trace element analysis in the serum and hair of antarctic leopard seal, hydrurga leptonyx, and...

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Trace element analysis in the serum and hair of Antarcticleopard seal, Hydrurga leptonyx, and Weddell seal,Leptonychotes weddellii

Rachael Graya,b,⁎, Paul Canfielda, Tracey Rogersb,c

aFaculty of Veterinary Science, The University of Sydney, NSW 2006 AustraliabAustralian Marine Mammal Research Centre PO Box 20 Mosman, NSW 2088 AustraliacEvolution&EcologyResearchCentre andSchool of Biological Earth andEnvironmental Sciences, TheUniversity ofNewSouthWales,NSW2052Australia

A R T I C L E I N F O

⁎ Corresponding author. JD Stewart Building/B2643; fax: +61 2 9351 6880.

E-mail address: [email protected]

0048-9697/$ – see front matter © 2008 Elsevdoi:10.1016/j.scitotenv.2008.03.039

A B S T R A C T

Article history:Received 3 December 2007Received in revised form25 March 2008Accepted 27 March 2008

Leopard seal, Hydrurga leptonyx, and Weddell seal, Leptonychotes weddellii, occupy an uppertrophic level within the Antarctic ecosystem and are useful indicator species in theSouthern Ocean of trace element concentrations. Reference values for the concentration of19 trace elements were determined in the serum and hair of leopard and Weddell sealssampled in Eastern Antarctica. These reference values can be used as ‘baseline’ levels formonitoring trace element concentrations in these species. Greater trace elementconcentrations were determined in hair compared to serum, indicating different timescales of trace element accumulation in these samples. For the majority of trace elements,except for Se in the leopard seal samples and Cr in the Weddell seal samples, significantregression relationships for trace element concentrations in hair and serum were notelucidated. Significant differences were determined in the concentrations of seven out of 15elements with hair type, moult and new, in the leopard seal; concentrations in moult hairwere determined to be greater than in new hair for all elements except Zn. Hair analysis wasdetermined to be useful for monitoring exposure to trace elements and when collected offthe ice from moulting seals, hair can be employed as a non-invasive sample for traceelement analysis in leopard and Weddell seals.

© 2008 Elsevier B.V. All rights reserved.

Keywords:AntarcticaHairLeopard sealSerumTrace elementsWeddell seal

1. Introduction

Leopard seal, Hydrurga leptonyx, and Weddell seal, Leptony-chotes weddellii, are long-lived mammals occupying an uppertrophic level within the Antarctic ecosystem. As such, theirpotential to accumulate trace elements make them useful‘indicator species’ of trace element bioaccumulation in themarine ecosystem they occupy, similar to other pinnipedspecies (Noda et al., 1993; Miyazaki, 1994; Ivanter et al., 1998;

O1, Faculty of Veterinary

(R. Gray)

ier B.V. All rights reserve

Beckmen et al., 2002; Ikemoto et al., 2004; Griesel et al., 2008).The primary source of trace elements in marine mammals isvia their diet (Yediler et al., 1993; Noda et al., 1995; Saeki et al.,1999; Brookens et al., 2007) and trace element accumulation inAntarctic species is thought to be a natural phenomenon, withnegligible impact from anthropogenic influences when com-pared to northern hemisphere species (Yamamoto et al., 1987;Noda et al., 1993; Andrade et al., 2007). With increasing humanimpacts in the Antarctic, including an increase in tourism and

Science, University of Sydney NSW 2006 Australia. Tel.: +61 2 9351

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http://dx.doi.org/10.1016/j.scitotenv.2008.03.039

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industry, the degree of anthropogenic contamination in theregion may change, thereby, altering the exposure of allspecies in this marine ecosystem to trace elements.

Studies detailing trace element concentrations in Antarcticpinnipeds are limited (Steinhagen-Schneider, 1986; Andradeet al., 2007); therefore monitoring trace element contaminationin Antarctic pinnipeds is difficult due to a lack of baseline valuesfor monitoring studies. Whilst several studies report traceelement concentrations in the tissues of the leopard seal (Beck,1956; Denton et al., 1980 and references therein; Wagemann andMuir, 1981;Wagemann,unpublisheddata; Szefer et al., 1993, 1994;Kemper et al., 1994; Gray, 2005) and Weddell seal (Beck, 1956;Denton, Marsh & Griffiths, unpublished data; Denton et al., 1980and references therein; Wagemann and Muir, 1981; Wagemann,unpublished data; Schneider et al., 1985; Steinhagen-Schneider,1986; Honda et al., 1987; Yamamoto et al., 1987; Noda et al., 1993;Szefer et al., 1993, 1994; Gray, 2005), the majority of these studiesare based on a small sample size and the trace elementconcentrations reported may not be useful as baseline levels formonitoring trace element concentrations in these species. To ourknowledge, apart from one study detailing trace elementconcentrations in the blood and hair of a small number ofWeddell seals (Yamamoto et al., 1987) and another detailingcopper (Cu) concentration in the blood of two Weddell seals andtwo leopardseals (Beck,1956), thereareno reports in the literatureof trace element concentrations in the serum and hair of thesespecies.

Typically, tissue samples such as liver, kidney, and muscleare the main samples utilised for trace element analysis inpinnipeds. However, opportunities for collecting tissue sam-ples from free-ranging leopard and Weddell seals are limitednecessitating the investigation of alternative and preferablynon-invasive samples for this analysis, for example hair, toobtain statistically significant sample sizes. Trace elementsare incorporated into hair during its period of growth (Jerviset al., 1977; Brookens et al., 2007), trace elements are deliveredto hair mainly via the blood supply (Ikemoto et al., 2004;Andrade et al., 2007) and trace elements remain unchangedonce incorporated into the hair (Phelps et al., 1980). Hair iseasy to collect and store and has the potential to reflect long-term accumulation of trace elements (Gallagher et al., 1984).Several studies detail trace element concentrations in pin-niped hair/fur (Medvedev et al., 1997; Ivanter et al., 1998; Saekiet al., 1999;Wiig et al., 1999; Ikemoto et al., 2004; Andrade et al.,2007), and in several of these studies, the concentration ofsome trace elements was greatest in hair when compared totheir concentrations in the other tissues analysed (Medvedevet al., 1997; Ivanter et al., 1998; Ikemoto et al., 2004; Gray, 2005;Brookens et al., 2008). Relatively high concentrations of sometrace elements such as lead (Pb), nickel (Ni), cobalt (Co) andchromium (Cr) have been determined in pinniped hair, whilstlow or non-detectable concentrations of these elements havebeen determined in tissue samples, such as liver and muscle,in previous studies (Denton et al., 1980 and references therein;Frank et al., 1992; Szefer et al., 1994; Watanabe et al., 1996,2002), most likely due to negligible accumulation of theseelements in the other tissues analysed. Therefore, for thosetrace elements for which appreciable tissue accumulationdoes not occur, hair may be the most useful sample todetermine trace element concentrations.

Several studies suggest that hair analysis is a useful tool fordetermining trace element concentrations in pinnipeds (Wenzelet al., 1993; Yediler et al., 1993; Watanabe et al., 1996; Wiig et al.,1999). Hair has a role as an excretory route for particular elementsin pinnipeds via the moult (Wenzel et al., 1993; Watanabe et al.,1996, 1998; Saeki et al., 1999;Wiig et al., 1999; Ikemoto et al., 2004;Brookens et al., 2007, 2008); andwhen collectedoff the ice or othersubstrate from moulting seals, hair can be regarded as a non-invasive sample for trace element analysis. Reports also suggestthat trace element concentrations in hair reflect the internaltissue concentrations of several elements (Watanabe et al., 1996;Ikemoto et al., 2004); that hair is a reliable indicator of exposure totrace elements (Wenzel et al., 1993) with one study suggestingthat hair may be a more reliable and sensitive indicator of traceelement concentrations when compared to liver, kidney andmuscle samples (Medvedev et al., 1997); that hair samples couldpotentially be employed to monitor several trace elements inpinnipeds (Ikemoto et al., 2004; Andrade et al., 2007; Brookenset al., 2007), and that trace element concentrations determined inmoult hair can be used as an indicator of the biomagnificationprocess of heavy metals in the food web (Andrade et al., 2007).However, the extent towhich trace element concentrations in thehair of marine mammals reflect internal tissue concentrations(Wiig et al., 1999) and body burden (Brookens et al., 2008) of traceelements has been questioned, a potential disadvantage for theutilisationofhair given that tissuesamples, for example, liver andmuscle, are generally regarded as the ʽgold standard' fordetermining trace element concentrations.

Few studies have investigated the relationship between traceelement concentrations in hair and serum/blood, or the effect ofsealgenderandhair type,or stageof thehair cycle (moultandnew)on trace element concentrations in hair. One study reportssignificant relationships between concentrations of trace ele-ments, specifically total mercury (THg), in hair and blood andsignificantly greater mean concentrations of THg in the hair ofadult male compared to adult female Pacific harbour seal, Phocavitulina richardii (Brookens et al., 2007). Apart from one other studydetailing significantly greater concentrations of cadmium (Cd) inthehairofadultmalecompared toadult femaleharbourseal,Phocavitulina (Wenzeletal., 1993), significantdifferences in traceelementconcentrations in the hair of male and female pinnipeds have notbeen demonstrated (Watanabe et al., 1996; Medvedev et al., 1997;Ivanteretal., 1998;Beckmenetal., 2002). Finally,onlyonestudyhascompared trace element concentrations inmoult and new hair ofpinnipeds (Wenzel et al., 1993). Therefore, inaddition to investigat-ing the effect of seal gender on trace element concentrations inhair, an important objective of the present study is to determinethe effect of hair type on trace element concentrations.

In the present study, trace element analysis by inductivelycoupled plasma mass spectroscopy (ICP-MS) was performedon serum and hair samples collected from leopard andWeddell seals in Antarctica to determine the concentrationsof aluminium (Al), arsenic (As), barium (Ba), bismuth (Bi),calcium (Ca), cadmium (Cd), cobalt (Co), chromium (Cr), copper(Cu), mercury (Hg), iron (Fe), magnesium (Mg), manganese(Mn), nickel (Ni), lead (Pb), selenium (Se), tin (Sn), vanadium(V), and zinc (Zn). The objectives of this study were (i) todetermine the relationship, if any, between trace elementconcentrations in hair and serum in these species; (ii) toestablish reference values for trace element concentrations in

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the serum and hair of the leopard andWeddell seal which canbe used as ‘baseline’ levels for future monitoring of traceelement concentrations in these species; (iii) to investigate theeffect of hair type and seal gender on trace elementconcentrations in the hair of the leopard seal; and (iv) toassess the usefulness of hair as a non-invasive sample fortrace element analysis in the leopard and Weddell seal.

2. Materials and methods

2.1. Leopard seals

Eight male and 17 female adult leopard seals were immobilisedfor sample collectionduring theAustral summer seasonsof 1999–2002 along the fast ice edge and on ice floes in Prydz Bay nearDavis Station (68°36'S, 78°02'E), Eastern Antarctica (Fig. 1). Six ofthese seals (three male and three female) were immobilised andsampled in both 1999/2000 and 2000/2001 seasons, therefore atotal of 31 immobilisations were undertaken for sample collec-tion. Leopard seals were accessed along the fast ice edge by all-

Fig. 1 –Map of the Antarctic coastline near Davis Station showingand the location of sampling of Weddell seals are shown. VestfoData Centre, Australian Antarctic Division, 2008. Map produced b

terrain vehicles or by boat when on ice floes. Seals wereapproached on foot and darted from a distance of 12–15 m (CO2

powered Telinject G.U.T 50 rifle, Telinject, Australasia). Immobi-lisation was achieved by either a combination of 0.18–0.27 mg/kgmidazolam (Roche Products Pty. Ltd., Australia; reconstituted to15mg/ml by Royal Hobart Hospital, Tasmania) and 1.0–1.5mg/kgpethidine (Sigma Pharmaceuticals Clayton, Victoria; reconsti-tuted to 150 mg/ml by Royal Hobart Hospital, Tasmania), or a 1:1ratio of 0.5–1.5 mg/kg tiletamine/zolazepam (Telazol, 100 mg/ml,Fort Dodge, Australia or Zolatil100, 100 mg/ml, Virbac, AustraliaPty. Ltd.). Atropine (AstraZeneca Pty. Ltd., Australia; reconstitutedto 16mg/ml by Royal Hobart Hospital, Tasmania) at a dose rate of0.015 mg/kg was administered with the immobilising drugs. Atotal of 30 serum samples from the leopard seals immobilisedwere available for trace element analysis in this study.

2.2. Weddell seals

Eleven adult lactating Weddell seals were immobilised forsample collection during the Austral summer season of 2002/2003 at several sites in Long Fjord near Davis Station (Fig. 1).

the study area. The GPS location of individual leopard sealslds coastline GIS data obtained from the Australian Antarcticy M. Terkildsen.


Weddell seals were immobilised with 0.3–0.5 mg/kg midazo-lam (midazolam hydrochloride 20 mg/ml, prepared by thePharmacy Department, Royal Hobart Hospital, Hobart, Tas-mania) by hand injection using a needle attached to 140 cmlength of sterile plastic tubing, induction with 5% isoflurane(100% vol./vol. Forthane, Abbott Australasia Pty Ltd. NSW,Australia) in 15 L/min oxygen by face mask, and maintenancewith isoflurane in 6–10 L/min oxygen (Bodley et al., 2005).Blood was collected from 10 of these seals; hair was collectedfrom all eleven seals. A hair sample was also collected fromone adult lactating Weddell seal found dead in Long Fjord,bringing the total number of Weddell seal hair samplescollected in 2002/2003 to 12.

2.3. Blood collection and sample preparation

Blood was collected from the extradural intravertebral sinus inthe lumbar region using 3 1/4 inch spinal needles (Yale® SpinalNeedle B-D Becton Dickinson Spain) into plain serum tubes (BDVacutainer Systems, Bellivier Industrial Estate, Plymouth, UK).Samples were left to clot, the tubes were centrifuged at10,000 rpm, and serumwas stored at −80 °C or in liquid nitrogenprior to analysis. Serumwas available for trace element analysisin the present study. Serum was thawed at room temperatureand mixed by gentle rotation. 50 μl of serum was mixed with4450 μl of diluent consisting of 2 g EDTA (Ethylenediaminete-traacetic acid, diammoniumsalt hydrate 97%, Aldrich ChemicalCompany Inc., Milwaukee, USA), 2 g TritonX-100 (t-Octylphe-noxypoly-ethoxyethanol Sigma® Sigma-Aldrich Co., St. Louis,MO, USA), 50ml Ammonia (di-Ammoniumhydrogen orthopho-sphate AnalaR® BDH Limited Poole, England), and 4 ml InternalStandard (Rhodium 1000 ppm, Atomic Spectroscopy Standard,Perkin-Elmer, USA). Serum samples were then analysed usingICP-MS. Sampleswith analyte concentrationsover-range for thehighest standard were diluted 1 in 10 and processed as above.

2.4. Hair collection and sample preparation

A total of 38 hair samples, moult (n=27) and new (n=11), werecollected from leopard seals in 2000/2001 and 2001/2002 eitherduring immobilisationorvia collectionoff the ice fromunknownseals. Of these 38 hair samples, both hair types, moult and new,were collected from five individuals. Pre-moult hair wascollected from immobilised lactating Weddell seals (n=11) andfrom one lactating adult seal found dead in Long Fjord in 2002/2003.Approximately1–5gofhairwas collected fromeachsealbyeither cutting hair with sterile stainless steel scissors, pluckinghair from moulting seals, or collection of hair off the ice from asite were a moulting seal had recently hauled out. Hair wasstored insterilepolyethylenebagsprior toanalysis. Themajorityof the leopard seal hair samples were washed twice in reagent-grade diethyl ether and then air-dried overnight for anotherstudy (Hall-Aspland et al., 2005). Diethyl ether has been used towash hair samples prior to trace element analysis (Jervis et al.,1977; Evans and Jervis, 1987; Caroli et al., 1992) and suitablerecovery rates (for example N80% for Pb) were obtained using awash of doubly distilled water, ethanol and diethyl ether(Chattopadhyay et al., 1977; Jervis et al., 1977).

Prior to analysis, all hair samples were washed in reagentgrade acetone (AnalaR Acetone, Merck Pty Ltd. Kilsyth, Victoria,

Australia) using the method recommended by the InternationalAtomic Energy Agency (Chatt and Katz, 1988). Samples werefreeze-dried at 760 mm mercury (Edwards High Vacuum Ltd,Manor Royal, Crawley, Sussex, Model # EF03) for 4 h. 50 mg ofwashedand freeze-driedhairwere digestedwith 400 μl of reagentgrade concentrated nitric acid (Suprapur® Nitric Acid 65% Merck,Darmstadt, Germany) in a sand bath at 80–100 °C for 8 h. Samplesweredilutedwith 1600μl of doubledistilledwater; 50μl of thehairdigest was mixed with 4450 μl of the diluent described above forserum samples and analysed using ICP-MS. The methodologyemployed in this study for the preparation of hair samples wasbased on the modification of the existing protocol used for thepreparation of tissue samples in the commercial laboratoryemployed for the analysis. Each hair sample was analysed induplicate, and the mean concentration was used in all statisticalanalyses. As the majority of trace element concentrations inpinniped hair/fur are reported on a μg/g dry weight basis, theseunits have been used in the present study to report trace elementconcentrations in hair to provide ease of comparisonwith resultsreported in the literature.

2.5. Analytical technique

Trace element concentrations were determined in serum andhair samples by ICP-MS (Varian Ultra-Mass SpectrometerSystem Varian Australia Pty Ltd Mulgrave, Victoria) and thesignal intensitieswere compared to a standard calibration curvefor each trace element. Blankswere analysed every ten samplesto check for contamination. The accuracy and precision of theanalytical procedurewas verified by the analysis of replicates ofstandardmaterial Normal Range Trace Element SerumToxicol-ogy Control (UTAK Lot # 66816, UTAK Labs Inc., Valencia,Ca 91355;n=10) and NIST (National Institute of Standardsand Technology) Standard Reference Material 1577b BovineLiver (U.S. Department of Commerce National Institute ofStandards andTechnology, Gaithersburg,MD;n=9), comparisonof observed and certified values (where available), and calcula-tion of the coefficient of variation of the replicates for each traceelement. For the serumcontrols, the coefficient of variationwasacceptable (b15 %) for Al, Ba, Ca, Cr, Cu, Fe, Mg, Se, Zn; thecoefficient of variationwas N15% in thosemetals inwhich therewere very low concentrations (Bi, Cd, Hg, Sn) and for those traceelements inwhich polyatomic spectral interferences are knownto occur (V). The observed concentrations of As, Co, Mn, Ni andPb were below the detection limit in the serum controls. For theNIST bovine liver standard referencematerial, the coefficient ofvariation was acceptable (b15 %) for Al, Cu, Fe, Mg, Mn, Zn; thecoefficient of variationwas N15% in thosemetals inwhich therewere very low concentrations (Bi, Cd, Co, Hg, Ni, Pb, Sn). Theobserved concentrations of As, Ba, Ca, Cr, Se, and V were belowthe detection limit in the NIST bovine liver controls.

2.6. Statistical methods

Data was analysed using the GenStat® statistical package (6thEdition; Rothamsted Experimental Station 2002). For thepurposes of statistical analysis, zero values for trace elementconcentrations were used where concentrations were belowthe detectable limit. Values more than three standard devia-tions from the mean were excluded from the statistical


analysis. To enable direct comparison of serum and hairresults in the tables, trace element concentrations in serum inμg/L were converted to an equivalent μg/g dry weight (wt)concentration by dividing the concentration in μg/L by thedensity of serum (1.025 g/ml). Reference values were devel-oped for each trace element using the estimated 2.5th and97.5th percentiles. Significance was taken to be pb0.05 for allstatistical analysis. For all statistical analyses, loge transfor-mations for normality of the data and homogeneity ofvariances were conducted as required, and the assumptionsof independence and random sampling were also met.

2.6.1. Comparison of trace element concentrations in serumand hairLinear regression was used to assess the association betweenthe concentration of each trace element in the serum (μg/L) andhair (μg/g) using animals where both measures were collected,and these were performed separately for leopard seals (n=19)andWeddell seals (n=10). For the leopard seal samples, the twohair types (moult and new hair) were analysed as a single group(averagingasnecessaryovermoult andnewhair concentrationswhen both were available on the one animal, n=5). This

Table 1 – Trace element concentrations in the serum of adult leAntarctica

Traceelement

Units Mean±standard dev(median)

Mg μg/L 17,964±2269 (17,77μg/g 17.5±2.21 (17.3)

Al μg/L 254±88.7 (267)μg/g 0.25±0.09 (0.26)

Ca μg/L 64,141±10,723 (62,6μg/g 62.6±10.5 (61.1)

Va μg/L 60.7±4.56 (61.6)μg/g 0.06±0 (0.06)

Cra μg/L 224±18.2 (228)μg/g 0.22±0.02 (0.22)

Mnb μg/L 4.09±1.01 (4.17)μg/g b0.005

Feb μg/L 3921±1753 (3397)μg/g 3.83±1.71 (3.31)

Nib μg/L 7.04±1.48 (6.63)μg/g 0.01±0 (0.01)

Cu μg/L 615±293 (626)μg/g 0.60±0.29 (0.61)

Zn μg/L 489±119 (471)μg/g 0.48±0.12 (0.46)

Asb μg/L 69.9±21.7 (65.0)μg/g 0.07±0.02 (0.06)

Se μg/L 606±154 (602)μg/g 0.59±0.15 (0.59)

Cd μg/L 0.53±0.55 (0.32)μg/g b0.005

Hgb μg/L 0.03±0.14 (0)μg/g b0.005

Pbb μg/L 0.01±0.05 (0)μg/g b0.005

Mean±standard deviation (median), developed reference range of values,Co were below detection limits and are not shown; Sn and Ba were not anmass.a n=28.b n=29.

combining was undertaken as the origin of the hair samplehas no significant effect on the regression relationship for traceelement concentrations in serumandhair as determined by thefitting of the regression model:

Serum ¼ β0 þ β1Hair þ HairTypeþ ε

where ‘Serum’ is the concentration of the trace element inserum; β0 is the y-intercept or constant; β1 is the slope of the line;‘Hair’ is the corresponding concentration of the trace element inhair; ‘HairType’ is a categorical variable indicating origin of thehair (moult, new, or averaged concentration), and ε is therandom error component for the model.

Based on this model, there was no significant effect ofHairType on Serum, therefore the single predictor linearregression model was used for this analysis:

Serum ¼ β0 þ β1Hair þ ε

Untransformed data, loge transformations of the x axis andboth x and y axes were employed, the choice of transformationbeing based on the data for each of the associations investigated.

opard seals (n =30) sampled in 1999/2000 and 2000/2001 in

iation Referencevalues

Observedvalues

3) 13,323–22,605 12,150–23,39013.0–22.1 11.9–22.873.0–436 56.1–4310.07–0.43 0.05–0.42

02) 42,210–86,073 43,235–95,76441.2–84.0 42.2–93.451.4–70.1 52.6–69.60.05–0.07 0.05–0.07187–262 178–2530.18–0.26 0.17–0.252.02–6.16 1.80–6.420–0.01 0–0.01331–7512 2025–82080.32–7.33 1.98–8.014.01–10.1 5.02–12.10–0.01 0–0.0114.6–1215 36.9–11690.01–1.18 0.04–1.14247–732 262–7490.24–0.71 0.26–0.7325.4–114 46.3–1400.02–0.11 0.05–0.14291–921 363–9640.28–0.90 0.35–0.940–1.67 0–2.07– –0–0.30 0–0.73– –0–0.11 0–0.27– –

and observed range of values in μg/L and μg/g drywt equivalent. Bi andalysed in serum. Trace elements are arranged in the table by atomic

Table 2 – Trace element concentrations in the serum (μg/L andμg/g dry wt equivalent, n =10) and hair (μg/g dry wt, n =12) ofWeddell seals sampled in 2002/2003 in Antarctica

Trace element Tissue(units)

Mean±standard deviation(median)

Referencevalues

Observedvalues

Mg Serum (μg/L) 22,407±2375 (22,859) 17,034–27,779 17,653–25,983Serum (μg/g) 21.9±2.32 (22.3) 16.6–27.1 17.2–25.3Hair (μg/g) 754±145 (781) 434–1074 531–933

Al Serum (μg/L) 81.4±204 (3.23) 0–542 0–651Serum (μg/g) 0.08±0.20 (0) 0–0.53 0–0.64Hair (μg/g) 9.13±4.69 (8.07) 0–19.5 0.09–17.3

Ca Serum (μg/L) 68,389±4719 (66,895) 57,714–79,065 63,489–77,465Serum (μg/g) 66.7±4.60 (65.3) 56.3–77.1 61.9–75.6Hair (μg/g) 604±69.0 (594) 453–756 457–704

V Serum (μg/L) 104±11.9 (99.3) 77.2–131 91.8–128Serum (μg/g) 0.10±0.01 (0.10) 0.08–0.13 0.09–0.12Hair (μg/g) 4.22±0.65 (4.37) 2.80–5.65 2.73–5.06

Cr Serum (μg/L) 379±43.6 (357) 280–477 332–458Serum (μg/g) 0.37±0.04 (0.35) 0.27–0.47 0.32–0.45Hair (μg/g) 5.87±0.47 (5.99) 4.84–6.89 4.93–6.42

Mn Serum (μg/L) 0.40±0.96 (0) 0–2.58 0–3.05Serum (μg/g) b0.001 – –Hair (μg/g) 1.15±0.41 (1.23) 0.26–2.04 0.52–1.80

Fe Serum (μg/L) 3417±781 (3316) 1650–5183 2508–4945Serum (μg/g) 3.33±0.76 (3.24) 1.61–5.06 2.45–4.82Hair (μg/g) 73.9±16.4 (72.0) 37.7–110 39.4–108

Co Serum (μg/L) 0.62±0.37 (0.52) 0–1.45 0.33–1.57Serum (μg/g) b0.001 – –Hair (μg/g) 0.04±0.04 (0.03) 0–0.13 0.01–0.13

Ni Serum (μg/L) BDLa

Hair (μg/g) 3.52±0.76 (3.50) 1.84–5.20 2.37–5.18Cu Serum (μg/L) 371±180 (323) 0–779 195–837

Serum (μg/g) 0.36±0.18 0–0.76 0.19–0.82Hair (μg/g) 15.1±13.2 (10.1) 0–44.2 3.87–51.3

Zn Serum (μg/L) 371±55.7 (365) 245–497 319–502Serum (μg/g) 0.36±0.05 (0.36) 0.24–0.49 0.31–0.49Hair (μg/g) 137±14.3 (134) 105–168 117–159

As Serum (μg/L) 46.2±23.5 (44.7) 0–99.4 9.43–96.4Serum (μg/g) 0.05±0.02 (0.04) 0–0.10 0.01–0.09Hair (μg/g) 2.51±2.11 (2.30) 0–7.14 0.11–7.14

Se Serum (μg/L) 233±78.6 (215) 55.1–411 137–440Serum (μg/g) 0.23±0.08 (0.21) 0.05–0.40 0.13–0.43Hair (μg/g) 3.12±0.93 (2.92) 1.08–5.16 2.04–4.71

Cd Serum (μg/L) 0.62±0.98 (0.36) 0–2.83 0–3.20Serum (μg/g) b0.001 – –Hair (μg/g) 2.81±0.58 (2.76) 1.54–4.07 1.93–3.82

Sn Serum (μg/L) BDLa

Hair (μg/g) 1.36±1.78 (0.60) 0–5.28 0–5.99Ba Serum (μg/L) 77.5±36.7 (86.6) 0–161 4.23–118

Serum (μg/g) 0.08±0.04 (0.08) 0–0.16 0–0.12Hair (μg/g) 4.82±9.22 (0.69) 0–25.1 0.19–25.7

Hg Serum (μg/L) 10.8±7.10 (11.3) 0–26.9 0–21.8Serum (μg/g) 0.01±0.01 (0.01) 0–0.03 0–0.02Hair (μg/g) 5.60±1.43 (6.01) 2.45–8.74 3.43–7.33

Pb Serum (μg/L) 0.21±0.36 (0.02) 0–1.02 0–0.91Serum (μg/g) b0.001 – –Hair (μg/g) 1.29±1.12 (0.80) 0–3.79 0.08–3.30

Mean±standard deviation (median), developed reference range of values, and observed range of values shown. Bi was below detection limitsand is not shown. Trace elements are arranged in the table by atomic mass.a Below detection limit=BDL.


2.6.2. Effect of hair type and seal gender on trace elementconcentrations in the leopard sealA general linearmodel analysis of variance (ANOVA)was used toinvestigate theeffect ofhair type (moult andnew)andseal genderon trace element concentrations in the hair of the leopard seal.

Backwardseliminationof factorswasemployed toeliminatenon-significant factors from the general linearmodel. Five individualswere sampled in successive seasons (1999/2000 and 2000/2001). Itwas not possible to include seal identification as a factor in thestatisticalmodel due to collinearity with other terms, thus seal to


seal variationwasnot explicitly taken into account.However, thisis unlikely to impact the statistical results due to the smallnumber of repeat samples employed, and the use of samplingtimes sufficiently far apart such that serial correlation would notbe expected for the trace element concentrationsmeasured in thehair.Where the assumptions of ANOVA (normal distribution andhomogeneity of variances) were not met by the data asdetermined by assessment of the histogram of residuals andthe fitted-value plot, the Mann–Whitney U test was employedand each factor compared separately. Where variables were logenormally distributed or where non-parametric statistical testswere employed, geometric means (back transformed from theloge scale) and 95% confidence intervals are reported instead ofthe mean and standard deviation. Although the Mann–WhitneyU test was used for the comparison of Pb concentrations inmoult and new hair, the mean±standard deviation is reportedinsteadof thegeometricmeanand95%confidence intervaldue tothe lowsample size for newhairwhen loge transformeddatawasemployed.

3. Results

3.1. Comparison of trace element concentrations in serumand hair

Table 1 displays descriptive statistics and developed referencevalues for trace element concentrations in the serum (μg/L andμg/g dry wt equivalent) of leopard seals (n=30) sampled in1999/2000 and 2000/2001. Table 2 displays descriptive statis-tics and developed reference values for trace element con-centrations in the serum (n=10) and hair (n=12) of Weddellseals sampled in 2002/2003.

Of the 14 trace elements compared in thehair and serumofthe leopard seal, namely Al, As, Ca, Cd, Cr, Cu, Fe, Hg, Mg, Mn,Ni, Se, V, and Zn, a significant regression relationship wasonly determined for the concentration of Se (t17=3.331;p=0.004; R2=39.5%) and the R2 value indicates that this isnot a strong regression relationship. The equation for thefitted regression line for Se is ySerum=397+65.0xHair. For all ofthe trace elements analysed, trace element concentrations

Table 3 – Test statistic, statistical test, and the mean concentratfemale leopard seals sampled in 1999/2000 and 2000/2001 in Anwere demonstrated with hair type and seal gender, respectivel

Trace element Test statistic and statistical test

As F=16.05(1,28); p=0.000 (ANOVA)Mg F=6.65(1,36); p=0.014 (ANOVA)Zn F=22.42(1,36); p=0.000 (ANOVA)Ca U:28.0(1,36); pb0.001 (Mann–Whitney)Cd U:61.0(1,36); p=0.004 (Mann–Whitney)V U:65.0(1,36); p=0.006 (Mann–Whitney)Ni U:78.0(1,36); p=0.023 (Mann–Whitney)As F=21.23(1,28); p=0.000 (ANOVA)Se F=5.64(1,30); p=0.024 (ANOVA)Hg F=6.92(1,30); p=0.013 (ANOVA)Mn F=6.87(1,30); p=0.014 (ANOVA)

All concentrations in μg/g dry wt.

were greater in hair when compared to serum for the leopardseal.

Of the 15 trace elements compared in the hair and serum oftheWeddell seal, namely, Al, As, Ba, Ca, Cd, Co, Cr, Cu, Fe, Hg,Mg,Pb, Se, V and Zn, a significant regression relationship was onlydetermined for the concentration of Cr (t8=−3.05; p=0.016;R2=53.7%) and the equation for the fitted regression line for Crwas ySerum=769–67.0xHair. For all of the trace elements analysed,trace element concentrations were greater in hair when com-pared to serum for the Weddell seal.

3.2. Effect of hair type and seal gender on trace elementconcentrations in the leopard seal

Table 3 displays the test statistic, statistical test and the meanconcentration of trace elements in moult and new hair, and inmale and female leopard seals, for trace elements in whichsignificant differences were demonstrated with hair type andseal gender respectively. Significant differences in the con-centration of trace elements with hair type (moult and newhair) were demonstrated for seven of the 15 trace elementsanalysed. The concentration of trace elements was greater inmoult hair compared to new hair for all of these elementsexcept Zn. Significant differences in trace element concentra-tions were also determined with seal gender for As, Se, Hg andMn. No significant differences were determined in theconcentration of Al, Cr, Pb, Cu and Fe with either seal genderor hair type in leopard seals. Table 4 displays descriptivestatistics and developed reference values for the concentra-tion of trace elements in moult and new hair of the leopardseal.

In five leopard seals in which moult and new hair werecollected from the same seal in the same season, greatermeanconcentrations of Mg, Ca, V, Co, Ni, As, Cd, Pb and Bi weredetermined in moult hair compared to new hair. In two sealsin which new hair was collected in one season and moult hairin the following season (thus essentially the same hair wascollected), the concentrations of all of the elements except forZn, Se and Hg were greater in moult hair compared to newhair, whilst Cu concentration in one of the seals was greater innew hair compared to the moult hair.

ion of trace elements in moult and new hair, and male andtarctica, for trace elements in which significant differencesy

Mean concentrations (μg/g dry wt)

Moult hair 1.63 (n =27) New hair 0.85 (n =10)Moult hair 894 (n =27) New hair 585 (n =11)Moult hair 103 (n =27) New hair 128 (n =11)Moult hair 896 (n =27) New hair 563 (n =11)Moult hair 1.19 (n =27) New hair 0.22 (n =11)Moult hair 1.97 (n =27) New hair 0.87 (n =11)Moult hair 1.35 (n =27) New hair 0.59 (n =11)Male seals 1.99 (n =10) Female seals 1.07 (n =21)Male seals 4.47 (n =11) Female seals 3.01 (n =21)Male seals 5.27 (n =11) Female seals 2.98 (n =21)Male seals 1.50 (n =11) Female seals 1.24 (n =21)


4. Discussion

4.1. Comparison of trace element concentrations in serumand hair

In the present study, no obvious relationship between traceelement concentrations in hair and serum was determinedand hair type had no significant effect on the regressionrelationship for trace element concentrations in serum andhair of the leopard seal. The lower trace element concentra-tions observed in serum reflect trace element concentrationsat the time of collection with no accumulation over time.These findings support those of previous studies in humanswhere hair was determined to represent trace elementconcentrations over a period of months, whilst trace elementconcentrations in blood/serum reflect current exposure, hoursor days (Laker, 1982). If there was a relationship between traceelement concentrations in hair and serum in seals, it would beexpected to be observed in serum and new hair, however, inthe only study detailing the relationship between hair andblood trace element concentrations in pinnipeds, a significantrelationship between THg concentrations in moult hair andblood of the Pacific harbour seal was determined (Brookenset al., 2007). In human studies, a high degree of correlationwasdetermined for Hg concentrations in new hair and blood,indicating that Hg concentrations in newly formed hair didreflect recent blood levels (Phelps et al., 1980). Significantcorrelations between blood and hair concentrations of Pb(Chattopadhyay et al., 1977; Jervis et al., 1977), and Cu (Klevay,1970) were also reported. However, in other studies, concen-trations of Cu, Zn, Pb, and Se in human hair were poorlycorrelated with tissue and blood levels (Gallagher et al., 1984;

Table 4 – Trace element concentrations in moult (n =27) and neand 2000/2001 in Antarctica

Traceelement

Geometric mean and 95% CI ormean±standard deviation (median)

Moult New

Mg 894 (739, 1081) 585 (446, 767)Al 7.62±4.73 (6.79) 8.87±2.45 (8.00)a

Ca 896 (774, 1037) 563 (464, 683)V 1.82 (1.62, 2.04) 0.91 (0.56, 1.48)Cr 3.81±0.64 (3.96) 4.12±0.64 (4.11)Mn 1.36 (1.28, 1.45) 1.36 (1.08, 1.68)Fe 73.1 (70.3, 76.0) 76.8 (66.9, 88.1) a

Ni 1.35 (1.17, 1.57) 0.76 (0.47, 1.25)Cu 3.37 (3.03, 3.75)b 3.66 (3.00, 4.47)Zn 103 (97.7, 108) 128 (117, 141)As 1.63±0.73 (1.52) 0.85±0.61 (0.75)a

Se 2.95±1.41 (2.90) 4.13±2.24 (3.95)Cd 1.12 (0.87, 1.42) 0.31 (0.14, 0.73)Hg 3.07±2.29 (2.78) 4.64±2.53 (5.45)Pb 0.06±0.10 (0.02) c 0.01±0.02 (0) a

All concentrations in μg/g dry wt. Mean±standard deviation (median) or geoof values, and observed range of values shown. Concentrations of Ba, Bi, Co aare arranged in the table by atomic mass.a n=10.b n=26.c n=25.

Taylor, 1986 and references therein) except in extremesituations such as toxicity (Taylor, 1986 and referencestherein), and where persistent and cumulative elementsincluding Pb, Cd and Hg are ingested in single large doses orirregularly, blood analysis was considered to erroneouslyreflect total body burden of those elements (Chattopadhyayet al., 1977). In other data collected in human studies,significant correlations between hair and plasma concentra-tions of Ca, Zn and Al were not determined (Taylor, 1986).

Comparison of trace element concentrations determined inthe serumof leopard andWeddell seals sampled in the presentstudy with previously published values is not particularlyinformative due to the lack of published trace elementconcentrations in pinniped serum, and the limited number ofstudies detailing trace element concentrations in whole bloodof pinnipeds (Beck, 1956;Yamamoto et al., 1987; Brookenset al.,2007; Griesel et al., 2008). However, similar orders ofmagnitudewere determined for the concentration ofMn, Cu, Cd, andHg inthewhole blood of two adultWeddell seals and in the serumoftheWeddell seals sampled in the present study, whilst greaterFe concentrations were determined in the previous study(Yamamoto et al., 1987), most likely due to the use of wholeblood for trace element determination in the earlier study. Cuconcentrations reported in the blood of two leopard and twoWeddell seals in a previous study (Beck, 1956) were also of thesame order of magnitude as those determined in the presentstudy. However, these comparisons are limited by the verysmall sample size used in the earlier studies (n=2) and thedifferent units employed to report trace element concentra-tions for the two adult Weddell seals sampled in the study byYamamoto et al., 1987.

Serumsamples aremore readily obtained from leopard andWeddell seals and other pinniped species when compared to

w hair (n =11) of adult leopard seals sampled in 1999/2000

Reference values Observed values

Moult New Moult New

333–2398 238–1435 232–1634 229–9040–17.3 3.32–14.4 0.15–22.7 6.04–12.5419–1915 297–1069 296–1804 331–7570.99–3.32 0.18–4.60 0.63–2.46 0.19–2.582.50–5.13 2.70–5.53 2.71–5.00 3.35–5.571.00–1.87 0.64–2.82 0.95–2.01 0.88–2.4559.7–89.5 49.7–119 64.5–96.6 60.9–1100.64–2.87 0.15–3.95 0.38–2.36 0.33–2.891.96–5.79 1.89–7.09 1.95–5.26 2.54–7.4279.0–134 93.8–176 77.6–130 101–1570.13–3.14 0–2.23 0.25–3.71 0–2.190.05–5.85 0–9.12 0.67–6.40 0.89–7.740.32–3.90 0.02–5.09 0.10–3.25 0.09–3.510–7.76 0–10.3 0–10.4 1.11–10.20–0.27 0–0.07 0–0.47 0–0.06

metric mean and 95% confidence interval, developed reference rangend Snwere below detection limits and are not shown. Trace elements


tissue samples and are frequently collected from marinemammals as part of other investigations. Our study hasshown that serum samples can be utilised for trace elementanalysis in the leopard and Weddell seal in the absence ofother samples being available for analysis, and the referencevalues developed in the present study will be useful for futurecomparative studies and for the assessment of trace elementtoxicity or deficiency in individual leopard and Weddell seals.However, there are limitations associated with the use ofserum for trace element determination including the inabilityof serum (and whole blood) concentrations to reflect long-term accumulation of trace elements (Laker, 1982). For thosetrace elements that accumulate in erythrocytes, for examplemethylmercury (Berlin, 1979), trace element concentrations inserumwill be less reflective of trace element concentrations inwhole blood. In addition, trace element concentrations inserumorwhole bloodwill fluctuate throughout the year due toa number of factors. These include prey availability and theamount of prey consumed by an individual, the trophic levelof the prey species which will affect the degree of traceelement accumulation in the prey species, individual nutri-tional status, as well as environmental factors such asvariation in the level of anthropogenic contamination. All ofthese factors need to be considered when trace elementconcentrations in serum or whole blood are comparedbetween individuals and over different sampling periods.

4.2. Effect of hair type and seal gender on trace elementconcentrations in the leopard seal

Previous studies in a number of pinniped species have reportedrelatively great concentrations of several trace elements in hair.Trace element concentrations in hair represent circulatinglevels in the blood during the period of hair growth with thetrace elements delivered to the hairmainly via the blood supply(Ikemoto et al., 2004; Andrade et al., 2007). Seal hair growsrapidly and not continuously (Ling, 1984). Phocids renew theirpelage during moult; and in the southern hemisphere, sealsincluding the leopard and Weddell seal, moult their hairannually during summer and autumn, December to April/May(Ling, 1970).The timeframe formoult andnewhairgrowth in theleopard seal has not been defined, but regardless of the exacttime period of hair growth in the leopard seal, the deposition oftrace elements is expected to be complete in moult hair. Haircollected soon after themoult or ʽnew' hair will reflect themostrecent exposure to trace elements with elements being depos-ited in this hair as it grows. Based on the different time periodsavailable for traceelementaccumulation inmoult andnewhair,it is not unexpected that the trace element concentrationsdetermined to be significantly different in this study weregenerally higher in moult hair compared to new hair, and thispattern of accumulation has been discussed in previous studiesof the polar bear,Ursus maritimus (Born et al., 1991) and harbourseal (Wenzel et al., 1993), the latter being the only studyreporting the effect of hair type on trace element concentrationsinpinnipedhair. In thepresent study, greaterZn concentrationswere determined in new compared to moult hair, perhapsreflecting the role of Zn in keratogenesis in newly forming hairand production of melanin pigments in hair. In human hair, adecrease in Zn andCu concentrations is thought to be related to

depigmentation of hair (Eads and Lambdin, 1973) and darkerhair has significantly greater Zn concentrations compared tolighter hair (Eads and Lambdin, 1973; Chatt and Katz, 1988).During the annual moult in seals, there is initially the presenceand then the loss of melanin in continuously growing hairs(Ling, 1970) and in the leopard seal, new hair is generally darkerthan moult hair, although the site of hair collection can affecthair colour.

The finding of significant differences in trace elementconcentrations with hair type for several of the trace elementsexamined, although based on a limited sample size, hasimportant practical implications for sample collection. It isparamount that the type of hair collected is noted at the timeof sampling to enable meaningful comparisons of traceelement concentrations in future studies and to furtherinvestigate the effect of hair type on trace element concentra-tions in pinniped hair.

In our study, hair type had a greater influence on traceelement concentrations in hair than seal gender. In previousstudies of marine mammals, significant differences were notdemonstrated for Hg concentrations in the hair of Baikal seal,Phoca siberica, with seal gender (Watanabe et al., 1996), nor forCd, Pb, Cu, Ni and Zn concentrations in the hair of maleand female ringed seal, Phoca hispida hispida, and bearded seal,Erignathus barbatus (Medvedev et al., 1997). However, signifi-cantly greater Cd concentrations were reported in the hair ofmale compared to female adult harbour seals (Wenzel et al.,1993), and significantly greater THg concentrations werereported in the hair of adult male compared to adult femalePacific harbour seal (Brookens et al., 2007). Lesser concentra-tions of Hg and Cd found in the tissues of male compared tofemale Baikal seal were attributed to a lower dietary intake asevidenced by less fat mass and blubber thickness of malecompared to female seals (Watanabe et al., 1998), and differ-ences in dietary intake and perhaps dietary preferences inmaleand female leopard seals, in addition to physiological influ-ences, could be contributing to the differences in trace elementconcentrations with seal gender in the present study. Thefinding of greater concentrations of both Hg and Se in malecompared to female leopard seals in the present study is notunexpected, as positive correlations between these two ele-ments in tissues have been commonly reported in marinemammals (Wagemann et al., 1983; Frank et al., 1992; Brookenset al., 2007).

4.3. Usefulness of hair for trace element analysis in pinnipeds

Hair has a role in the accumulation of trace elements andserves as an excretory route for trace elements via the moult.In a study of trace element concentrations in the hair, liver,kidney, and muscle of several seal species, the greatestconcentrations of Pb, Ni and Zn were demonstrated in hair,whilst hair and liver contained the greatest concentrations ofHg and Cu, and kidney and hair the greatest concentrations ofCd (Medvedev et al., 1997). In the Baikal seal, notableconcentrations of Cu were determined in moult hair andliver Cu concentrations were less than expected, suggestingthat a significant excretion of Cu occurred during the moultresulting in less tissue accumulation of this element in thisspecies (Watanabe et al., 1996). In a later study of trace

Table 5 – Trace element concentrations in the hair of leopard and Weddell seal in the present study and those reported in the literature for other pinniped species

Source Species Hair type V Mn Fe Ni Cu Zn Cd Hg Pb

Present study Leopard sealHydrurga leptonyx

Moult 1.82 1.36 73.1 1.35 3.37 103 1.12 3.07±2.29 0.06±0.10n =27 (1.62, 2.04) (1.28, 1.45) (70.3, 76.0) (1.17, 1.57) (3.03, 3.75) (97.7, 108) (0.87, 1.42) 0–10.4 0–0.47

0.63–2.46 0.95–2.01 64.5–96.6 0.38–2.36 1.95–5.26 77.6–130 0.10–3.25New 0.91 1.36 76.8 0.76 3.66 128 0.31 4.64±2.53 0.01±0.02n =11 (0.56, 1.48) (1.08, 1.68) (66.9, 88.1) (0.47, 1.25) (3.00, 4.47) (117, 141) (0.14, 0.73) 1.11–10.2 0–0.06

0.19–2.58 0.88–2.45 60.9–110 0.33–2.89 2.54–7.42 101–157 0.09–3.51Weddell sealLeptonychotes weddellii

Pre-moult 4.22±0.65 1.15±0.41 73.9±16.4 3.52±0.76 15.1±13.2 137±14.3 2.81±0.58 5.60±1.43 1.29±1.12n =12 2.73–5.06 0.52–1.80 39.4–108 2.37–5.18 3.87–51.3 117–159 1.93–3.82 3.43–7.33 0.08–3.30

Andrade et al., 2007 Southern elephant sealMirounga leonina

Moultn =27Juveniles – – – b0.02–0.92 11.3±3.95 164±63.6 b0.04–0.12 – b0.05n =17Adult ♀ – – – 1.02±0.61 12.9±4.48 168±55.8 0.38±0.18 – b0.05

Ikemoto et al., 2004 Baikal seal Pusa sibirica – 1.0±0.8 1.81±0.91 – – 5.37±1.97 105±13 0.09±0.07 3.6±1.7 13.4±15.3n =20 (except Cdn =19)

0.06–2.5 0.50–3.60 3.61–13.3 81.4–127 b0.001–0.23 0.69–7.6 2.57–58.0

Caspian seal Pusa caspica – 0.71±0.39 1.75±1.55 – – 33.8±59.7 98.1±26.4 0.39±0.35 1.6±0.9 3.53±2.14n =16 (except Hgn =18)

0.03–1.8 0.40–6.65 3.30–219 58.7–169 0.04–1.44 0.56–3.5 0.05–6.72

Northern fur sealCallorhinus ursinus

– 3.1±1.0 0.35±0.30 – – 6.13±1.79 186±55 0.64±0.25 4.9±1.1 7.68±5.60n =20 1.5–5.5 0.09–1.05 4.05–10.7 150–401 0.37–1.37 2.9–7.6 2.38–26.1

Saeki et al., 1999 Northern fur seal – – – – – – – – – –n =2 0.77–1.5

Wiig et al., 1999 Atlantic walrusOdobenus rosmarus rosmarus

Moult – – – – – – 0.86±0.32 0.24±0.10 –n =15 0.35–1.51 0.12–0.42

Watanabe et al., 1998 Baikal seal Moult – – – – – – – 4.5±1.3 –n =18 adult 3.0–7.6

Ivanter et al., 1998 Ladoga ringed sealPhoca hispida ladogensis

n =23 – – – 4.11±0.82 22.5±6.02 324±59.0 0.96±0.11 17.5±4.00 6.34±1.900.01–15.0 4.6–148 111–1466 0.32–2.00 4.80–79.5 0.34–40.0

(n=18)Medvedev et al., 1997 Lake Ladoga ringed seal – – – – 4.11±3.92 22.5±28.9 324±283 0.96±0.53 17.5±17.0 6.34±9.09

n =23 (except Hg,n =18)

0.01–15.0 4.60–148 111–1466 0.32–2.00 4.80–79.5 0.34–40.0

Ringed sealPhoca hispida hispida

– – – – 2.32±1.26 14.1±9.99 178±44.4 1.45±0.79 4.26±1.37 1.58±1.33n =15 (except Hg,n =9)

0.66–5.02 1.05–34.6 121–290 0.70–3.44 2.50–6.60 0.36–5.14

Bearded sealErignathus barbatus

– – – – 3.11±1.83 5.77±4.22 146±65.9 1.30±0.43 0.78±0.21 1.42±0.80n =3 2.05–4.17 3.33–8.20 108–184 1.00–1.80 0.54–0.92 0.56–2.11

Watanabe et al., 1996 Baikal seal Moult – – – – – – – – –n =2 2.1–8.2 130–200 6.2–6.8 120–130 3.0–4.3

Yediler et al., 1993 Mediterranean Monk sealMonachus monachus

Moult – – – – 12.6±4.3 129±17.2 0.20±0.08 22.4±15.4 0.78±0.31n =7 9.93–20.4 109–159 0.01–0.36 5.2–54.3 0.5–1.42

Hyvärinen and Sipilä, 1984 Saimaa ringed sealPhoca hispida saimensis

– – – – 5.7±2.54a – – 0.7±0.13a 20.7±4.11a 6.4±2.25a

n =8 – – – –Beckmen et al., 2002 Northern fur seal – – – – – – – – 7.84±1.78 –μg/g wet wt n =12 5.89–12.1

Adult ♀Wenzel et al., 1993 Harbour seal Phoca vitulina Both – – – – – – 0.12±0.09 33.5±38.5 0.6±0.4

n =47 – – –μg/g wet wt Adults and pupsYamamoto et al., 1987 Weddell seal – – – – – – – – – –

n =2 0.35–1.29 22.7–341 5.08–5.26 78.4–91.3 0.36–1.18 0.55–0.89μg/g wet wt Adult

Data is presented as mean and standard deviation or geometric mean and 95% confidence interval, followed by the observed range of values. All concentrations are in μg/g dry wt unless otherwise indicated. Hair type is indicated whenknown from other studies. Trace elements are arranged in the table by atomic mass.a Standard error of the mean. 211

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element concentrations in the Baikal seal, it was suggestedthat Hg was mainly excreted by moulting and parturition(Watanabe et al., 1998), and in a number of pinniped species,high concentrations of Zn, Pb, Ni, Mn, Cu, Hg, V and Cd werereported in hair (Yamamoto et al., 1987; Wenzel et al., 1993;Watanabe et al., 1996; Medvedev et al., 1997; Ivanter et al.,1998; Saeki et al., 1999; Ikemoto et al., 2004). Table 5 displaystrace element concentrations in the hair of the leopard andWeddell seal in the present study and trace element concen-trations in the hair of other pinniped species.

Comparable concentrations of several trace elements Mn, Fe,Ni, Zn, V, Cd and Cu, were determined in our study compared totrace element concentrations reported in pinniped hair inprevious studies. However, lesser Cu concentrations weredemonstrated in our study compared to in the ringed seal(Medvedev et al., 1997; Ivanter et al., 1998), Caspian seal, Pusacaspica (Ikemoto et al., 2004), and Southern elephant seal, Mir-ounga leonina (Andrade et al., 2007). Hg concentrations weresimilar to those reported in several northern hemisphere speciessampled in Lake Baikal, Russia, the Caspian Sea, and inwaters offSanriku, Japan (Ikemoto et al., 2004) but were less than thosereported in several other studies of seals from northern waters(HyvärinenandSipilä, 1984;Wenzel et al., 1993;Yediler etal., 1993;Medvedev et al., 1997; Ivanter et al., 1998), perhaps reflectingdiffering levels of environmental contamination of the food webin the two regions. Pb concentrations in the hair, particularly ofthe leopard seal in the present study, were less than thosepreviouslydetermined inotherseal species (HyvärinenandSipilä,1984; Medvedev et al., 1997; Ivanter et al., 1998; Ikemoto et al.,2004), whilst the concentration of Cd in the hair of leopard andWeddell seals in the present study were greater than thosereported in several northern hemisphere species (Ivanter et al.,1998; Ikemoto et al., 2004) and in the Southern elephant seal(Andrade et al., 2007). Cr concentrations in both leopard andWeddell seals sampled in the present study were also greaterthan those reported in other species (Ikemoto et al., 2004). Thesedifferences in trace element concentrationsmay reflect anumberof factors including species variability in the intake, storage andexcretion of trace elements, environmental influences andvariation in the level of anthropogenic contamination, physiolo-gical influences, dietary preferences and prey availability, time ofsampling with regard tomoult status, sample collection, storage,preparation, and analytical technique.

There are no reports in the literature of trace elementconcentrations in the hair of the leopard seal, however, traceelement concentrations have been determined in the hair oftwo adult and one newborn Weddell seal (Yamamoto et al.,1987). High concentrations of Pb and Ni were determined inthe adult seals compared to the results of the present study,and greater concentrations of Cd and Hg were determined inthe present study compared to the previous study, whilstcomparable Fe, Mn, Zn and Cu concentrationswere seen in thetwo studies. Again, different units were used to report traceelement concentrations in the two studies.

The paucity of data in the literature for trace elementconcentrations in the hair of these species illustrates theimportance of the reference values developed in our study formonitoring trace element concentrations in these species andfor enabling comparative studies of trace element concentra-tions in species occupying northern hemisphere waters. With-

out species-specific ‘baseline’ levels for comparison,monitoringchanges in trace element concentrations is not possible. Byestablishing baseline levels of trace element concentrations inthe hair of the leopard and Weddell seal, more effective non-invasive monitoring of trace element concentrations in thesespecies can be undertaken and future comparative studies candetermine if changes in contaminant status have occurred. Ifchanges do occur, investigations can be directed towardselucidating the cause of such changes, whether due to naturalfluctuations or anthropogenic influences. Alterations in traceelement concentrations in pinnipeds can have implications forhealth and susceptibility to disease, thus monitoring of traceelement concentrations is an important component of anystudy evaluating individual and population health status.

If trace element concentrations in hair reflect internaltissue concentrations, the usefulness of hair as a non-invasivesample for trace element analysis would be improved. Whilstqualitative differences were demonstrated in the concentra-tion of trace elements in hair and tissues of the leopard seal(Gray, 2005) with greater concentrations of Mg, Ca, Cr, Ni, V, Pb,and similar concentrations of Zn and As observed in the haircompared to the tissue samples analysed, due to the smallsample size it was not possible to determine statistically ifpositive correlations or regression relationships existedbetween the concentration of trace elements in hair andinternal tissues of the leopard seal. There are contrary reportsin the literature as to the degree to which hair and tissue traceelement concentrations are correlated. Previous studies inmarinemammals have elucidated significant positive correla-tions for hair and tissue concentrations, with Hg the mainelement studied (Watanabe et al., 1996; Ikemoto et al., 2004;Brookens et al., 2007). Significant relationships have also beenreported for Cd (Medvedev et al., 1997; Ivanter et al., 1998), Zn,and Pb concentrations (Ikemoto et al., 2004). Hair was reportedto be an indicator of the extent of contamination and could beutilised for non-invasive monitoring of some trace elements,for example, Hg (Watanabe et al., 1996). However, a recentstudy of the Pacific harbour seal recommended the use ofmuscle samples obtained from live or dead seals, instead ofliver or hair for Hg biomonitoring as Hg concentrations inmuscle better reflect internal tissue concentrations andoverallbody burden of trace elements when compared to hair(Brookens et al., 2008). It has been suggested that a clearrelationship between trace element concentrations in hair andinternal tissues (not necessarily serum) may be demonstratedafter the new hair has formed, with Hg concentrations in thehair postulated to reflect Hg concentrations in internal tissuesat the time of the last moult (Watanabe et al., 1996). Althoughsignificant relationships betweenHg, Zn, Pb and Cd in hair andtissues of marine mammals have been reported, few othercorrelations between trace element concentrations in hair andtissue samples have been reported. Thismay, in part, be due tothe limited number of studies of the hair in marine mammalsor because of the use of small sample sizes in the studies thathave been undertaken, however, it is more likely that fewcorrelations exist. A reviewof trace element analysis in humanhair concluded that there were no useful correlations betweenthe concentration of trace elements inhair and internal tissues(Zn, Cd, with equivocal results for Cu) and that instead of usinghair to ascertain body burden, as has been done in previous


studies, itmay bemoremeaningfully applied tomonitoring forexposure (Taylor, 1986 and references therein). In anotherhuman study, it was stated that hair analysis represents pastexposure but does not necessarily reflect body burden (Bush etal., 1995). Finally, for elements that accumulate with age, suchas Hg and Cd, tissue trace element concentrations will reflectexposure over the lifetime of an animal; thus, animal age willhave an important bearing on the relationship between hairand tissue trace element concentrations.

Hair samples were only collected from adult seals of bothspecies in the present study; therefore it was not possible toinvestigate the affect of seal age on trace element concentra-tions in hair. Previous studies have reported significantdifferences in trace element concentrations in hair with age,mainly with respect to Hg. Hg concentrations increased withage in both Ladoga ringed seal, Phoca hispida ladogensis, andringed seal of the White Sea, whilst no significant differenceswere seen with age for Cd, Pb, Cu, Ni and Zn in the same study(Medvedev et al., 1997). THg concentrations in the fur of adultfemale Northern fur seal, Callorhinus ursinus,were significantlygreater than in their pups (Beckmen et al., 2002), and THgconcentrations were also greater in adult Pacific harbour sealscompared to concentrations in pups and juveniles (Brookenset al., 2007). The concentration of Hg also increasedwith age inLadoga ringed seal, whilst for the remainder of the traceelements determined, Cd, Pb, Cu, Ni and Zn, no age-relateddifferences were determined (Ivanter et al., 1998). Finally, in astudy of Southern elephant seals, adult females had greater Cdand Ni concentrations compared to juveniles, whilst nosignificant differences were determined for Cu and Znconcentrations in adult and juvenile hair (Andrade et al.,2007). These findings indicate that age may impact on traceelement concentrations in hair, similar to the findings fortissue samples such as liver and muscle. Most likely thesedifferences are related to the different foraging behaviour andtrophic level of prey species consumed by the age cohortscompared (Brookens et al., 2007); in another study, the timingof themoult in different age cohortswas also determined to beimportant in the accumulation of trace elements with age(Wenzel et al., 1993). Whilst age may impact on trace elementconcentrations in hair, it is unlikely that age differences arethe cause of the significant differences in trace elementconcentrations determined in moult and new hair in thepresent study. The differences in trace element concentra-tions with hair type determined in the present study werebased solely on the analysis of hair from adult leopard seals;therefore, differences in foraging behaviour and the timing ofthe moult are not likely to have significantly affected traceelement concentrations in moult and new hair. In addition,the difference in age of the seals with respect to body burdenof trace elements, between the time trace elements weredeposited in the moult hair and the new hair, would not beexpected to be sufficiently long for this to be reflected asstatistically significant differences in trace element concen-trations in moult and new hair.

Our finding of comparatively great concentrations of certaintrace elements in the hair of leopard andWeddell seal indicatesthat hair may be a useful non-invasive sample for determiningthe concentration of selected trace elements in these twospecies. Hair analysis has several advantages over blood and

tissue trace element analysis, particularly in species such as theleopard and Weddell seal in which tissue and blood samplesmay be difficult to obtain in sufficient numbers. These include:non-invasive collection of hair off the ice and other substratesduring the moult, minimising the impact of handling onindividual seals; greater trace element concentrations com-pared to blood (Chattopadhyay et al., 1977; Laker, 1982) enablingeasier detection of trace elements; a better assessment of‘normal’ trace element concentrations as short-term fluctua-tions (such as would be determined in the serum/whole blood)are averaged out (Laker, 1982); assessment of longer termexposure to trace elements compared to blood samples whentissue samples are unavailable; and monitoring of annualfluctuations and, therefore, annual exposure to trace elementconcentrations by repeated sampling of moult hair oversuccessive seasons.

Limitations of hair analysis include the inability of hair (likeserum/whole blood) to provide an indication of long-termexposure (years) compared to tissue samples; hair concentra-tions may not be indicative of serum/whole blood or internaltissue trace element concentrations and overall body burden oftrace elements; exogenous contamination of hair samples maycontribute to the trace element concentrations determined; theincreased likelihood of contamination due to the many stepsinvolved in the washing procedure; the effect of hair type ontrace element concentrations and therefore the need todetermine hair type at the time of collection; the affects ofage, sex, site of growth, and hair colour on trace elementconcentrations in hair (Taylor, 1986); and the comparativelylarge volume of hair required for this analysis which may limitthe use of hair collected off the ice from seals during themoult.Despite such limitations, monitoring exposure to trace ele-ments via regular analysis of hair, a non-invasive sample, maybe a useful means of assessing exposure to trace elementconcentrations in species such as the leopard andWeddell sealin which tissue samples are difficult to obtain.

5. Conclusion

We report trace element concentrations in the serum and hairof two upper trophic level Antarctic pinnipeds. The establish-ment of reference values for trace element concentrations inthese two populations will enable more effective monitoringof variations in trace element concentrations in these speciesand will facilitate the investigation of the importance of thesetrace elements to the health status of individual seals. Wehave also demonstrated that serum can be used to assesstrace element concentrations circulating in the blood at thetime of collection in the leopard and Weddell seal, whilstannual monitoring of trace element concentrations utilisingmoult hair can be undertaken, providing a non-invasivemeans of assessing trace element exposure in species inwhich opportunities for collecting tissue samples for traceelement analysis are limited.

Thepresent studyhasdemonstrated thathair haspotential asa non-invasive sample for trace element analysis in the leopardand Weddell seal and that hair can be employed for monitoringtrace element concentrations in these species. However, furtherinvestigationneeds tobeundertaken toelucidate the relationship


between trace element concentrations in serum/whole blood,hair and tissue samples, utilising bothmoult andnewhair,with alarger samplesizeoverseveral years, to fullydetermine theextenttowhichhair can be employed as anon-invasive sample for traceelement analysis in these species.

Acknowledgements

We are grateful for the field assistance provided by Sophie Hall-Aspland, Damien Higgins, Andrew Irvine, Julie Barnes, SophieConstable, Claire Holland, Birgit Buhleier, and other members ofthe Australian Antarctic Program 1999–2002 particularly BobJones, Brett Hill, Brendan Hill, Ben Patrick, Glenn Robertson,Michael Terkildsen, boat drivers and helicopter pilots. Weddellseal samples from2002/2003werekindly collectedbyTamaravanPolanen Petal, Marjolein van Polanen Petal and Kate Bodley.Logistical support for this work was provided by the AustralianAntarctic Division, Kingston, Tasmania. Funding for this workwas provided by the Antarctic Science Advisory Committee,Australian Research Council, Scott Foundation, National Geo-graphic, Sea World Research and Rescue Foundation, ZoologicalParks Board of NSW and the University of Sydney. The ClinicalBiochemistry Department, especially the Trace and Toxic Ele-ment Unit at Royal Prince Alfred Hospital Camperdown, NSWAustralia provided trace element analysis with particular thanksto Robert McQuilty who was involved in all facets of samplepreparation and analysis, Associate Professor Peter Stewart,Georgina Chronopoulus, and Aileen Wing-Simpson. Expertstatistical advice was provided by Dr. Peter Thomson, Faculty ofVeterinary Science, The University of Sydney. We gratefullyacknowledge Roche Products Pty. Ltd, Sigma Pharmaceuticals,Fort Dodge, Virbac Australia Pty. Ltd, AstraZeneca and Parke-Davis for donating their products and the Pharmacy Department,Royal Hobart Hospital, Hobart Tasmania for the reconstitutedproducts. Thisworkwas conducted under theAntarctic ScientificAdvisoryCommitteeProjects 1144and1148.TheAnimalCareandEthics Committees of the Australian Antarctic Division and TheUniversity of Sydney approved the activities undertaken for thisresearch. We also thank the two anonymous reviewers for theirvaluable comments on the manuscript.

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