aragonite) pairs of otoliths of juvenile herring from the Celticand Clyde Sea populations. Significant differences are highlighted in bold. td, statistic of

the t-test for paired samples; L, left; R, right; A, aragonite; V, vaterite

Area (mm2) Perimeter (mm) Length (mm) Width (mm) Mass (mg)

Normal pairs �xxL¼ 2 276 106 �xxL¼ 7566�6 �xxL¼ 2504�8 �xxL¼ 1307�5 �xxL¼ 1�218n¼ 101 �xxR¼ 2 277 341 �xxR¼ 7237�1 �xxR¼ 2490�6 �xxR¼ 1310�6 �xxR¼ 1�214

td¼�0�16 td¼ 1�41 td¼ 2�57 td¼ 0�67 td¼ 0�52P> 0�05 P> 0�05 P< 0.05 P> 0�05 P> 0�05

Vaterite pairs �xxA¼ 2 408 805 �xxA¼ 7654�8 �xxA¼ 2582�8 �xxA¼ 1338�6 �xxA¼ 1�298n¼ 26 �xxV¼ 2 536 751 �xxV¼ 9140�1 �xxV¼ 2643�5 �xxV¼ 1354�7 �xxV¼ 1�073

td¼�3�18 td¼�6�71 td¼�2�56 td¼�1�41 td¼ 7�58P< 0.05 P< 0.05 P< 0.05 P> 0�05 P< 0.001



the proportion of the total otolith area occupied by vaterite and the differencein weight between the vaterite and its aragonite pair (ANOVA, d.f.¼ 1 and 23,P< 0�05; Fig. 4). Overall, 66% of the difference in otolith mass was explained bythe extent of the vaterite area. The absolute size and outer boundary of thearagonite inner area of vaterite otoliths varied between individuals, indicatingthat the shift from aragonite to vaterite deposition occurred at different timesfor each individual fish. Based on the calculated relationship between the otolitharea and fish age for fish with normal otoliths, vaterite deposition commencedbetween 4–10 months of age (Fig. 5). No vaterite otoliths were found in 3 monthold fish that were examined. There was no relationship between the size of theinner aragonite sector and the total size of vaterite otoliths, so total otolithgrowth was independent of when the shift to vaterite deposition occurred.Regardless of the differences in the dimensions of vaterite otoliths whencompared with their respective aragonite pairs, the overall shape of the vateriteotolith was the same as that of the aragonite otolith, indicating that otolithshape and polymorph precipitation were not controlled by the same mechan-isms. The dimensions of the aragonite otoliths from each vaterite pair were alsocompared to the dimensions of aragonite otoliths from normal pairs to estalishwhether the presence of a vaterite otolith had any effect on the growth anddevelopment of its aragonite partner. There were no significant differences indimension between the aragonite otoliths from normal pairs compared to thosefrom vaterite pairs (Table IV) except for the otolith perimeter, probably becausethe outer edge was more crenulated in some otoliths than in others. The shiftfrom aragonite to vaterite deposition was a local phenomenon since vateritedeposition in one otolith did not affect the dimensions of the otolith growingnormally in the other sacculus.

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00 100.00

Per cent of vaterite replacement

Dif

fere

nce

in m

ass

betw

een

the

vat

erit

icot

olit

h an

d it

s n

orm

al p

air

(mg)

FIG. 4. Relationship between the extent of vaterite areas and the difference in mass between the vaterite

otolith and its aragonite pair. The curve was fitted by y¼ 0�0036x� 0�0176 ( r2¼ 0�66).



COMPOSITION OF VATERITE AND NORMAL OTOLITHPAIRS

The concentrations of Ca, Sr, Co, Ni, Mn and Sc were measured usingSB-ICPMS and compared between vaterite and normal otoliths. There wereno significant differences in composition between the two otoliths of normalpairs. The normal and vaterite otoliths within vaterite pairs had similar con-centrations of Ca, Co, Ni and Sc, but Sr concentrations were much lower andMn concentrations much higher in the vaterite otoliths compared to the normalaragonite otoliths (Fig. 6). These results indicate the relevance of the crystalstructure in determining the final composition of the otolith.

0

50

100

150

200

250

300

350

400

450

0.00E + 00 5.00E + 05 1.00E + 06 1.50E + 06 2.00E + 06 2.50E + 06 3.00E + 06 3.50E + 06

Otolith area (µm2)

Age

(in

day

s af

ter

hatc

hin

g)

FIG. 5. Relationship between the total otolith area and the age of juvenile herring from the Clyde Sea

population only. ^, aragonite otoliths; &, inner aragonite area in vaterite otoliths. This relationship

was used to estimate the age at which the vaterite starts precipitating in Clyde Sea fish. The curve was

fitted by y¼ 0�0001xþ 94�66 (r2¼ 0�83).

TABLE IV. One-way ANOVA between the different otolith dimensions of the aragoniteotoliths from normal pairs and from vaterite pairs. The data used to characterise

aragonite pairs came from the left otoliths. A, aragonite; V, vaterite

d.f. source MS source d.f. error MS error F P SNK test

Area 1 364 101� 106 125 181 253� 106 2�00 >0�05 –Perimeter 1 3 117 559 125 767 860 4�06 <0�05 V>ALength 1 125 867 125 82 429 1�52 >0�05 –Width 1 20 068 125 12 681 1�58 >0�05 –Mass 1 0�13 125 0�08 1�48 >0�05 –



SPATIAL VARIATION IN THE ELEMENTAL COMPOSITIONOF VATERITE OTOLITHS

The chemical transition between the aragonite part and the vaterite part ofthe otoliths was sharply delineated. WDS analyses along transects from the coreto the outer edge of the otolith showed conclusive evidence that Sr, Na and Kconcentrations were much lower in the vaterite part of the otolith. Only 51% ofthe Sr measurements and 86% of the K measurements were above the analyticaldetection limits (Table II) in the vaterite area of the otoliths (Figs 7 and 8). Theconcentrations of Ca, Sr, Na and K at 15 randomly chosen spots within each ofthe aragonite and vaterite parts of the otoliths were compared. Vaterite areas ofthe otoliths had significantly lower concentrations of Sr, Na and K thanaragonite areas (ANOVA, Sr: d.f.¼ 1 and 58, P< 0�001; Na: d.f.¼ 1 and 58,P< 0�001; K: d.f.¼ 1 and 58, P< 0�001) whereas Ca concentrations did notshow significant differences (d.f.¼ 1 and 58, P> 0�05) (Fig. 9). There were nosignificant differences in Ca, Sr, Na or K concentrations between the inneraragonite part of the vaterite otoliths and the equivalent inner part of thenormal otoliths (ANOVA, d.f.¼ 1 and 61, all P> 0�05), indicating that theshift from aragonite to vaterite is independent of the elemental composition ofthe inner otolith area.

Cal

cium

(µg

g–1

)

Normalotoliths

0

5000

10 000

15 000

20 000

25 000

30 000

35 000

40 000

45 000

50 000

Left Right

Vateriteotoliths

Left Right

Normalotoliths

Left Right

Vateriteotoliths

Left Right

Normalotoliths

Left Right

Vateriteotoliths

Left Right

Normalotoliths

Left Right

Vateriteotoliths

Left Right

Normalotoliths

Left Right

Vateriteotoliths

Left Right

Normalotoliths

Left Right

Vateriteotoliths

Left Right

Stro

nti

um 8

8 (µ

g g–1

)

0

200

400

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1000

Nic

kel 6

0 (µ

g g–1

)Sc

andi

um 4

5 (µ

g g–1

)

0

10

20

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50

60

Cob

alt

59 (

µg g

–1)

0.00

0.01

0.02

0.03

0.04

0.0

0.1

0.2

0.5

0.4

0.3

0.8

0.7

0.6

Man

gan

ese

55 (

µg g

–1)

0

1

2

3

4

5

6

7

(a) (b) (c)

(f)(e)(d)

FIG. 6. SB-ICPMS results of the analysis of aragonite and vaterite herring otoliths (means� S.D.).

Vaterite samples are presented as left otoliths for convenience of representation. (a) Calcium, (b)

strontium, (c) nickel, (d) cobalt, (e) manganese and (f) scandium.



DISCUSSION

The occurrence of different CaCO3 polymorphs in otoliths of teleosts is welldocumented (Gauldie, 1993), but this is the first report of vaterite otoliths inC. harengus. Vaterite and calcite otoliths have been observed both in reared

Sr, N

a an

d K

con

cen

trat

ion

s (p

pm)

Ca

con

cen

trat

ion

(pp

m)

0

200

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2000

0

200

400

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800

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1200

1400

1600

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2000

0 100 200 300 400 500 600 700 800

0 100 200 300 400 500 600 700 800

Distance from the nucleus (µm)

0

25 000

50 000

75 000

100 000

125 000

150 000

175 000

200 000

225 000

250 000

275 000

300 000

325 000

350 000

375 000

400 000

0

25 000

50 000

75 000

100 000

125 000

150 000

175 000

200 000

225 000

250 000

275 000

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FIG. 7. WDS results of the analysis ( &, Na; &, Sr; &, K; ~, Ca) of an otolith pair from a reared herring

from the Celtic Sea population. (a) Aragonite and, (b) vaterite otoliths. Horizontal lines represent

the limits of detection (LOD) of the WDS measurements for Ca, Sr, K and Na from top to bottom

under the analytical conditions detailed in Table II.



species such as chinook salmon Oncorhynchus tshawytscha (Walbaum) (Gauldie,1986, 1996) and lake trout Salvelinus namaycush (Walbaum) (Bowen II et al.,1999), and in fishes from wild populations (Morales-Nin, 1985; Strong et al.,1986; Gauldie, 1993; Brown & Severin, 1999). In the present study, the frequen-cies of vaterite otoliths of juvenile herring (13�9% in the Celtic Sea populationand 7�8% in the Clyde Sea population) were lower than those reported instudies of other reared species [34% for juvenile chinook salmon (Gauldie,1986, 1996), 26–41% for stocked lake trout (Bowen II et al., 1999)] but still

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Distance from the nucleus (µm)

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pm)

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FIG. 8. WDS results of the analysis ( &, Na; &, Sr; &, K; ~, Ca) of an otolith pair from a reared herring

from the Clyde Sea population. (a) Aragonite and, (b) vaterite otoliths. Horizontal lines represent

the limits of detection (LOD) of the WDS measurements for Ca, Sr, K and Na from top to bottom

under the analytical conditions detailed in Table II.



higher than the range reported for samples of fishes caught in the wild [2–5% inpollock Pollachius virens (L.) (Strong et al., 1986), 3% in Genypterus capensis(Smith) (Morales-Nin, 1985) and between 0�4 and 14% in a variety of species(Gauldie, 1993)]. Vaterite otoliths were found in reared red drum Sciaenopsocellatus (L.), but not in wild juveniles (David et al., 1994). In the present study,only two of the 36 (5�5%) adult herring from the wild caught in the Celtic Seahad vaterite otoliths. This was a smaller percentage than found in the juvenileherring of the same population reared in the laboratory (13�9%), but couldsimply be due to the selective mortality between juvenile and adult stages ratherthan the result of rearing conditions.Otolith malformations ultimately express otic malformations, which could

also be the expression of malfunctioning genes (Gauldie, 1986). Complicationsduring the development of the neural crest could result in otolith malformationstogether with deformities in the head of the fish. Gene loci mutations that causemalformations in the vestibular and auditory apparatus, including the otoliths,have been mapped on the zebrafish genome (Malicki et al., 1996; Whitfield et al.,1996). These zebrafish malformations were expressed during embryonic devel-opment, but in this herring study most vaterite otoliths had an aragonite coreand juveniles sampled 93 days after hatching all had normal otoliths. Theindividual variation in age at the shift to vaterite deposition indicates that itis not the result of a pre-programmed disruption associated with ontogeny.External factors including stress, food quality, or viral infection might inducethe expression of mutated genes. The fact that the composition of the aragoniteportion of the vaterite otoliths did not differ from the same sector in normal

K (

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50 0001e5

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FIG. 9. Comparisons of the composition (a) K, (b) Ca, (c) Sr and (d) Na (means� S.D.) of the inner

aragonite area and the outer vaterite area in the otolith pairs analysed with the WDS. *, Significant

differences (P¼ 0�05) between means.



otoliths strongly suggests that the development of the otolith had been normalup to that point.

EFFECT ON FISH

The vaterite otoliths were bigger in area, perimeter and length than theiraragonite pairs, probably because the vaterite crystal system is larger than thearagonite crystal system. Wardlaw et al. (1978) calculated that a molecule-by-molecule replacement of aragonite by calcite in the shells of a marine gastropod,Strombus gigas resulted in an 8% increase in volume. The vaterite otoliths stillheld the characteristic herring shape, as observed in the vaterite and calciteotoliths of other species (Morales-Nin, 1985; Gauldie, 1986; Strong et al., 1986).The different CaCO3 polymorphs may affect the functioning of the inner earsince otolith function is determined by density, size and shape. Vaterite deposi-tion resulted in lighter, but larger, otoliths. A normal otolith has a density ofc. 3 g cm�3 (Fay & Simmons, 1999), corresponding to the density of aragonite.Inorganic vaterite is less dense (2�54 g cm�3) than inorganic aragonite(2�94–2�95 g cm�3) and the results of this study confirm that biovaterite is alsoless dense than bioaragonite. The lower density of vaterite otoliths could pro-duce a delayed response of lower intensity compared to an aragonite otolith.Density differences between the left and right otoliths could impair the detectionof sound (Popper & Lu, 2000).In reality, there was little influence of aberrant otoliths on the growth of

juvenile herring used in this study, since the growth of deformed fish withnormal otoliths was no different from that of deformed fish with aberrantotoliths. Deformed fish grew more slowly that the normal looking fish, butthe frequency of occurrence of vaterite otoliths was not different betweennormal and deformed fish. This indicates that the crystal form of the otolithwas not directly linked to fish growth, nor did skull deformities directly result invaterite deposition. Low growth was caused by the deformities in the headrather than by otolith deformities, probably because the deformities in themouth, gills and jaws prevented the fish from eating properly. Herring useboth visual and acoustic signals for feeding and predator avoidance (Blaxter& Fuiman, 1990). Schooling behaviour may also counteract any effects ofasymmetry in the otolith density. Herring schools in the wild display a direc-tional avoidance away from a sound source (Olsen, 1976). Individual herringwith vaterite otoliths may rely on visual cues and the response of the school toaccess food and avoid danger.It may be that fish survival is not dependant on the saccular otoliths, and the

fishes have other sensory organs to sense the surrounding environment and maylearn to compensate for the differences in the information received by one of thetwo ears (Lombarte et al., 1993). In some fish species there is a significantrelationship between the area of the otolith and the area of the sensory epithelia(Lombarte & Fortuno, 1992; Arellano et al., 1995), indicating that certainotolith dimensions are important for the correct functioning of the inner ear.Moreover, the heterogeneity of hair cells in the sensory epithelia has led someauthors to consider that different parts of the otolith are involved in theperception of sound of different frequencies (Popper et al., 1993). Vaterite



otoliths have probably more ‘to say’ about the mechanisms of otolith mineral-ization than about functional anatomy.

DIFFERENCE BETWEEN VATERITE AND ARAGONITE

In terms of the microchemistry of the otoliths, there was a dramatic change incomposition between the aragonite and vaterite areas of the otoliths. Theconcentrations of Na, Sr and K were significantly depleted in the vateriteareas, though the decreases in Na were less marked. These elements were alsodepleted in the vaterite otoliths of Stenodus leucichthys (Guldenstadt) (Brown &Severin, 1999) and chinook salmon (Gauldie, 1996). The change in compositionof the vaterite compared to aragonite otolith sections could be explained by thegeometry of the crystal, resulting in molecular conformations with little roomfor inclusion of other elements (Curti, 1999). Sr (1�13 A) and K (1�33 A) havelarger ionic radiuses than Ca (0�99 A) and would be included less readily,whereas Na (0�95 A) has a smaller ionic radius and its inclusion may be lessaffected by the shift from aragonite to vaterite. Sodium ions may be trapped invaterite crystals defects (Gauldie, 1986; Brown & Severin, 1999) rather thansubstituting for Caþ ions (Busenberg & Plummer, 1985). Manganese was moreconcentrated in the vaterite otoliths than in the aragonite otoliths and has beenobserved to be strongly associated with vaterite precipitation and is used as atracer of vaterite to calcite transformation (Nassrallah-Aboukais et al., 1996,1998). Manganese is incorporated into the vaterite crystal lattice (Brecevic et al.,1996) by adsorption followed by surface precipitation, forming a ‘manganesecoating’ that decreases the solubility of the vaterite (Nassrallah-Aboukais et al.,1998) preventing its dissolution. It is soluble at low pH (Fraser & Harvey, 1982;Moreau et al., 1983) and its presence in the vaterite otoliths may indicate pHand osmoregulatory changes that could also facilitate a reduction in Ca influxand in Na efflux (Reader & Morris, 1988).There was no evidence to suggest that changes in the composition of the

aragonite areas of the otolith caused the shift to vaterite deposition since theconcentrations of Ca, Sr, Na and K in the inner aragonite sector of vateriteotoliths were the same as the equivalent sector of the aragonite pair. The shiftwas a localized phenomenon because it happened generally only in one ear andsometimes only in a certain region of the otolith. The physico-chemical condi-tions of the endolymph may be variable around the otolith and allow vateriteand aragonite to precipitate concurrently at the small spatial scale revealed bythe Raman analyses. In most fishes with aragonite otoliths the asteriscii areentirely vaterite (Chesney et al., 1998; Campana, 1999), indicating that theshared endolymph (Gauldie, 1993) is capable of producing the aragonite sagittaand vaterite asteriscus simultaneously, even though controlled by independentmechanisms (Riley & Grunwald, 1996). In conditions of high solubility vateriteis precipitated in preference to aragonite or calcite (Dalas et al., 2000). Loca-lized changes in the composition of the endolymph may affect the solubility ofthe solution at the surface of the growing otolith. In vitro experiments (Faliniet al., 1996) have shown that vaterite co-precipitates with aragonite in thepresence of aragonite-coding proteins, when the ion flux at the surface of



precipitation is reduced resulting in small scale spatial differences similar inscale to those observed in the otolith using the Raman spectrometer.Since the shape of the otoliths was unaltered by polymorph replacement, the

possibility that the vaterite replacement had started from the outside of theotolith to the inside was considered, so that diagenesis (a process of recrystal-lisation) explained the presence of vaterite or calcite. Diagenesis (of aragoniteinto calcite) occurs in fossils subject to pressure, temperature or pH influenceover a geological time scale (Wardlaw et al., 1978), but also as an instantaneousprocess as demonstrated by immersing fossil and living coral in a solution ofsodium chloride (Yoshioka et al., 1985). Nonetheless, since diagenesis can betraced by the unequivocal high Sr concentrations that remain in the aragonitewhen it changes to calcite (Wardlaw et al., 1978) and the present results showedthat vaterite areas had very low Sr concentrations, diagenesis was not likely tobe the cause of vaterite in the herring otoliths.The comparisons between vaterite and aragonite otolith areas also demon-

strated problems inherent with WDS sampling of CaCO3 of different densities.Vaterite and aragonite otoliths differed in density, yet the WDS was not able toshow this difference in absolute Ca concentrations. This is explained by the factthat concentrations are reported in ppm in mass not in volume, and so theamount of calcium in the vaterite is the same as in the aragonite (40%), as is theamount of carbonate (60%). For a given accelerating voltage, electronspenetrate deeper into a vaterite sample than in an aragonite sample and thevolume sampled is greater in vaterite than in aragonite. The method of correc-tion of count rates of the WDS used in this study (the Pouchou & Pichoirmethod and by extension any ZAF correction procedure) cancels out any effectsof sample density, and thus is insensitive to differences between vaterite andaragonite. Consequently, the analytical results from surface analysis techniquessuch as the WDS cannot be used to study differences in otolith density.

We are indebted to P. Hill (Faculty of Geology and Geophysics of the University ofEdinburgh, U.K.) for access to the NERC funded WDS facility and to the Faculty XRDfacility, and for valuable discussions on the use of the WDS. We thank R. Johnston (StrixLtd., Castletown, Isle of Man) for the Raman analyses. Thanks are also due toN. Fullerton for supplying the reared fish from the Larval Rearing Centre of the PortErin Marine Laboratory and to F. McArdle (Royal Hospital, University of Liverpool)for the ICPMS analyses. The manuscript was considerably improved by the constructivecomments made by G. Pilling.

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morphometry and composition of aragonite and vaterite otoliths of

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