ORIGINAL PAPER

Detection of transgenerational barium dual-isotope marksin salmon otoliths by means of LA-ICP-MS

Gonzalo Huelga-Suarez & Beatriz Fernández &

Mariella Moldovan & J. Ignacio García Alonso

Received: 12 July 2012 /Revised: 19 September 2012 /Accepted: 21 September 2012 /Published online: 17 October 2012# Springer-Verlag Berlin Heidelberg 2012

Abstract The present study evaluates the use of anindividual-specific transgenerational barium dual-isotopeprocedure and its application to salmon specimens fromthe Sella River (Asturias, Spain). For such a purpose,the use of laser ablation inductively coupled plasmamass spectrometry (LA-ICP-MS) in combination withmultiple linear regression for the determination of theisotopic mark in the otoliths of the specimens is pre-sented. In this sense, a solution in which two barium-enriched isotopes (137Ba and 135Ba) were mixed at amolar ratio of ca. 1:3 (NBa137/NBa135) was administeredto eight returning females caught during the spawningperiod. After injection, these females, as well as theiroffspring, were reared in a governmental hatchery locat-ed in the council of Cangas de Onís (Asturias, Spain).For comparison purposes, as well as for a time-monitoring control, egg and larva data obtained bysolution analysis ICP-MS are also given. Otoliths (9-month-old juveniles) of marked offspring were analysedby LA-ICP-MS demonstrating a 100 % marking efficacyof this methodology. The capabilities of the molar frac-tion approach for 2D imaging of fish otoliths are alsoaddressed.

Keywords LA-ICP-MS . Otoliths . Transgenerationalmarking . Barium isotopes

Introduction

During the last years, the use of enriched stable isotopes hasbecome a useful means of fish marking [1–3] and, therefore,a very important tool for fisheries management and researchas it offers a faster and more efficient alternative to physical[4] and chemical (fluorescent/elemental) [5, 6] marking. Inthis sense, the use of one barium isotope spike as trans-generational marking procedure has demonstrated its effica-cy since Thorrold et al. [7] reported that it was possible tomark thousands of larvae with a single maternal parentinjection. The detection of the isotopic mark in fish otolithsby laser ablation inductively coupled plasma mass spec-trometry (LA-ICP-MS) was the preferred analytical proce-dure in these studies [7]. Unfortunately, this single-isotopeapproach does not allow differential marking [8]. Nonethe-less, this issue was recently solved by using a barium dual-isotope procedure [9]. Thus, by means of this methodology,it is possible to clearly identify the offspring of an individualor a group of fishes, as one single fish can be specificallymarked based on the molar ratio of two barium isotopes:137Ba and 135Ba. As a result, this individual-specific trans-generational marking procedure offers a reliable solution tofisheries issues such as mortality, migration movements orrestocking success. The dual-isotope mark was easily iden-tified in eggs and larvae of the offspring, but its unequivocaldetection in fish otoliths after total digestion of the samplerequired the use of a multi-collector ICP-MS instrumentbecause of the high fraction of natural abundance bariumin these samples [9].

Otoliths, also known as earstones, are paired calcifiedstructures (mainly aragonite) used for balance and/or hear-ing in all teleost fishes. The fact that the otolith is acellularand metabolically inert means that any element or com-pound accreted onto its growing surface is permanentlyretained, whereas the continued growth of the otolith from

Published in the topical collection Isotope Ratio Measurements: NewDevelopments and Applications with guest editors Klaus G. Heumannand Torsten C. Schmidt.

G. Huelga-Suarez : B. Fernández :M. Moldovan :J. I. G. Alonso (*)Department of Physical and Analytical Chemistry,University of Oviedo,Julián Clavería 8,33006 Oviedo, Spaine-mail: jiga@uniovi.es

Anal Bioanal Chem (2013) 405:2901–2909DOI 10.1007/s00216-012-6452-2

before the time of hatch to the time of death implies that theentire lifetime of the fish has been recorded [10]. A core isformed initially, around which different calcareous layers(annuli) grow, such that the observations of otolith annulihave been used as indicators of fish age for more than acentury. Recently, Martin et al. [11] demonstrated, by meansof LA-ICP-MS, that the core of salmon otoliths retains thematernal isotopic information. Laser ablation allows a spa-tial analysis within the otolith and, in this case, a cleardiscrimination between the freshwater and marine habitatscould be achieved based on strontium isotope ratios [11].

Therefore, the use of laser ablation as sample introduc-tion system for the detection of transgenerational bariumdual-isotope marks is evaluated in this study. Its capabilityof direct analysis of the otoliths, as well as the possibility ofspatially resolved isotopic analysis, makes LA-ICP-MS asuperior approach than solution ICP-MS. Moreover, the useof LA-ICP-MS permits to specifically analyse the region ofthe otolith wherein barium exhibits a non-natural signatureobtained from the maternal influence, which would be thecore in the samples subjected to analysis. So, the presentwork is focused on the development of a methodologywhich permits the unequivocal detection of the bariumdual-isotope mark in fish otoliths of juvenile salmon bymeans of LA-ICP-MS. A mathematical tool based on mul-tiple linear regression had to be developed for the treatmentof the time-resolved data.

Experimental

Reagents and materials

Two barium carbonates, enriched in 135Ba (94.85 %) and137Ba (82.23 %), respectively, were purchased from Isoflex(San Francisco, CA, USA). These enriched tracers will bereferred to as ‘Ba135’ and ‘Ba137’ throughout the paper.Barium standard stock solutions of natural isotope abun-dance were supplied by Merck (Darmstadt, Germany). Theisotopic abundances of natural barium [12] and the twoisotopic tracers used for the marking of salmons are pre-sented in Table 1. The isotope composition of the two

enriched barium spikes was determined by multi-collectorICP-MS using a natural abundance barium solution (Merck,Darmstadt) for mass bias correction [9]. Ultrapure water wasobtained from a Milli-Q Gradient A10 water purificationsystem (Millipore, Molsheim, France). Pro Analysis HNO3

65 % (v/v), Pro Analysis HCl 37 % (v/v), Suprapur H2O2

30 % (v/v) and Suprapur HF 40 % (v/v) were purchased fromMerck (Darmstadt, Germany). All materials employedthroughout this work were thoroughly cleaned before theiruse following a three-step cleaning procedure consisting ofsuccessive immersions (24 h each) in sub-boiled HCl 10 %(v/v), sub-boiled HNO3 10 % (v/v) and Milli-Q water baths.The embedding of the otoliths was performed with a poly-ester resin (Crystic R115 PA) purchased from Resinas Cas-tro (Porriño, Spain). An MD-Piano 1200 diamond disc(Struers, Ballerup, Denmark) of 15 μm in grain size wasused for the smoothing of the embedded otoliths, whilelapping films of 45, 15 and 1 μm from the same companywere employed in order to polish the samples.

Instrumentation

Egg, larva and otolith measurements were carried out on anElement2 sector field ICP-MS (Thermo Electron Corporation,Bremen, Germany). This is a double-focusing magnetic sectormass spectrometer of reverse Nier–Johnson geometry. In theconfiguration used for liquid samples, the instrument wasequipped with a Meinhard nebuliser, a double-pass Scott-type spray chamber operating at room temperature and asecondary electron multiplier with discrete dynode detector.All isotope ratio measurements were performed at low-resolution setting (m/Δm0300) using the electrostatic scan-ning (E-scan) mode keeping the magnetic field constant. Inorder to obtain optimum precision and accuracy, the operatingparameters were adjusted to the values listed in Table 2.

Otolith measurements were performed by coupling theElement2 instrument to a CETAC LSX-213 laser system(Cetac Technologies, Omaha, NE, U.S.). The optimisedconditions for such measurements are also listed in Table 2.The laser-generated aerosol was transported through a high-purity tube (Teflon-lined Tygon tubing) into the ICP torch. Itis worth stressing that LA-ICP-MS coupling was optimised

Table 1 Natural and enrichedbarium isotopic abundancesexpressed in atom percent

The isotope composition of nat-ural abundance xenon at thebarium isotopes is also given

Isotope Xe nat Ba nat Ba135 Ba137

130 4.0710±0.0013 0.106±0.001 0.0024±0.0001 0.0017±0.0001

132 26.9086±0.0033 0.101±0.001 0.0114±0.0004 0.0098±0.0002

134 10.4357±0.0021 2.417±0.018 0.1973±0.0002 0.0324±0.0009

135 – 6.592±0.012 94.8488±0.0014 0.084±0.016

136 8.8573±0.0044 7.854±0.024 2.9932±0.0004 0.3182±0.0034

137 – 11.232±0.024 0.6752±0.0002 82.234±0.039

138 – 71.698±0.042 2.2717±0.0011 17.321±0.020

2902 G. Huelga-Suarez et al.

daily using an SRM National Institute for Standards andTechnology (NIST) 612 glass standard (NIST, Gaithersburg,MD, USA) for high sensitivity and low background inten-sity. The 238U/232Th signal ratio, that should be close to 1,was checked in order to ensure a low fractionation effect.Moreover, 248ThO/232Th signal ratio was also monitored forcontrolling oxide formation, being always below 0.4 % atthe selected conditions.

The isotope composition of the two enriched bariumspikes was measured using the Neptune multi-collectorICP-MS instrument (Thermo Electron Corporation, Bre-men, Germany). This instrument provides double focusingwith a Nier–Johnson geometry and was operated in low-resolution mode (m/Δm0400). The sample introductionsystem consisted of an auto-aspirating low-flow (100 μL

min−1) PFA nebuliser (ESI Scientific, Omaha, NE, USA)mounted onto a combined cyclonic/double-pass spraychamber made of quartz glass. The instrument settings,cup configuration and data acquisition parameters used forthe determination of barium isotope abundances are alsosummarised in Table 2.

Salmon eggs and larvae were freeze-dried in a Lyolab3000 instrument (Heto-Holten A/S, Allerod, Denmark). Fortheir digestion, a CEM automated research microwave re-actor, purchased from CEM Explorer (Matthews, NC,USA), was used. Two-dimensional images of fish otolithswere created, using MatLab mathematical software, frommolar fraction data calculated in Excel employing the math-ematical procedure developed.

Samples

All the measurements carried out in the present study wereperformed with the offspring of eight returning salmonfemales caught in October 28, 2010 in a fishway located atthe Sella River in the council of Cangas de Onís (Asturias,Spain) for restocking purposes. Thus, an isotopic mixturewith a molar ratio of ca. 1:3 (NBa137/NBa135) was deliberatelyprepared to mark these specimens. Salmons were first anaes-thetised in a bucket containing ethylene glycol–monophenylether diluted in water and then weighed to determine theinjection volume of marking isotopic mixture for each fish,so that 0.3 mg of Ba per kilogram of body weight dosagewas delivered. The barium solution was intramuscularlyadministered using an insulin syringe. Each fish was after-wards gently revived and transported in oxygenated tanks toa governmental hatchery located in the proximities of thefishway, in which the salmons were kept and reared. Eggsfrom each individual female were collected for analyticalpurposes during the spawning period (between November29, 2010 and December 30, 2010). The rest of the eggs werefertilised with the sperm of two to three non-marked salmonmales and were kept in frames until hatching. Larvae(9 weeks old) and juveniles (9 months old) were eventuallycollected for analysis. In this sense, eggs and larvae wereanalysed after total digestion while the otoliths from juve-niles were extracted to perform LA-ICP-MS measurements.

Sample preparation

Eggs and larvae

Eggs and larvae were freeze-dried for 8 hours. Subsequently,1 mL sub-boiled HNO3, 0.5 mL Suprapur H2O2 30 % (v/v)and 50μL Suprapur HF 40% (v/v) were added as acid mixtureto digest these samples. The microwave-assisted digestionprogramme employed was the following: (a) 2 min ramp to50 °C; (b) 2 min hold at 50 °C; (c) 2 min ramp to 70 °C; (d)

Table 2 Instrument settings and acquisition parameters for the Ele-ment2 SF-ICP-MS, CETAC LSX-213 laser ablation (LA) and theNeptune MC-ICP-MS units

Instrument settings for the Element2 SF-ICP-MS unit

RF power 1,275 W

Cool gas flow rate 16 Lmin−1

Auxiliary gas flow rate 0.87 Lmin−1

Sample gas flow rate 0.98 Lmin−1 (nebulisation)

Sample gas flow rate 0.50 Lmin−1 (laser ablation)

Acquisition parameters for the Element2 SF-ICP-MS unit

Settling time 0.001 s

Mass window 5 %

Sample time 0.001 s

Points per peak 200

Runs 10

Passes 600

Instrument settings for the LA CETAC LSX-213 unit

Laser energy 100 % (5 mJ)

Repetition rate 20 Hz

Spot size 50 μm

Scan speed 5 μms−1

Ablation mode Single line scan

Carrier gas (He) 1.0 Lmin−1

Instrument settings for the Neptune MC-ICP-MS unit

RF power 1,200 W

Plasma gas flow rate 15 Lmin−1

Nebuliser gas flow rate 1.0 Lmin−1

Auxiliary gas flow rate 0.8 Lmin−1

Acquisition parameters for the Neptune MC-ICP-MS unit

Integration time 4.2 s

Number of cycles 50 per block

Number of blocks 1

Cup configuration for the Neptune MC-ICP-MS unit

L3 L2 L1 C H1 H2 H3130Ba+ 132Ba+ 134Ba+ 135Ba+ 136Ba+ 137Ba+ 138Ba+130Xe+ 132Xe+ 134Xe+ 136Xe+

Detection of transgenerational barium dual-isotope marks 2903

1 min hold at 70 °C; and (e) cooling. Steps (a) and (b) wereperformed at 50-W power, while a power of 70 W was usedfor steps (c) and (d). Samples were diluted with Milli-Q waterprior to their measurement by SF-ICP-MS.

Otoliths

Sagittal otoliths were extracted at the Department of Func-tional Biology of the University of Oviedo. Upon extraction,otoliths were thoroughly rinsed with Milli-Q water to re-move possible adhering tissue, dried on filter paper andstored in 1.5-mL microcentrifuge tubes. Afterwards, theotoliths were embedded in a polyester resin at the Facultyof Geology of the University of Oviedo. Subsequently, a 15-μm grain size diamond disc was used to slightly smooth thesurface of the otoliths. Finally, a three-step polish using a45-, 15- and 1-μm lapping film, respectively, was doneleaving the samples ready for their analysis by LA-ICP-MS.

Measurement of barium isotope compositions in the spikesand the enriched mixture by ICP-MS

Barium has seven stable isotopes of masses: 130, 132, 134,135, 136, 137 and 138. Their natural isotope abundance, astabulated by the IUPAC, is given in Table 1. For ICP-MSmeasurements, isobaric interferences at masses 130, 132,134 and 136 may occur due to xenon impurities in the argonplasma gas. The natural isotope composition of xenon isalso given in Table 1 for comparison purposes. Therefore,isotopes 135, 137 and 138 are free from isobaric interfer-ences (the interference from low abundance 138La and 138Ceon 138Ba was considered negligible in fish tissues). This factexplains why enriched 135Ba and 137Ba isotopes were selectedfor fish marking.

The isotope composition of the enriched spikes used inthis study was measured by MC-ICP-MS using a bracketingprocedure in which a blank, a natural abundance barium, theenriched spike and a second natural abundance barium weremeasured in succession. The blank was employed to sub-tract the xenon contribution to all barium masses measured,while natural barium was used for mass bias correction(exponential model) [13]. The final isotope composition ofboth Ba135 and Ba137 is shown in Table 1. The sameprocedure was employed for the measurement of the isotopecomposition of the enriched mixture.

Measurement of the molar fraction ratios of Ba135/Ba137in the samples by solution ICP-MS

The procedure is described in detail in reference [9]. In brief,the measured isotope ratios in the sample Ri

iBa/138Ba werecorrected for mass bias using the exponential function andan arbitrary initial value for the mass bias factor (e.g. k00).

The mass bias-corrected isotope ratios (Ri) were then calcu-lated and the isotope abundances in the sample:

Ais ¼

RiPni¼1 Ri

ð1Þ

The mass bias-corrected isotope abundances were thendeconvoluted by multiple linear regression to calculate themolar contributions of each isotope signature in the sampleusing the following equation:

A130s

A132s

A134s

A135s

A136s

A137s

A138s

2

666666664

3

777777775

¼

A130Xe nat A130

Ba nat A130Ba137 A130

Ba135A132Xe nat A132

Ba nat A132Ba137 A132

Ba135A134Xe nat A134

Ba nat A134Ba137 A134

Ba135A135Xe nat A135

Ba nat A135Ba137 A135

Ba135A136Xe nat A136

Ba nat A136Ba137 A136

Ba135A137Xe nat A137

Ba nat A137Ba137 A137

Ba135A138Xe nat A138

Ba nat A138Ba137 A138

Ba135

2

666666664

3

777777775

�XXe nat

XBa nat

XBa137

XBa135

2

664

3

775þ

e130

e132

e134

e135

e136e137

e138

2

66666664

3

77777775

ð2ÞPlease note that Eq. (2) takes into account the contribution

of natural abundance barium and natural abundance xenon tothe final isotope abundances measured, so blank correction isnot necessary here. Then, the mass bias correction factor kwasadjusted in order to provide the minimum sum of squareresiduals for the multiple linear regression [13] using theSOLVER application in Excel. The final molar ratio NBa137/NBa135 in each sample was obtained from the molar fractionratios xBa137/xBa135 determined from Eq. (2).

Measurement of the molar fraction ratios of Ba135/Ba137in the otoliths by LA-ICP-MS

The time-resolved signals at masses 130, 132, 134, 135, 136,137 and 138 were obtained while performing a line scan of theotolith through its core using the operating conditions shownin Table 2. Next, all isotope ratios xxxBa/138Ba were calculatedfrom the intensity data, corrected for mass bias and trans-formed to isotope abundances using Eq. (1). The time-resolved molar fractions for natural Xe, natural Ba, Ba135and Ba137 were obtained by applying the function LINEST inMicrosoft Excel to the time-resolved isotope abundances and,finally, the ratio of molar fractions xBa137/xBa135 calculated forthe whole line scan. Additionally, the residuals of the multiplelinear regression were calculated for all measured masses.Subsequently, the square sum of residuals for all data pointsat all the measuredmasses was calculated. This value was thenminimised using the SOLVER application in Excel by chang-ing the mass bias correction factor applied to all measuredisotope ratios. The final molar fraction ratios Ba135/Ba137 inthe otoliths were calculated from all the data points of the linescan which fulfilled the criterion that the molar fractions forBa135 and Ba137 were higher than 0.05. An additional testbased on robust statistics using the median was employed toeliminate outliers.

2904 G. Huelga-Suarez et al.

Results and discussion

Analysis of eggs and larvae by solution ICP-MS

A 0.3-mg Ba per kilogram of body weight dosage wasdelivered intramuscularly to each female salmon as thisamount of barium has no significant effect on the physiol-ogy of fish [14]. Moreover, the risk to humans who mayconsume treated fish is minimal as defined by the Interna-tional Programme for Chemical Safety [15] and the U.S.Department of Health and Human Services [16]. Thus,enriched barium solutions can be used at low dosages tomark female fishes and, therefore, to mark larvae of fishessubject to potential human consumption.

Once the enriched mixture of barium was injected to thefemale salmon, they were kept in frames until the spawningperiod, in which 25 eggs were randomly collected, before theaddition of sperm, and analysed by using an Element2 SF-ICP-MS. After spawning, and subsequent fertilisation with thesperm of non-marked male salmon, eggs were mixed togetherand kept in frames until they hatch. Thus, 9-week-old larvae(20 specimens), reared in indoor tanks, were collected andanalysed by solution SF-ICP-MS to monitor the behaviour intime of the mark. The results obtained for these two kinds ofsamples, using the calculation methodology described in theprocedures, are compiled in Fig. 1. The xBa137/xBa135 molarfraction ratios are plotted versus the normalised molar frac-tions of natural barium in the sample [(xBa nat/(xBa nat+xBa135+xBa137))]. The horizontal line corresponds to the molar ratioNBa137/NBa135 obtained for the prepared enriched mixture. Ascan be observed, the data obtained for eggs and larvae(squares and triangles, respectively) are in good concordancewith the expected value for the enriched mixture of 0.3202±0.0008 (1 s) with some variability. Moreover, there is also an

excellent agreement among all the different marked samples(eggs and larvae) irrespective of the amount of the naturalbarium contribution.

Theoretically, the administered spike solutions will be di-luted with natural barium as the fish grow. In this sense, eggsmust have higher relative concentrations of the spike solutions(with reference to natural barium) than larvae or otoliths.However, this does not agree with what it is shown in thegraph. In this case, eggs show a higher and more variablenatural barium contribution than larvae. We have observedpreviously that it was possible to incorporate some naturalabundance barium contamination when doing digestions anddilutions for ICP-MS measurements and that this contamina-tion was variable on a day to day basis. Nonetheless, a small‘contamination’with natural barium is not worrying as long asthe artificial mark is not diluted so much that cannot bedetected using ICP-MS. In fact, the point is that all thedifferent samples show good agreement among them irrespec-tive of the natural barium contribution. As a result, differences

0.20

0.25

0.30

0.35

0.40

0.45

0.50

0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0

x Ba nat normalized

x Ba1

37 /

x Ba1

35

Fig. 1 Molar fraction ratiosxBa137/xBa135 found in the eggs(squares) and larvae (triangles)spawned from injected salmonfemales. Uncertainty is given as1 standard deviation

Table 3 Average molar fraction ratios xBa137/xBa135 obtained for eggand larva samples

Sample xBa137/xBa135 Combined standarduncertainty

Eggs 0.3208 0.0005

Larvae 0.3246 0.0010

Parameter Tracer mixture Uncertainty (2 s)

Molar ratio: NBa137/NBa135 0.3202 0.0016

Uncertainties are given as combined standard uncertainties (n025 foreggs and n020 for larvae). Moreover, the molar ratio, NBa137/NBa135,obtained for the enriched mixture (NBa137/NBa13500.3202) is also giv-en. Uncertainty for the tracer mixture is given as 2 standard deviations

Detection of transgenerational barium dual-isotope marks 2905

in the natural barium molar fractions (x-axis in Fig. 1) are notcritical for the identification of the marked fishes. What ismore, this fact can be considered as an advantage of thebarium dual-isotope procedure. The average results for allsamples (eggs and larvae) are shown in Table 3 in comparisonwith the expected value. As can be observed, a very goodagreement is found for the average values.

Detection of the isotopic signature in otoliths by laserablation ICP-MS

The main experiment to control the mark evolution wasperformed with 9-month-old juveniles (nine specimens)from which otoliths were extracted and analysed by LA-ICP-MS. In theory, once the maternal isotope signature isdeposited in the core of the otoliths, it should remain intactthroughout the life of the fish, so a proper detection of thebarium dual-isotope mark would permit, with little doubt,the specific identification of an individual marked fish. Themethodology developed for the detection of the bariumdual-isotope marks by LA-ICP-MS is described below andexpands on the methodology described previously for theisotope analysis by solution ICP-MS [9].

Figure 2 shows a typical time-resolved signal (raw data)obtained by LA-ICP-MS at masses 135, 137 and 138 whenperforming a line scan on one of the marked otoliths. Inten-sity data for masses 130, 132, 134 and 136 were alsoobtained to cover the whole barium isotope profile and toaccount for the xenon contribution to the different bariummasses. As can be observed, background data before andafter laser ablation were acquired to better define the xenoncontribution to the final isotopic profile. Please note thatincreased signals at masses 135 and 137 are observed in the

core of the otolith, which indicate the presence of theenriched isotopes used for maternal labelling.

The next step is the calculation of the time resolvedisotope ratios. Thus, all isotope ratios xxxBa/138Ba werecalculated on a point by point basis from the intensity datashown in Fig. 2. The results obtained for the ratios 135Ba/138-

Ba and 137Ba/138Ba are shown in Fig. 3. In this sense, it ispossible to observe that the isotope ratios varied during thelaser ablation scan showing a maximum in the core of theotolith. However, these isotope ratios do not clearly definethe isotopic mark because of the variable contribution fromnatural abundance barium. The measured isotope ratioswere corrected for mass bias and, for the multiple linear

0

5000

10000

15000

20000

25000

30000

35000

40000

45000

50000

0 50 100 150 200

Time (s)

Sig

nal

(cp

s)

Fig. 2 Raw intensities obtained for a marked otolith (sample 3 fromTable 4) by LA-ICP-MS for 135Ba (grey circle), 137Ba (black circle)and 138Ba (empty circle)

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0 50 100 150 200

Time (s)

Iso

top

e ra

tio

s

Fig. 3 Isotope ratios 135Ba/138Ba (grey circle) and 137Ba/138Ba (blackcircle) obtained for a marked otolith (sample 3 from Table 4) by LA-ICP-MS

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0 50 100 150 200

Time (s)

Iso

top

e ab

un

dan

ces

Fig. 4 Isotope abundances for 135Ba (grey circle), 137Ba (black circle)and 138Ba (empty circle) in a marked otolith (sample 3 from Table 4) byLA-ICP-MS

2906 G. Huelga-Suarez et al.

regression, point by point isotope abundances were calcu-lated using Eq. (1). Those results for isotopes 135, 137 and138 are shown in Fig. 4. Next, we applied Eq. (2), using theLINEST function in Excel, to the point by point isotopeabundances in order to obtain the variation of the molarfractions (xi) with time for the different components presentin the mixture: natural abundance xenon, natural abundancebarium, 135-enriched barium and 137-enriched barium. Thedata obtained for the molar fractions of natural abundancebarium and the two enriched isotopes are shown in Fig. 5.As can be seen, the contribution of both Ba135 and Ba137in the core of the otolith is clearly observed. Finally, themolar fractions for Ba137 were divided by those of Ba135

to obtain the molar fraction ratios shown in Fig. 6. Now, thedata obtained in the core of the otolith should be in agree-ment with the expected ratio of 0.3208. When the otolithsample was not isotopically labelled, the same data disper-sion was obtained for the whole line scan including theotolith core.

For the internal correction of mass bias, the residuals ofthe multiple linear regression were calculated for all massesand at all measured data points. Then, a single value for thesum of squared residuals was calculated for the whole dataset. This value was minimised by changing the mass biascorrection factor employed using the SOLVER applicationin Excel. The data shown in Figs. 3, 4, 5 and 6 are alreadycorrected with the optimum mass bias factor.

For the data treatment of the molar fraction ratios shownin Fig. 6, two additional criteria were employed. First, onlydata points for which both the molar fractions for Ba135 andBa137 were higher than 0.05 were taken into considerationfor each sample. As we can see in Fig. 5, only data points atthe otolith core fulfil this criterion. Second, for the remain-ing data points, a robust statistics test was employed toremove outliers [17]. The median of all data points wascalculated and, subsequently, the absolute deviations of eachindividual data point from the median were calculated.Then, the median of the absolute deviations (MAD) wascomputed and a ‘z score’ for each data point was computedusing the equation:

z score ¼ median

1:483�MAD

Data points which obtained a z score value higher than ±3were eliminated from the data set while the remaining values

0.01

0.1

1

10

100

0 50 100 150 200

Time (s)

XB

a137

/ X

Ba1

35

Fig. 6 Molar fraction ratiosx137Ba/x135Ba (grey circle andempty circle) for a markedotolith (sample 3 from Table 4)by LA-ICP-MS. The line showsthe expected molar fraction ra-tio and the white data pointsindicate those retained for thefinal calculations in this partic-ular otolith

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

0 50 100 150 200

Time (s)

Mo

lar

frac

tio

ns

Fig. 5 Molar fractions for ‘135Ba’ (grey circle), ‘137Ba’ (black circle)and natural Ba (empty circle) in a marked otolith (sample 3 fromTable 4) by LA-ICP-MS

Detection of transgenerational barium dual-isotope marks 2907

were employed to calculate the final molar fraction ratio forthat particular otolith. Such remaining data points are shownin white in Fig. 6. All remaining data points obtained in thecore of the nine otoliths analysed were averaged and the finalnumerical data are displayed in Table 4, including the numberof data points employed in the final calculation. As can beobserved, the measured molar fraction ratios are in agreementwith the theoretical expected ratio within the standard uncer-tainty of the measurements. Please note that no smoothing orany other data treatment was performed to the data shown inall figures indicating the robustness of the labelling proceduredeveloped.

2D imaging of fish otoliths

In order to study the capabilities of the dual-isotope mark forimaging purposes, one of the otoliths was subjected to 2Dlaser ablation. The ablation area was defined as a square of28 data points in the horizontal line and 21 data points in thevertical line with a spot diameter of 50 μm and a separationof 60 μm between the centre of each data point. So, the scanarea was ca. 1.53 mm wide and 1.30 mm high. A photo-graph of the otolith after laser ablation sampling is shown inthe centre of Fig. 7. The acquired data were subjected to themathematical procedures described before and the final

731aB531aB

Ba nat Xe nat

Fig. 7 Imaging of fish otoliths performed by 2D LA-ICP-MS scanning. The photograph of the otolith after laser ablation is shown in the centre ofthe figure. Molar fraction images for natural abundance Xe, natural abundance Ba, Ba135 and Ba137 are shown

Table 4 Average molar fractionratios xBa137/xBa135 obtained fornine otolith samples afterremoving of outliers usingrobust statistics

Uncertainties are given as stan-dard uncertainties

Otolith Number of data points employed xBa137/xBa135 Standard uncertainty

1 90 0.336 0.047

2 45 0.329 0.057

3 48 0.343 0.060

4 56 0.344 0.076

5 87 0.342 0.081

6 68 0.325 0.060

7 35 0.367 0.076

8 25 0.341 0.035

9 39 0.360 0.074

2908 G. Huelga-Suarez et al.

molar fraction data for natural Xe, natural Ba, Ba135 andBa137 were plotted as image with the help of MatLab. Theresults obtained are also presented in Fig. 7. The molarfractions for natural abundance Xe are higher outside ofthe otolith because of the low signals for barium in this partand the background contribution of xenon impurities in theargon plasma gas. These xenon data, together with the datafor natural abundance barium, help in defining the edge ofthe otolith which is in agreement with the photographshown. It is of interest to observe that the contribution ofnatural barium in the core of the otolith shows a minimum,while the contribution of Ba135 and Ba137 show, asexpected, a maximum in this area. Moreover, the ratio ofmolar fractions Ba137/Ba135 is constant in the core of theotolith and, therefore, provides the final information on thedual-isotope mark. The use of enriched isotopes and molarfraction data could be employed in other cases of imaging ofbiological materials where enriched isotopes are employedto study kinetic metabolic effects.

Conclusions

Laser ablation in combination with sector field ICP-MS iscapable of detecting dual barium isotopic marks present in theotoliths of 9-month-old salmon. In comparison with previousresults using total digestion of the otolith and multi-collectorICP-MSmeasurements [9], the proposed methodology is easierand more affordable providing a 100 % detection efficiencyeven with a noisy instrumental set-up and no data filtering.Although LA-ICP-MS provides a more modest precision thansolution ICP-MS, it clearly suffices as, due to the possibility ofanalysing an specific region of the otolith, it is not possible tomistake the natural signature with the artificial mark and, there-fore, discrimination between marked and non-marked speci-mens can be easily done at any stage of the life the fish, as oncedeposited in the core the otolith the mark remains intact untildeath. Based on the results shown in Table 4 for the molarfraction ratios and their standard uncertainties, it will be possi-ble to distinguish easily between salmon marked with differentisotopemixtures (e.g. molar fraction ratios of 1:10, 1:5, 1:3, 1:2,2:3, 3:4, 1:1, 4:3, 3:2, 2:1, 3:1, 5:1 and 10:1) for advancedsalmon population studies.

Acknowledgments The authors would like to thank Jerónimo de laHoz and the Dirección General de Recursos Naturales y ProtecciónAmbiental (Gobierno del Principado de Asturias) for their collabora-tion and care of the marked salmons. Dr. America Garcia-Valiente isacknowledged for the extraction of fish otoliths and Emilio J. Ariñoand Joaquín Vázquez for their help with the otolith sample preparation.Financial support through the project MICINN CTQ2009-12814, aswell as the provision of FEDER funds for the purchase of the multi-collector ICP-MS instrument, is gratefully acknowledged.

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