The effect of ablation pattern on LA-ICPMS analysis of otolith element composition in hake,...

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Environmental Biology of Fishes ISSN 0378-1909Volume 95Number 4 Environ Biol Fish (2012) 95:509-520DOI 10.1007/s10641-012-0065-7

The effect of ablation pattern on LA-ICPMS analysis of otolith elementcomposition in hake, Merlucciusmerluccius

Mei-Yu Chang, Audrey J. Geffen, JanKosler, Siv Hjorth Dundas & GregoryE. Maes

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The effect of ablation pattern on LA-ICPMS analysisof otolith element composition in hake, Merluccius merluccius

Mei-Yu Chang & Audrey J. Geffen & Jan Kosler &

Siv Hjorth Dundas & FishPopTrace Consortium &

Gregory E. Maes

Received: 12 October 2011 /Accepted: 25 May 2012 /Published online: 4 August 2012# Springer Science+Business Media B.V. 2012

Abstract Laser ablation ICPMS (inductively coupled

plasma mass spectrometry) analysis is a powerful tool

for studies of fish ecology, based on measurement of the

chemical composition of otoliths (ear stones). A key

trade-off for this analysis is selecting the size of the

ablation area to maximize the resolution for discrete

temporal intervals during the life of an individual fish,

vs the amount of otolith material required to produce

reliable data. Three different widths of ablation lines

were used to analyze the otoliths of European hake

(Merluccius merluccius). The best temporal resolution

was produced by ablation lines of 10 μm width,

corresponding to less than 2 weeks in the fish’s life,

but the data from this configuration were variable and

often below the detection limit for many elements. Ab-

lation lines of 20 and 30 μm width produced accurate

and precise data corresponding to approximately 20 and

30 days in terms of temporal resolution. When tested on

hake otoliths, the measured element concentrations dif-

fered significantly between the 20 and 30 μm lines. The

30 μm ablation line resulted in a better multivariate

model for discrimination between populations, with

higher classification success and higher probability of

individual assignment to source location.

Keywords Fish otoliths . Temporal resolution .

Laser ablation inductively coupledmass spectrometry .

Microchemistry . Stock discrimination

Introduction

Otoliths are aragonite structures that constitute one of

the components of the acoustico-lateralis system of

teleost fish. Otoliths form during embryo development,

Environ Biol Fish (2012) 95:509–520

DOI 10.1007/s10641-012-0065-7

M.-Y. Chang :A. J. Geffen (*)

Department of Biology, University of Bergen,

PO Box 7803, 5020 Bergen, Norway

e-mail: [email protected]

M.-Y. Chang


A. J. Geffen

Institute of Marine Research,

Bergen, Norway

J. Kosler : S. H. Dundas

Department of Earth Science/Center for Geobiology,

University of Bergen,

Allegaten 41, Bergen N-5007, Norway

J. Kosler


S. H. Dundas


G. E. Maes

Laboratory of Biodiversity and Evolutionary Genomics,

Katholieke Universiteit Leuven,

Leuven, Belgium


Present Address:

M.-Y. Chang

Biodiversity Research Center, Academia Sinica,

128 Academia Road Sec.2, Nankang, Taipei 11529, Taiwan

Author's personal copy

and continue to grow, in incremental layers of CaCO3 in

a protein matrix, throughout the life of the individual

(Morales-Nin 2000). Each layer is a permanent feature,

and can be identified in relation to daily, seasonal, and

annual cycles of growth. The pattern of fish growth is

indeterminate, i.e. fish grow continuously throughout

their life, but the rate of growth decreases with age of

the fish. The same pattern is seen in the otoliths, such

that the innermost increments, which are deposited

when the fish is young, are usually larger than the

outermost increments, which are those most recently

deposited. For example, the daily increments at the

otolith edge in a young fish (less than 1 year old) may

be 3–5 μm in width, whereas similar growth increments

at the otolith edge in a 5 year old fish are generally less

than 1 μm in width (Li et al. 2008; Neat et al. 2008).

Many elements are incorporated into the otolith

during the biomineralisation process (Campana

1999), and often in direct relation to their relative

concentrations in surrounding waters (Milton and

Chenery 2001; Elsdon and Gillanders 2004). The con-

centrations of these elements are influenced by envi-

ronmental conditions such as temperature and salinity,

and physiological conditions such as metamorphosis

and reproductive activity (Kalish 1991). Otolith chem-

ical composition, or microchemistry, can be analyzed

as an indicator of where an individual fish has lived

(Elsdon et al. 2008) and for population discrimination

(Swan et al. 2006). The time-recording property of the

otolith increments means that multi-element informa-

tion can be retrieved from distinct growth zones and

used to reconstruct a life history transect for an indi-

vidual fish (Morales-Nin et al. 2005; Elsdon et al.

2008). The challenge is to combine a measurement

tool that can operate at a sufficiently high spatial

resolution on the otolith to give the desired temporal

resolution of the fish’s life. If the area analyzed on the

otolith is too small, then the amount of material sam-

pled will not be sufficient to obtain data with required

precision and accuracy, but if it is too large, then the

material will integrate over too long a period of time.

We selected laser-ablation inductively coupled mass

spectrometry (LA-ICPMS) for analysing small-scale

variations across fish otoliths. The ICPMS enables

multi-element analysis over a wide range of sample

concentrations, and has been tested for otolith applica-

tions world-wide (Campana et al. 1997). Most instru-

ment configurations allow the adjustment of the size and

shape of the ablated and analyzed area, which translates

into control over the temporal resolution in terms of the

fish’s life history. Our goal was to utilize the technique

for high-throughput measurements of fish otoliths, and

so we analyzed analytical reference standards and oto-

liths in a real-life application situation. The standard and

certified reference materials (SRMs and CRMs) were

analyzed in a controlled experiment to test the accuracy

and precision of the measurements using ablation raster

lines of 10, 20, or 30 μm in width. We then tested these

ablation lines on the otoliths of the European hake

(Merluccius merluccius), and evaluated the effect on

fitness-for-purpose, by comparing population discrimi-

nation models using data from the different line widths.

Hake is one of the most valuable and heavily

exploited demersal species in Europe, and is currently

managed as two stocks (north and south) in the Atlan-

tic and one in the Mediterranean Sea (Lo Brutto et al.

2004; Mattiucci et al. 2004; Swan et al. 2006; Tomas

et al. 2006). These stock designations may hide sepa-

rate populations associated with different geographical

areas. Estimating the exploitation rates of different

populations, clarifying the habitat use of the fish dur-

ing different life stages and finally tracing back indi-

vidual fish to the locations from which they were

caught are important aspects for setting management

policies and developing conservation measures.

The growth rate of hake otoliths is likely to be ap-

proximately 1–1.5 μm · day−1 (de Pontual et al. 2006)

and the laser patterns we tested correspond to temporal

periods of 10–15 days, 20–30 days, and 30–45 days. We

evaluated the trade-off between data quality and tempo-

ral resolution by comparing the precision and accuracy

of elemental measurements and the suitability of the

data for determining the source location of the fish.

Material and methods

Reference materials and otolith samples

One glass reference material (NIST 610, (National

Institute of Standards and Technology, USA)) and

two fish otolith powder reference materials (FEBS-1

(National Research Council, Canada; Sturgeon 2005)

and NIES No.22 (National Institute for Environmental

Studies, Japan; Yoshinaga et al. 2000)) were used for

laser ablation as calibration standards and quality con-

trol samples. The fish otolith CRMs were pressed into

pellets at 750 MPa (10 T) with a laboratory press.

510 Environ Biol Fish (2012) 95:509–520


Sagittal otoliths were collected from adult hake,

Merluccius merluccius, from different locations in

the eastern Mediterranean; from the Aegean (n030,

total length 195–403 mm), the north Adriatic (n019,

total length 245–370 mm) and the Turkish coast (n0

30, total length 170–270 mm). The fish were sampled

as part of a larger study of population structure and

traceability throughout European waters (FishPop-

Trace: https://fishpoptrace.jrc.ec.europa.eu/). After be-

ing extracted from the fish, the otoliths were stored

separately in plastic tubes. The left otolith was chosen

for the microchemistry analysis, although the right

otoliths were used when the left one was broken or

missing. The otoliths were first washed with MilliQ

water (18.2 MΩ) in an ultrasonic bath for 1 min, and

then soaked in a 3 % H2O2 (ACS reagent, Sigma-

Aldrich, Germany) solution for 15 min in order to

remove any residual tissue. After washing in MilliQ

water in the ultrasonic bath for a further 5 min, the

otoliths were cleaned with 2 % HNO3 (redistilled 1x, >

99.999 % trace metal basis, Aldrich, Germany) for

15 s, and rinsed with MilliQ water. The otoliths were

then processed through further 1 min ultrasonic bath

washes to remove any residual HNO3. The cleaned

otoliths were then dried at 50 °C for at least 1 h.

The dried otoliths were embedded inNMLaminering

275 resin (Nils Malmgren, Sweden) and sectioned in the

transverse plane with a slow speed diamond blade cir-

cular saw. The 500 μm thick slices containing the core

region were then fixed to glass slides with the same NM

resin and polished with silicon carbide grinding papers

(P2500/4000, Buehler, Germany), followed by a

0.25 μm diamond suspension (Buehler, USA) to reveal

the otolith core with a smooth surface, free of any

irregularities that could complicate the laser ablation

sampling. The otoliths of fish from different regions

were randomly distributed on the slides to reduce the

preparation or analytical artifacts. The slides were

cleaned with 2 % HNO3 for 15 s and rinsed with MilliQ

water to remove remaining surface contamination.

LA-ICPMS analysis

The elemental concentrations in CRMs and otoliths

were determined by LA-ICPMS using an Element2

HR-ICP-MS (Thermo Scientific, Bremen, Germany)

coupled with a RESOlution M-50 193 nm laser system

(Resonetics, Nashua, NH) equipped with a double

volume Laurin sample cell (Laurin Technic,

Australia). The normal sensitivity of the ICPMS in

standard solution mode is around 1.6 · 106 cps (counts

per second) for 115In in a 1 μg In L−1 solution using a

standard H skimmer cone. In LA mode, the sensitivity

of the ICPMS was monitored by the 232Th signal in

the NIST610 glass CRM. The typical sensitivity in

laser mode is 1.5 · 106 cps using an X skimmer cone

(Thermo Scientific, Bremen, Germany), laser spot di-

ameter of 64 μm, 5 Hz laser repetition rate and 45 mJ/

pulse laser energy output. All measurements were

done in the low resolution mode.

The glass NIST 610 SRM signal was primarily

used for tuning the ICPMS parameters, namely for

adjusting the torch position and focusing the ion beam.

However, NIST 610 is a silicate standard and the

concentrations are not in the same range as in the

otoliths, hence the use of the two fish otolith CRMs.

Three ablation patterns—lines of different widths

(10 μm, 20 μm and 30 μm) not more than 10 μm

deep, were compared, with five replicate measure-

ments in each of the SRM and CRMs, and single

measurements at the edge of each otolith (Fig. 1).

Otoliths from the Aegean and Turkish Coast hake

populations were analyzed with all three different

widths, but only the 20 μm and 30 μm lines were

used to measure otoliths from the North Adriatic hake.

The laser parameters were set to 5 Hz repetition rate,

100 µm

Fig. 1 Position of laser ablation rasters along the proximal

margin of a hake otolith. Lines shown are the 10, 20, and

30 μm ablation paths. Inset picture is the whole transverse

section of the same otolith, with proximal surface of the otolith

facing to the upper left. Scale bar in main picture is 100 μm, and

1 mm in inset picture

Environ Biol Fish (2012) 95:509–520 511


https://fishpoptrace.jrc.ec.europa.eu/

45 mJ/pulse energy and 40 s ablation (dwell) time for

approximately 200 μm ablation length (Table 1). Ten

isotopes were measured for each otolith and line

width: 7Li, 23Na, 24Mg, 25Mg, 43Ca, 44Ca, 55Mn,63Cu, 88Sr and 138Ba. All the reference materials were

measured at the beginning and the end of the session,

and also after every 20 otolith sample measurements,

for calibration and drift correction.

The data reduction software GLITTER (GEMOC,

Macquarie University, Australia) was used to calculate

the element concentrations, expressed as μg · g−1 based

on natural isotope composition. The raw output is pro-

cessed by first designating the drift and calibrations

standards. 43Ca was used as an internal standard to

correct for differences in the ablation yield between

samples and standards. Not all of the elements of interest

were certified in a single otolith CRM so FEBS-1 and

NIES 22 were both used for the calibration of the

element concentrations. 7Li and 55Mn were calibrated

by FEBS-1 and the remaining elements were calibrated

by NIES 22. The data processing proceeds by identify-

ing the background and signal windows for each mea-

surement. Each measurement is defined here as the

acquisition of data from one complete raster line. The

background signal is defined as the period during which

only the carrier gas composition ismeasured, prior to the

laser firing. This section of the signal is used to calculate

the lower limit of detection (LOD) for each measure-

ment, by Poisson counting statistics (GLITTER Manu-

al, Version 4.0, using IDL version 5.3.1, GEMOC Laser

ICP-MS Total Trace Element Reduction):

LOD ¼ 2:3�p2� total background interval countsð Þ

This procedure produces a separate LOD value for

each measurement because each measurement contains a

background, firing, and washout period. To maintain a

balanced dataset, the values of measurements below the

limit of detection were replaced with the individual LOD

calculated for that measurement (Gillespie et al. 2010).

Statistical analysis

The sensitivity of the different ablation line widths for

reference material and otolith samples was compared

based on the number of measurements where the con-

centration calculated from the data acquired fell below

the limits of detection. Next, the reproducibility of the

measurements in the standards was evaluated by com-

paring the precision of the measurements of the three line

widths, based on the coefficient of variation (CV%, stan-

dard deviation/mean x 100) for (n05) replicates of the

different ablation line widths for each reference material.

Finally, the effect of the different line widths in the

otoliths was compared to demonstrate “fitness-for-

purpose”. Ultimately, only the 20 μm and 30 μm line

widths produced sufficient reliable measurements (see

Results), and so the element concentrationsmeasured by

these line widths were compared within each fish pop-

ulation using a t-test for dependent samples (paired

t-test). A standard discriminant analysis model (DFA)

was calculated separately from the concentrations of Li,

Na, Mg, Ca, Mn, Cu, Sr, and Ba measured by each line

width. The individual fish were assigned to designated

groups (locations of collection) by canonical classifica-

tion analysis. The classification success was not cross-

validated because of the small sample sizes and the

objective of comparing data derived from the two meas-

urements of the same individuals. The posterior classi-

fication probabilities were calculated as the reciprocal of

the distance to the assigned group centroid, and these

probabilities represent how closely the model is able to

place that individual to the assigned group. The suitabil-

ity of the different line widths, in terms of the applica-

tion of distinguishing the location source of each fish,

Table 1 The laser and ICP-MS configuration and operating parameters for comparison of ablation line widths in the analysis of

standards and hake otoliths

Ar gas flows

(L/min)

RF power

(Watt)

Isotopes Laser

energy

Frequency Ablation

(Dwell)

Time (sec)

Line size (μm)

(spot size x

length)

Carrier gas

type

Carrier gas

flow (L/min)

Cool Aux Sample

16 1.2 0.65 1358 7Li, 23Na, 24Mg, 25Mg,43Ca, 44Ca, 55Mn,63Cu, 88Sr and 138Ba

45 mJ 5 Hz 40 10×200 He 0.65

20×200

30×200



was determined by direct comparison of the probabili-

ties of assignment that were achieved with the 20 μm

versus 30 μm lines. In this way, we included all aspects

of the quality of the data produced by the different line

widths, and evaluated their performance in a real-life

application.

Statistical analyses were performed using Statistica

v.10 (Statsoft, Inc. USA 1984–2011).

Results

Line width comparisons

The performance of the ablation line widths was eval-

uated for analytical standards and for fish otolith sam-

ples. The concentration data for eight elements were

compared; Li, Na, Mg, Ca, Mn, Cu, Sr, and Ba. For

the analytical standards, which are expected to be

homogenous and well characterized, the number of

measurements that fell below the LOD was lower for

the more abundant elements, as expected. More of the

10 μm line width measurements fell below the LODs,

in comparison to the 20 μm and 30 μm line width

results (Table 2). This was especially the case for the

FEBS measurements, where virtually no data were

retrieved from the 10 μm lines. Measurements of Li

in the standards and Li and Mn in the otoliths were

often below LODs.

The precision of measurement was calculated from

replicate measurements of the standards: NIST610,

FEBS, and NIES. Precision was usually lowest (higher

CV%) for the 10 μm ablation lines, and highest for the

30 μm lines (Table 3). Exceptions were measurements

of Sr in NIST610, Mg and Ca in FEBS, and Cu and Ba

in NIES. The 20 and 30 μm ablation line measure-

ments were also more accurate, being closer to the

certified values of the analytical standards, compared

to the measurements made with the 10 μm laser abla-

tion lines (Fig. 2). In general, the reliability of the data,

based on precision and accuracy, improved with in-

creasing line width (Table 3).

Because so many of the 10 μm line measurements

were below the LODs, and because of the poor accu-

racy and precision in the 10 μm line width data from

the reference standards, only the 20 μm and 30 μm

line otolith measurements were used to evaluate the

fitness-for-purpose in population discrimination and

individual assignment. Paired t-tests of 20 μm and

30 μm line width measurements showed significant

differences in the measured concentrations of Ca, Mn,

Cu, and Ba in the Aegean (AG) fish otoliths, Na, Mn,

Table 2 The average LOD as μg · g−1 (and number of measurements falling below the LODs) for the different line widths used on

standards and hake otoliths. N0number of line measurements

N 7Li 23Na 24Mg 44Ca 55Mn 63Cu 88Sr 138Ba

CRMs

NIST610

10 μm 5 59.9 (0) 420.0 (0) 18.0 (0) 920 (0) 9.2 (0) 9.2 (0) 1.6 (0) 0.8 (0)

20 μm 5 13.0 (0) 86.4 (0) 3.6 (0) 200 (0) 1.9 (0) 1.8 (0) 0.3 (0) 0.1 (0)

30 μm 5 7.0 (0) 45.4 (0) 1.8 (0) 110 (0) 1.0 (0) 0.9 (0) 0.1 (0) 0.1 (0)

FEBS

10 μm 5 45.7 (5) 2000.0 (4) 57.2 (4) 4800.0 (2) 130.0 (2) – 9.9 (1) 3.9 (3)

20 μm 5 2.1 (5) 91.4 (0) 1.9 (2) 220.0 (0) 0 (0) – 0.4 (0) 0.1 (0)

30 μm 5 0.9 (5) 39.4 (0) 0.7 (0) 96.9 (0) 0 (0) – 0.2 (0) 0.1 (0)

NIES

10 μm 5 – 860.0 (1) 9.3 (2) 2200.0 (0) – 2.7 (0) 4.4 (0) 1.7 (0)

20 μm 5 – 75.8 (0) 0.8 (1) 190.0 (0) – 0.2 (0) 0.4 (0) 0.1 (0)

30 μm 5 – 32.7 (0) 0.3 (0) 84.9 (0) – 0.1 (0) 0.2 (0) 0.1 (0)

Otoliths

10 μm 58 30.2 (35) 140.0 (23) 12.5 (34) 610.0 (6) 0.4 (46) 1.9 (51) 4.7 (12) 0.8 (36)

20 μm 77 4.3 (33) 30.9 (1) 2.1 (3) 200.0 (0) 0.3 (16) 0.9 (70) 2.4 (0) 0.1 (3)

30 μm 77 1.8 (15) 12.8 (0) 0.8 (2) 80.6 (0) 0.1 (10) 0.4 (58) 1.7 (0) 0.1 (1)



Table 3 The certified and measured concentrations (μg · g-1) in reference standards and hake otoliths collected from three different regions analyzed with 10, 20 and 30 μm ablation

line widths. Values shown are means±standard deviation (CV%) of five replicate measurements on standards and up to 30 individual otoliths from each population

7Li 23Na 24Mg 44Ca 55Mn 63Cu 88Sr 138Ba

Nist 610 482.2 97190.21 455.00 81905.31 436.40 432.90 525 446

10 μm 508±32 (6 %) 96000±7000 (7 %) 440±90 (20 %) 79000±1000 (1 %) 452±15 (3 %) 467±8 (2 %) 490±20 (4 %) 430±20 (5 %)

20 μm 486±8 (2 %) 95000±1000 (1 %) 480±50 (10 %) 82000±1000 (1 %) 438±6 (1 %) 425±9 (2 %) 500±90 (18 %) 422±4 (1 %)

30 μm 483±3 (1 %) 94900±700 (1 %) 465±3 (1 %) 81900±700 (1 %) 430±5 (1 %) 431±5 (1 %) 498±5 (1 %) 425±3 (1 %)

FEBS 0.305 2594 23.6 383000 0.686 – 2055 5.09

10 μm 37±12 (32 %) 2400±300 (13 %) 70±30 (43 %) 400000±300000 (7.5 %) 2.6±0.7 (27 %) – 1300±900 (69 %) 5±2 (40 %)

20 μm 1.5±0.2 (13 %) 2500±200 (8 %) 21±9 (43 %) 360000±40000 (11 %) 0.9±0.2 (22 %) – 2000±200 (10 %) 4.6±0.6 (13 %)

30 μm 0.67±0.03 (4 %) 2600±200 (8 %) 24±3 (13 %) 400000±20000 (5 %) 0.64±0.08 (13 %) – 2100±100 (5 %) 5.3±0.3 (6 %)

NIES – 2230 21 388000 – 0.74 2360 2.89

10 μm – 2500±1500 (60 %) 80±30 (38 %) 370000±20000 (5 %) – 2.4±0.6 (25 %) 2000±400 (20 %) 3.7±0.3 (8 %)

20 μm – 2300±200 (9 %) 25±6 (24 %) 390000±20000 (5 %) – 1.1±0.5 (45 %) 2400±100 (4 %) 2.5±0.2 (8 %)

30 μm – 2210±70 (3 %) 20±4 (20 %) 390000±10000 (3 %) – 0.6±0.2 (43 %) 2400±100 (4 %) 3±1 (33 %)

Aegean

10 μm 0.5±1.2 (240 %) 1600±400 (25 %) 27.54±3.33 (12 %) 390000±20000 (5 %) 3±4 (133 %) 6±1 (17 %) 1600±200 (13 %) 1.0±0.9 (90 %)

20 μm 1.1±0.3 (27 %) 2600±100 (4 %) 14.94±0.96 (6 %) 394000±5000 (1 %) 8±1 (13 %) 1.1±0.3 (27 %) 1690±60 (4 %) 2.5±0.3 (12 %)

30 μm 0.9±0.3 (33 %) 2500±100 (4 %) 16.68±0.95 (6 %) 412000±5000 (1 %) 5±1 (20 %) 0.9±0.3 (33 %) 1600±50 (3 %) 2.1±0.3 (14 %)

Turkish coast

10 μm 1.1±2.8 (255 %) 2200±200 (9 %) 351±3 (1 %) 370000±10000 (3 %) 1.2±0.2 (17 %) 5.6±0.6 (11 %) 1600±200 (13 %) 2.9±0.4 (14 %)

20 μm 2.5±1.3 (52 %) 2150±80 (4 %) 15±1 (7 %) 367000±6000 (2 %) 0.43±0.08 (19 %) 1.2±0.3 (25 %) 1450±70 (5 %) 1.2±0.2 (17 %)

30 μm 1.6±1.3 (81 %) 2320±70 (3 %) 15±2 (13 %) 390000±6000 (2 %) 0.56±0.08 (14 %) 0.6±0.3 (50 %) 1470±70 (5 %) 1.2±0.2 (17 %)

North Adriatic

20 μm 0.6±0.3 (50 %) 2500±100 (4 %) 14±1 (7 %) 389000±3000 (1 %) 0.93±0.03 (3 %) 0.6±0.5 (83 %) 1270±70 (6 %) 1.1±0.1 (9 %)

30 μm 0.3±0.2 (67 %) 2700±100 (4 %) 15±1 (7 %) 392000±3000 (1 %) 0.62±0.03 (5 %) 1.0±0.5 (50 %) 1230±70 (6 %) 1.2±0.1 (8 %)

514

Enviro

nBiolFish

(2012)95:509–520

Au

tho

r's perso

na

l cop

y

and Sr in the north Adriatic (NA) fish otoliths, and Na,

Ca, and Cu in the Turkish coast (TC) fish otoliths

(Table 4).

The DFA models derived from using either 20 μm

or 30 μm line width data were slightly different. Data

from the 20 μm line measurements resulted in a DFA

model with Na, Mn, Cu, and Ba contributing to the

first discriminatory axis, which explained 89 % of the

group variation, and Li, Na, Mg, Ca, and Sr forming

the second discriminatory axis, which explained the

remaining 11 % of the variation. Mn and Ba were the

most important elements for the first axis, and Na and

Sr for the second axis. The DFA model that was

calculated from the 30 μm line measurements con-

sisted of Ca, Mn, Sr, and Ba contributing to the first

axis, explaining 85 % of the variation, and Li, Na, Mg,

10 20 30

NIST 610

440

480

520

560

600

Li

10 20 30

NIES

10 20 30

FEBS

0

100

200

300

400

500

10 20 30

NIST 610

80000

85000

90000

95000

100000

105000

Na

10 20 30

NIES

10 20 30

FEBS

0

1000

2000

3000

4000

5000

10 20 30

NIST 610

300

350

400

450

500

550

600

Mg

10 20 30

NIES

10 20 30

FEBS

0

100

200

300

400

10 20 30

NIST 610

76000

78000

80000

82000

84000

Ca

10 20 30

NIES

10 20 30

FEBS

200000

400000

600000

800000

1000000

Fig. 2 Variability in measurements of element concentrations

(μg · g−1) when analyzed by laser ablation lines of 10, 20, and

30 μm width. Symbols show replicate measurements on stan-

dard reference material, lines show certified concentrations:

circles, solid line: NIST610, squares, dashed line: NIES, trian-

gles, dotted line: FEBS. NIST is shown on the left axis, the

otolith CRMs on the right axis



and Cu contributed to the second axis, which

explained the remaining 15 % of the group variation.

Ba was the most important element defining the first

axis, and Na the most important for the second axis.

With slightly more explained variation taken up in

the second discriminatory axis, the DFA model based

on the 30 μm line width data produced more distinct

groupings for the three geographic locations (Fig. 3),

and the overall classification success was better for the

30 μm line measurements (84 % for 30 μm vs 79 %

for 20 μm). The correct classification rate (the per-

centage of individuals correctly assigned to their col-

lection location) for fish collected in AG was 90 %

using 30 μm line measurements compared to 87 %

using 20 μm line measurements, for NA fish it was

79 % with the 30 μm line measurements compared to

10 20 30

NIST 610

420

430

440

450

460

470

480

Mn

10 20 30

NIES

10 20 30

FEBS

0

50

100

150

600

10 20 30

NIST 610

400

420

440

460

480Cu

10 20 30

NIES

10 20 30

FEBS

0

20

40

60

80

1001100

1150

1200

10 20 30

NIST 610

460

480

500

520

540

Sr

10 20 30

NIES

10 20 30

FEBS

0

500

1000

1500

2000

2500

3000

10 20 30

NIST 610

410

420

430

440

450

460

Ba

10 20 30

NIES

10 20 30

FEBS

2

4

6

8

10

Fig. 2 (continued)



84 % with the 20 μm line measurements, and for TC

fish the classification success was 84 % based on the

30 μm line measurements compared to 67 % using the

20 μm line measurements.

The performance of the population discrimina-

tion model relies on the quality of the data used in

its construction, as well as the extent of the differ-

ences between the different groups. Models that

describe well-characterized groups should achieve

high probability of assignment for classifying indi-

viduals. To test this, the probabilities for assigning

each individual fish to its collection location using

data from the 20 μm width line were compared

against the probabilities associated with the 30 μm

width measurements. The distribution of the indi-

vidual posterior probabilities suggests that the

model and assignment using the 30 μm laser ab-

lation line data was better, since these measure-

ments resulted in more individuals with higher

probabilities (Fig. 4).

Discussion

Laser ablation ICPMS is often used to analyze the

elemental composition of different parts of the otolith

to characterize the environment experienced at earlier

times. The multi-element signals in recently deposited

material at the otolith edge can be used for examining

population structure (Svedäng et al. 2010; Chang and

Geffen 2012) and habitat use (Morales-Nin et al.

2005); the signals at the core region can give the

information of nursery ground (Vasconcelos et al.

2008); and comparing the elemental composition of

larval otoliths and the core region of the adult otoliths

can clarify the connectivity between nursery and re-

cruitment (Cuveliers et al. 2010; Wright et al. 2010).

The physical spacing of the growth increments relative

to the size of the area sampled has a direct bearing on

how well the chemical information can be linked to

specific ages or dates (Weidel et al. 2007; Morales-Nin

et al. 2012). A minimum amount of material needs to

Table 4 Paired t-test comparison of element concentrations measured in the otoliths of hake from three different populations. t test

statistic, df degrees of freedom * indicates significant differences at α00.05

Population Li Na Ca Mn Cu Sr Ba

t df t df t df t df t df t df t df

AG −0.90 29 −0.77 29 2.51* 29 −4.34* 29 −5.37* 29 −1.64 29 −2.59* 29

NA −0.80 18 3.48* 18 1.20 18 −11.21* 18 0.69 18 −2.47* 18 0.90 18

TC 0.08 28 2.31* 29 3.46* 20 −1.58 29 −5.66* 29 −0.36 29 0.13 29

Fig. 3 Comparison of dis-

crimination (DFA) models

calculated from elemental

concentration data produced

with laser ablation lines of

either 20 μm width (left

panel) or 30 μm width (right

panel). The points represent

fish samples from the east-

ern Mediterranean which

were classified to geograph-

ic locations as indicated:

solid circles0AG Aegean,

open squares0NA North

Adriatic, solid triangles0TC

Turkish coast



be sampled in order to obtain useful data, and this

limits the temporal resolution of the analysis. Different

ablation area shapes (spots or lines) or sizes of the

ablation region sample larger or smaller areas of the

otolith and this may affect the resolution of habitat

classification, but the precision of such temporal res-

olution has not been studied previously.

It is a challenge to analyze multi-element signals in

heterogeneous otoliths. Therefore it was important to

use the technique not only on the glass standards but

also to apply it to a natural biomineralized tissue, such

as the fish otolith. The silicate glass standard is con-

sistent and important for tuning and control of the

instrument performance before starting any further

analysis. When tested on standard reference materials

and hake otoliths, the 10 μm laser ablation lines pro-

vided enough material for some of the analyzed ele-

ments, even for the trace elements such as Cu and Ba.

However, reproducibility is also an important consid-

eration for otolith chemical analysis. The high CVs

and low accuracy demonstrated the lower reliability of

the 10 μm ablation line measurements in this study.

The spatial/temporal resolution achieved with the

10 μm ablation line was not an advantage, because

of the high variability among individuals, which may

represent individual variation in growth and element

incorporation, but more likely, based on the results of

the reference materials, the result of analytical limita-

tions in the amount of ablated material. Even with the

20 and 30 μm ablation lines, it was difficult to obtain

accurate measurements of some elements, notably Li,

Mg, and Mn, although the results of the 30 μm line

measurements were best.

The elemental composition might vary among dif-

ferent otoliths, but the standards were homogeneous

(Yoshinaga et al. 2000; Sturgeon 2005). Comparing

the mean concentrations of the SRM/CRMs measured

using the three ablation widths, the 20 and 30 μm

ablation line data were a better match to the certified

values for most of the elements. And the CV% of the

elemental concentrations of the standards from 10 μm

ablation line was much larger compared to the other

two ablation line widths (Table 3).

The patterns of reproducibility and reliability of

data from three different ablation line widths were

similar between otolith sample and standards. When

applied to the hake otoliths, the 30 μm ablation lines

performed best by producing more accurate and repro-

ducible data, capable of characterizing groups from

different geographic locations. The data from the

Fig. 4 Comparison of the

probabilities of assignment

for individual hake based on

otolith elemental composi-

tion as measured using laser

ablation lines of 20 or

30 μm widths. Symbols

represent hake collected

from three Mediterranean

locations: solid circles0AG

Aegean, open squares0NA

North Adriatic, solid trian-

gles0TC Turkish coast



20 μm ablation lines led to better classification success

for fish collected in the north Adriatic, but not for fish

collected in the Aegean or from the Turkish coast. The

overall classification success was higher when using

the data from the 30 μm ablation lines, and individual

probability of assignments were higher for the 30 μm

line data. Although a 10 or 20 μm ablation line would

give better temporal resolution than 30 μm ablation

lines, there would be less usable data. The growth rate

of hake otoliths has been measured directly from

marked and recaptured hake in the Bay of Biscay (de

Pontual et al. 2006). Growth in the ventral radius

averages 3.5 μm daily, and is likely to be approxi-

mately 1–1.5 μm · day−1 at the ventro-proximal edge

which is where our measurements were made (Fig. 1).

Applying these estimates to our study, the 20 μm

ablation lines corresponded to a 20–30 day time peri-

od, whereas the 30 μm ablation lines measured con-

centrations representing 30–45 days.

The relative elemental composition of the otoliths is

often used in a multivariate analysis to determine dif-

ferences between fish sampled from different loca-

tions. Previous studies of hake otolith microchemistry

have demonstrated significant differences between

hake from different regions (Swan et al. 2006; Tomas

et al. 2006) as well as ontogenetic variation in chemical

composition (Morales-Nin et al. 2005; Tomas et al.

2006). The 30 μm ablation line gave at least twice

the signal compared to the 20 μm ablation line, which

should increase the precision of the data. For some

elements in our analysis, notably Li, Mg, Mn and Cu,

the accuracy was poor with either ablation pattern,

although previous certification studies of otolith refer-

ence material showed that Li, Na, Mg, Mn, Sr and Ba

were stable in solution based ICPMS measurements

(Sturgeon 2005), and Li, Na, K, Mn, Sr and Ba were

stable in LA-ICPMS measurements (CETAC Applica-

tion notes, http://www.cetac.com/literature/application_

notes.asp.). The poor accuracy of Li, Mn and Cu may be

due to their low concentrations in the otoliths, which

also resulted in the high percentage of measurements

falling below LODs. During the analysis, the signals

fromMgwere not as stable as other elements such as Sr.

The original counting signal of Mg fluctuated under

different analyses, which might depend more on the

operating conditions. Even though the operating param-

eters were consistent during all the measurements, the

detection of Mg concentration might be more sensitive

to mass drift. In our study, the effect of the

precision and accuracy of these elements on the

DFA was minimal because they were not the most

important in the discrimination model.

Although the 10 μm width ablation line provides the

best resolution for otolith microchemistry analysis using

LA-ICPMS, the reproducibility was poor. The reproduc-

ibility and spatial differences among locations detected

with both the 20 and 30 μm ablation widths were

similar, but the multi-element compositions were differ-

ent between two different ablation widths. The data

from the 30 μm ablation width also resulted in higher

probabilities for the group assignments, but the 20 μm

line measurements produced higher classification suc-

cess for one of the three populations. These results

provide an informed option in choosing the best analyt-

ical conditions for analyzing otoliths from different spe-

cies with different otolith sizes and growth rates.

Acknowledgements This study was undertaken as part of the

FishPopTrace project (KBBE-212399) funded by the European

Community’s Seventh Framework Programme in the area of

“Food, Agriculture and Fisheries, and Biotechnology”. G.E.M.

is a post-doctoral researcher funded by the Fund for Scientific

Research-Flanders (FWO Flanders). M-Y Chang was supported

by CalMarO, a Marie Curie Initial Training Network funded by

the European Community’s Seventh Framework Programme.

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