Elemental composition of otoliths as a discriminator of life stage and growth habitat of the European eel, Anguilla anguilla.

Elemental composition of otoliths as a discriminator of life stage

and growth habitat of the European eel, Anguilla anguilla

W. N. Tzeng

, K. P. Severin

, C. H. Wang

and H. Wickström

A

_{Institute of Fisheries Science, College of Life Science, National Taiwan University, Taipei 106, Taiwan.}

_{Department of Geology and Geophysics, University of Alaska Fairbanks, Alaska 99775-0760, USA.}

C

_{National Board of Fisheries, Institute of Freshwater Research, SE-178 93 Drottningholm, Sweden.}

_{Corresponding author. Email: wnt@ccms.ntu.edu.tw}

Abstract. The hypothesis that elemental composition of otoliths of the eel (Anguilla spp.) changes with life stage

and growth habitat was tested in the present study. The minor elements Cl, Na, K, Mg, Ca, Sr and P in otoliths

of European eels (Anguilla anguilla) were examined by using an Electron Probe Microanalyser (EPMA) equipped

with wavelength dispersive spectrometers (Cameca SX-50). Yellow-stage eels were collected from coastal waters

and lakes of Sweden in 1987, 1988, 1991, and 1994, with ages ranging from 5 to 18 years old. Strontium maps

and profiles of Sr : Ca ratio, as well as the elver check in otoliths, were used to classify life history stages of

the eels as leptocephalus, and freshwater- and seawater-resident yellow eels. Canonical score plots of the otolith

elemental compositions of the freshwater-resident yellow eel were completely separated from those of leptocephalus

and seawater-resident yellow eel, but the latter two partially overlapped. Strontium is the primary component in

determining the discrimination, but the nutrient-related (S and P), and the physiologically controlled elements

(Na and Cl), may also play an important role in the discrimination. These results indicate that multiple-elemental

information can provide additional insight into the migratory environmental history of diadromous fishes.

Extra keywords: electron probe microanalysis, microchemistry.

Introduction

The European eel, Anguilla anguilla (L.), is a diadromous

fish, widely distributed in freshwater and marine littoral areas

of North Africa, the Mediterranean Sea, the British Isles,

Iceland, and the western and northern European continent

(Tesch 2003). After spawning in the Sargasso Sea, the

lepto-cephali are transported by the North Equatorial Current, Gulf

Stream and North Atlantic Current to the continental shelf of

northern European countries (Bertin 1956). The larvae

meta-morphose into glass eels in coastal waters. Glass eels become

pigmented elvers when they enter estuaries. Their migration

from the Sargasso Sea to the estuaries requires 6–9 months

(Lecomte-Finiger 1992), or 14–16 months (Wang and Tzeng

2000). Male eels grow in rivers for 3–7 years, whereas female

eels grow from 4 to 15 years in rivers (Vollestad and Jonsson

1986). In late autumn, they transform from the yellow eel

into silver eel stage and start the downstream migration back

to the Sargasso Sea. Recent studies on Sr (strontium) : Ca

(calcium) ratios in otoliths indicate that a part of the eel

popu-lation may skip the freshwater life of the yellow eel phase and

can complete their entire life history in the seawater (Tzeng

et al. 1997, 2000; Tsukamoto et al. 1998; Tsukamoto and

Arai 2001).

Ratios of Sr : Ca in the otolith of anguillid eels

dramati-cally change at metamorphosis from leptocephalus to glass

eel during their migration from the ocean to the river (Otake

et al. 1994; Tzeng and Tsai 1994; Arai et al. 1997). This

drastic change in Sr/Ca ratios was proposed to be related to

metamorphosis rather than the transition of habitat from

sea-water to fresh sea-water (Tzeng 1996). In contrast, the Sr : Ca

ratios in otoliths of yellow eels changed significantly when

the eel migrated between fresh water and seawater (Tzeng

et al. 1997, 2002, 2003a, 2003b; Jessop et al. 2002, 2004;

Kraus and Secor 2003; Limburg et al. 2003; Shiao et al. 2003;

Cairns et al. 2004). These indicated that the changes in otolith

microchemistry throughout their life history of the eel were

more complicated than our current understanding.

There-fore, it is important to examine how elemental composition

of eel otoliths is influenced by both ontogenetic

develop-ments and habitat shift before using the elemental signature

to reconstruct their migratory environmental history.

In the present study, elements in addition to Ca and Sr

were measured to further understand the change in elemental

composition of otoliths in European eels during

metamor-phosis from leptocephalus to glass eel and during migration

of yellow-phase eels between fresh water and seawater.

Materials and methods

Ten otoliths of European eels collected from three freshwater lakes and two brackish estuaries in Sweden were used for microchemical analysis. These samples were classified into four groups according to sampling sites (Tzeng et al. 1997). The origin of groups 1 through 3 was clear: these eels were captured from areas where the restocking programme is well known. The individuals of group 4 were collected from a site some kilometres away from Eastern Lake Mälaren in 1994, and the origin of this group was unknown. They were derived either from a natural population that had migrated from brackish Baltic Sea, or from a stocked population caught from brackish waters on the west coast of Sweden and released at the yellow eel stage at∼40 cm in total length, or from a stocked population released at the elver stage imported from France or the British Isles. The stocking and sampling dates and biological characteristics of the four groups of eels (including sampling date, mean (± s.d.) salinity of sampling sites, stage, age, total length, bodyweight, and the stage and year at stocking) are given in Table 1.

Microchemical analysis

After removal from the eel, the otoliths were cleaned with distilled water, dried in air, embedded in thermo-epoxy (Petropoxy 154; Palouse Petro Products, Pullman, WA) and cured for 1 h at 135◦C. Embedded otoliths were ground from the proximal side of the sagittal plane of the fish until the primordium of the otolith was revealed. For microprobe analysis, the polished otoliths were coated under vacuum with a 30 nm layer of carbon to increase electron conductance.

The elemental composition of the eel otolith was measured with an electron probe microanalyser (EPMA) equipped with wavelength dispersive X-ray spectrometer (Cameca SX-50, Paris, France). Mea-surements were made at∼18 µm intervals along a transect from the primordium to the edge of the otolith for each of the 10 yellow eels in Table 1. The beam condition of the EPMA used in the measurement of these elements was 15 kV, 4 nA, and a 15µm diameter beam. The EPMA has minimum detection limits of a few hundred ppm (Statham 1981, 1982) and is not sensitive enough to detect trace elements. As a result of the detection limit of EPMA, only calcium and seven minor elements (Na, Mg, Cl, P, S, K and Sr) were selected for analysis. The counting time, standard for calibration, detection limit and analytical error for each of the eight elements are listed in Appendix 1.

Data analysis

The element : Ca concentration ratios on each sampling spot of the otoliths were calculated by weight (%) and grouped by stage and habitat use, which determined the life-history stages of leptocephalus of both

Table 1. Life history of the four groups of yellow stage, female European eels used in the present study

Group Specimen no. Sampling date Sampling site Salinity Age Total Bodyweight Stage and year

(psu) (years) length (mm) (g) at stocking

1 11538 9 Jul 1987 Strömstad (West Coast) 25.54± 3.38 5+ 393 96 Natural

11550 9 Jul 1987 Strömstad (West Coast) 25.54± 3.38 7+ 410 90 Natural

11906 20 Aug 1987 Björkö (West Coast) 23.12± 3.71 9+ 403 96 Natural

2 13743 31 May 1988 Eastern Lake Mälaren Fresh water 8+ 562 442 Elver, 1980

18589 7 Jun 1991 Eastern Lake Mälaren Fresh water 11+ 717 566 Elver, 1980

3 18793 1 Jul 1991 Lake Ången Fresh water ≥14+ 760 820 Yellow eel, 1979

19315 16 Jul 1991 Lake Ången Fresh water ≥18+ 728 668 Yellow eel, 1979

4 25106 30 Aug 1994 Lake Mälaren Fresh water 13+ 723 597 Unknown origin

25108 30 Aug 1994 Lake Mälaren Fresh water 18+ 721 535 Unknown origin

25110 30 Aug 1994 Lake Mälaren Fresh water 14+ 680 461 Unknown origin

freshwater- and seawater-resident yellow eels. Leptocephalus and yel-low eel were separated by the elver check on the otolith (Shiao et al. 2003) and yellow eels were further divided into freshwater and seawater residents according to the Sr concentration on the Sr map and the Sr : Ca ratio profile in their otoliths (Tzeng et al. 1997, 2002, 2003a, 2003b). Freshwater-resident yellow eel referred to the individuals with a Sr : Ca ratio less than 4× 10−3in between the elver check and the otolith edge. Seawater-resident yellow eel had Sr : Ca ratios higher than 4× 10−3 indicating that the individuals did not migrate upstream and lived in high salinity seawater during the yellow eel phase. The estuarine-resident yellow eel migrated between fresh water and seawater.

The significant difference of the mean values of the Ca element concentration ratios among stages of the eel (leptocephalus, freshwater-and seawater-resident yellow eels) were tested by non-parametric Tukey-type multiple comparisons. The significance of the correlation among different Ca element ratios on each sampling spots was tested for each life history stages by Pearson product-moment correlation coefficient (Zar 1984). The contribution of the elemental composition to the group-ing of the above mentioned life history stages was analysed by usgroup-ing stepwise Canonical Discriminant Analysis. The classification success of the life history stages by the elemental composition was calculated with a discriminant function analysis. All the statistics were preformed by using the software SPSS (SPSS Inc., Chicago, IL).

Results

Otolith elemental composition changed with life history

stage and habitat use

The mean value of the seven elements : Ca ratios within

otoliths of the yellow eel were ranked in descending order by

life history stages and habitat use (Table 2). The Sr : Ca ratios

constituted the first ranking in the elemental composition in

the leptocephalus stage, but dropped to second rank in those

of seawater-resident yellow eel stage and to the sixth rank in

freshwater-resident yellow eel stage. On the contrary, Na : Ca

ratios rose to the first rank in both seawater- and

freshwater-resident yellow eels. This indicated that many elements other

than Sr in otoliths of the eel changed with developmental

stage and habitat use.

Each of the mean (± s.e.) element : Ca ratios in otoliths

were also compared among life history stages and

habi-tat use (Table 2). Tukey’s honestly significantly different

(HSD) tests indicated that Sr : Ca ratios in otoliths of the

eel were the highest at marine leptocephalus stage (1.16

×

10

± 5.56 × 10

), followed by seawater-resident yellow

eel (4.75

× 10

± 1.04 × 10

), and it was the lowest in

freshwater-resident yellow eels (3.36

× 10

± 3.32 × 10

)

(Table 2). Similarly, the ratios of K : Ca, Cl : Ca, P : Ca, and

S : Ca were higher in the leptocephalus stage compared with

the freshwater- and seawater-resident yellow eels (Table 2).

The ratios of Na : Ca and Mg : Ca were higher in

lepto-cephalus than seawater-resident yellow eels. The ratio of

Na : Ca was higher in freshwater than seawater-resident

yel-low eels (Table 2). These indicated that except for Sr : Ca

ratios, the other six element : Ca ratios also changed with life

history stages and habitat use of the yellow eels.

Table 2. Ranking of the seven otolith elements to calcium concentration ratios by life history stages of the eel Ratios were compared among stages

Me/Ca Leptocephalus (n= 99) Seawater-resident Yellow eel (n= 392) Freshwater-resident Yellow eel (n = 304) Comparison by

Mean s.e. Rank Mean s.e. Rank Mean s.e. Rank Tukey’s HSD test

Sr/Ca 1.16× 10−2 5.56× 10−4 1 4.75× 10−3 1.04× 10−4 2 3.36× 10−4 3.32× 10−5 6 a > b > c Na/Ca 8.94× 10−3 3.82× 10−4 2 7.85× 10−3 1.1× 10−4 1 8.39× 10−3 9.57× 10−5 1 a= c > b Cl/Ca 1.48× 10−3 1.70× 10−4 3 1.05× 10−3 4.13× 10−5 3 9.27× 10−4 4.46× 10−5 2 a > b= c S/Ca 1.07× 10−3 4.49× 10−5 4 7.97× 10−4 1.74× 10−5 4 7.92× 10−4 2.07× 10−5 3 a > b= c P/Ca 8.98× 10−4 6.07× 10−5 5 5.52× 10−4 2.28× 10−5 6 5.95× 10−4 2.76× 10−5 5 a > b= c K/Ca 8.22× 10−4 5.80× 10−5 6 6.9× 10−4 1.59× 10−5 5 6.39× 10−4 1.48× 10−5 4 a > b= c Mg/Ca 2.76× 10−4 2.78× 10−5 7 2.02× 10−4 1.03× 10−5 7 2.14× 10−4 1.8× 0−5 7 a > b; c= a, b HSD, Honestly significantly different.

Table 3. Pearson correlation coefficient among seven elements : Ca ratios in weight % within otoliths of the eel by life history stage/habitat use

Sr : Ca K : Ca Cl : Ca P : Ca S : Ca Na : Ca Mg : Ca Leptocephalus Sr : Ca 1 K : Ca 0.15 1 Cl : Ca 0.19 0.62*** 1 P : Ca 0.38*** 0.19 0.05 1 S : Ca 0.31** 0.27** 0.27** 0.16 1 Na : Ca 0.03 0.39*** 0.86*** −0.07 0.14 1 Mg : Ca 0.09 0.21* 0.24* −0.01 0.13 0.22* 1

Seawater-resident yellow eel

Sr : Ca 1 K : Ca 0.24*** 1 Cl : Ca 0.35*** 0.14** 1 P : Ca 0.03 0.05 −0.12* 1 S : Ca −0.11* 0.01 0.01 −0.02 1 Na : Ca 0.32*** 0.14** 0.72*** −0.12* 0.03 1 Mg : Ca −0.04 0.04 0.01 0.06 0.05 –0.03 1

Freshwater-resident yellow eel

Sr : Ca 1 K : Ca 0.12* 1 Cl : Ca 0.03 0.02 1 P : Ca −0.04 0.02 0.13** 1 S : Ca 0.02 0.09 0.20*** 0.07 1 Na : Ca 0.01 −0.04 0.62*** 0.17** 0.25*** 1 Mg : Ca −0.07 0.07 0.06 0.07 0.06 0.02 1 *P < 0.05; **P < 0.01; ***P < 0.001.

Stage/habitat use mediated correlation among

element : Ca ratios in otoliths

The correlations matrix of element : Ca ratios in otoliths of

the eel were different in different life history stages and

habi-tat use (Table 3), for example, Sr : Ca ratios were positively

correlated with K : Ca, Cl : Ca and Na : Ca, and negatively

with S : Ca in seawater-resident yellow eel stage, but

posi-tively correlated with P : Ca and S : Ca in the leptocephalus

stage, and only slightly positively correlated with K : Ca

in freshwater-resident yellow eels. Similarly, the other

ele-ment : Ca ratios also have different combinations of cross

correlation. This may indicate that uptake of elements was

discriminated among different life history stages of the eel.

Classification of the life history stage and habitat

use of the eel by otolith multiple elements

Forward stepwise discriminant function analysis indicated

that the other five element : Ca ratios (except K : Ca and

Mg : Ca) contributed significantly to the discrimination of

the life history stage and habitat use of the eel (Table 4). The

relative contribution of the element : Ca ratio to the groupings

are listed in decreasing order as Sr : Ca, Na : Ca, P : Ca, S : Ca

and Cl : Ca (Table 4).

The plot of the first and second components of the

canonical discriminant functions indicated that

freshwater-resident yellow eels can be clearly separated from the

lepto-cephali and seawater-resident yellow eels by the five selected

element : Ca ratios in otoliths of the eel, but the latter two

partially overlapped (Fig. 1). The first component of the

canonical discriminant function contributed 96.3% of the

variance for the grouping of the eels whereas the second

component only contributed 3.7% of the variance. These two

components significantly contributed in the discrimination

(χ

-square test, P < 0.001). In the first component, the Sr : Ca

ratios contributed the greatest, followed by Na : Ca and S : Ca.

In the second component of the canonical discriminant

func-tion, the Na : Ca ratios were the most important, followed by

Cl : Ca, P : Ca and S : Ca (Table 4). The cross-validated

cor-rect classification percentage in the prediction of the three

different eel groups by the five element : Ca ratios was the

highest in freshwater-resident yellow eels (98.0%), followed

by seawater-resident yellow eel (84.4%), with leptocephalus

being the lowest at 69.7% (Table 5).

Discussion

In earlier studies of the elemental composition of fish otoliths,

it was believed that elemental composition reflects the

ele-mental concentration of ambient water that the fish lives in.

Thus, the Sr : Ca ratio in otoliths was widely used to study the

eel migration between freshwater and marine environments

(e.g. Tzeng et al. 1997, 2000, 2002, 2003a, 2003b; Tsukamoto

and Arai 2001; Jessop et al. 2002, 2004; Shiao et al. 2003).

Table 5. Cross-validated classification of the eel of different stage/habitat use by leave-one-out method using otolith elemental signature (Sr : Ca, Cl : Ca, P : Ca, S : Ca, and

Na : Ca)

Correctly classified percentages in bold Stage/habitat use nA Correct classification (%)

Leptocephalus Yellow eel

Seawater-resident Freshwater-resident

Leptocephalus 99 69.7 24.2 6.1

Yellow stage eel

Seawater-resident 392 2.3 84.4 13.3

Freshwater-resident 304 0.0 2.0 98.0

A_{The number of sampling spots for the measurement of elemental composition on the otoliths of the} 10 yellow eels in Table 1.

Table 4. Standardised canonical discriminant function coefficients of element : Ca ratios for discriminating the eel among different stage/habitat use as leptocephalus and freshwater- and

seawater-resident yellow eels in Fig. 1

Elemental ratio Function

1 2 Sr : Ca 1.011 −0.077 Cl : Ca −0.047 −0.607 P : Ca −0.041 0.594 S : Ca 0.092 0.426 Na : Ca −0.093 0.997 Eigenvalue 2.148 0.082 % of variance 96.3 3.7 Cumulative % 96.3 100.0

Fig. 1. Anguilla anguilla. Canonical discriminant scores plot of otolith element : Ca ratios for the European eel. The different stages assayed were those present in the adult eel otoliths, and classified by life stage and habitat use as leptocephalus, freshwater- and seawater-resident yellow eels. The elliptical circles indicate 95% confidence limit of each group.

In the present study, we explored elements other than Sr

and found that they also changed with life stage and habitat.

The Sr : Ca ratios ranked first in the leptocephalus stage, but

the top rank changed to Na : Ca in freshwater- and

seawater-resident yellow stage eels. In other words, the ranking of the

relative importance of the element : Ca ratios were different

between leptocephalus and both freshwater- and

seawater-resident yellow stage eels (Table 2). This demonstrated that

the elemental composition in otoliths of the eel significantly

changed with life stage and habitat transition. The

mecha-nism of the change of elemental composition of the otolith

with life stage is not clear in most fishes, but may relate to

the elemental concentration of ambient water and osmotic

pressure regulation of the diadromous eel when they change

from osmoconformer in the leptocephalus stage to

osmoreg-ulation in the yellow stage eel. The body fluid of a

lepto-cephalus is isotonic to seawater and its body tissue is known

to contain extensive amounts of a gelatinous extracellular

matrix composed of sulphated glycosaminoglycans (GAGs),

which have an affinity for alkali elements, particularly Sr

(Comper and Laurent 1978; Nishizawa 1978; Hascall and

Hascall 1981; Toole 1981). Sulphated glycosaminoglycans

breakdown during metamorphosis from leptocephalus to

glass eel may possibly reduce the absorption of Sr

and other

elements. Thus, the Sr : Ca ratios in otoliths of leptocephali

significantly decreased when they metamorphose into glass

eels (Otake et al. 1994; Arai et al. 1997). However, the effect

of ambient environment on the otolith Sr : Ca ratios of the eel

cannot be excluded because the growth habitat shifted from

high to low saline water when the eel metamorphoses from

leptocephalus to glass eel and Sr concentration is

∼100 fold

higher in seawater than in fresh water (Campana 1999). On the

contrary, the Na : Ca ratios in otoliths of the eel were higher

in freshwater- compared with seawater-resident yellow stage

eels, although Na concentration is higher in seawater than

fresh water. This may indicate that sodium uptake is

physio-logically controlled by the eel. Accordingly, except with the

exception of Sr, the other elements in fish otoliths are also

very important in the linking study between otolith

micro-chemistry and the life and migratory environmental history

of the fish.

This is the first paper to use multiple elements in addition

to Sr to study the life history and migratory environment of

the eel. The eight elements selected in the present study were

reported to have a positive correlation in abundance between

otoliths and ambient water (Goldberg 1965; Thresher 1999).

With the exception of the Sr : Ca and Na : Ca ratios, no

sig-nificant difference was found among leptocephalus and both

freshwater- and seawater-resident yellow eels (Table 2). This

indicates that it is difficult to use a single element : Ca ratios to

discriminate the eel stage and habitat use. However, the cross

correlation matrix of the seven element : Ca ratios in otoliths

was found to be different among stage and habitat use of the

eel (Table 3). This may indicate that the elements incorporated

into otoliths of the eel were conditionally selected. The

canon-ical plot and stepwise canoncanon-ical discriminant analysis also

indicated that along with Sr : Ca, the other four element : Ca

ratios (Cl : Ca, P : Ca, S : Ca and Na : Ca) have obviously

con-tributed to the grouping of the eels (Fig. 1). The classification

success of the different stage and habitat use of the eel reached

70.7–98.0% (Table 5). This suggested that multiple-elemental

information could provide additional insight into

environ-mental life history traits of the facultative catadromous eels.

A total of 31 elements have been detected in fish otoliths to

date (Campana 1999). Future studies are needed to assess the

use of elements that are below the detection limits of EPMA

for more detailed fingerprints regarding eel otoliths.

Acknowledgments

The present study was conducted with financial support from

the National Science Council (contract no. NSC

91–2313-B002–291, awarded to WNT). We thank Ms Y. C. Tsai for

otolith preparation, and Mr Brian M. Jessop, Dr Steven

Cam-pana and the anonymous referee for their helpful comments

on an early draft of this paper.

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Manuscript received 16 July 2004; revised 22 March 2005; and accepted 6 April 2005.

Appendix 1. Electron probe microanalyser counting times (peak and background), standards, detection limits, and analytical errors

Detection limits (wt %) and typical analytical errors (wt %, 1 σ) calculated after Scott et al. (1995)

Element Counting time Standard Detection limit Typical analytical error

(sec) Na 60 Halite (CM Taylor) 0.029 0.023 Mg 60 Osumilite (USNM 143967) 0.019 0.022 P 60 Apatite (Wilberforce) 0.036 0.027 S 60 Gypsum (CM Taylor) 0.023 0.017 Cl 46 Halite (CM Taylor) 0.027 0.015 K 46 Osumilite (USNM 143967) 0.019 0.012 Ca 20 Calcite (NMNH 136321) NA 0.245 Sr 120 Strontianite (Smithsonian R-10065) 0.036 0.019