trace element concentrations in the pacific harbor seal (phoca vitulina richardii) in central and...

ent 372 (2007) 676–692www.elsevier.com/locate/scitotenv

Science of the Total Environm

Trace element concentrations in the Pacific harbor seal(Phoca vitulina richardii) in central

and northern California

Tiffini J. Brookens a,⁎, James T. Harvey a, Todd M. O'Hara b

a Moss Landing Marine Laboratories, Moss Landing, CA 95039, USAb Institute of Arctic Biology, University of Alaska Fairbanks, Fairbanks, AK 99775-7000, USA

Received 19 June 2006; received in revised form 29 September 2006; accepted 4 October 2006Available online 28 November 2006

Abstract

To determine concentrations of trace elements (THg, MeHg, Se, and Pb) in tissues of the Pacific harbor seal (Phoca vitulinarichardii), live (n=186) and dead seals (n=53) were sampled throughout central and northern California from March 2003 toJanuary 2005. There were significant differences in THg concentrations in blood and hair based on age ( pb0.001). Adult maleharbor seals had greater THg concentrations in their hair than adult female harbor seals ( pb0.003). THg concentrations in liverincreased linearly with age and δ15N ( pb0.001); whereas, MeHg concentrations in liver increased exponentially untilapproximately 5 years of age with an asymptote at 1.3 μg/g wet weight. MeHg expressed as a percentage of THg (%MeHg) wasbest described by a decay function (r2=0.796, pb0.001), decreasing to a minimum at 4 years of age. Hepatic Se increased with ageand was in equimolar ratios with THg in adults; whereas, molar ratio of Se:THg in pups deviated from a 1:1 ratio. Significantdifferences among study locations in THg concentrations in blood and hair were not detected. Assessing the possible effect ofsampling location on Hg concentrations, however, was confounded and limited by lack of equal sample sizes for basic age and sexcohorts, a common dilemma in pinniped research.© 2006 Elsevier B.V. All rights reserved.

Keywords: Mercury; Selenium; Lead; Stable isotopes; Harbor seals; Liver; Hair; Blood

1. Introduction

Increasing concern about environmental pollutionhas stimulated many studies regarding heavy metalcontamination in marine mammals. The Pacific harborseal (Phoca vitulina richardii) generally inhabits near-shore areas associated with productive waters that pro-

⁎ Corresponding author. Tel.: +1 831 236 7541; fax: +1 831 6324403.

E-mail address: [email protected] (T.J. Brookens).

0048-9697/$ - see front matter © 2006 Elsevier B.V. All rights reserved.doi:10.1016/j.scitotenv.2006.10.006

vide adequate prey (Roffe and Mate, 1984; Harveyet al., 1995). Piscivorous harbor seals, situated near thetop of the marine food web, accumulate significantamounts of mercury, Hg (Smith and Armstrong, 1978;Himeno et al., 1989). Because they primarily eat fishand squid (Anas, 1974; Cappon and Smith, 1982), Hgconcentrations in some marine mammals often areorders of magnitude greater than concentrations foundin terrestrial carnivorous mammals (Skaare et al., 1994)and non-piscivorous marine mammals (Woshner et al.,2001a). Harbor seals, therefore, are useful mammalian

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677T.J. Brookens et al. / Science of the Total Environment 372 (2007) 676–692

integrators for Hg biomonitoring because they areendemic to the nearshore coastal environment and arehigh-level trophic consumers (Miles et al., 1992).

Since the industrial revolution, anthropogenic Hgemissions have increased atmospheric Hg concentrationsby three- to five-fold (Mason et al., 1994) and have causedcorresponding increases in Hg concentrations in aquaticand marine ecosystems (Wiener et al., 2003). Productionof monomethylmercury (MeHg) via methylation ofinorganic mercury, Hg+2 (IHg), by microbial sulfatereduction in the environment (Berlin, 1979) is a keyprocess affecting Hg concentration in fish and otheraquatic or marine biota. Nearly all Hg bioaccumulated infish isMeHg (Bloom, 1992), a highly neurotoxic form thatbiomagnifies to high concentrations in aquatic food webs(Wiener et al., 2003). Consumption of fish is the primaryroute of MeHg exposure for harbor seals (Koeman et al.,1975; Reijnders, 1980). It is efficiently absorbed acrossthe gut, crosses intercellular membranes, distributes viablood stream to all internal organs and tissues, andaccumulates in exposed organisms (Clarkson, 1994).

Mammals, including pinnipeds, can demethylateMeHg into IHg via intestinal flora (Norseth andClarkson, 1971), tissue macrophages, and liver (Na-tional Research Council, 2000). IHg and MeHg areretained by the liver, recirculated throughout the body,or excreted slowly in the feces and urine as part of acomplicated distribution and excretion process. MeHgalso can be sequestered directly into keratinized struc-tures and eliminated by hair/feather loss or epidermalsloughing (Scheuhammer, 1991; Clarkson, 1994; Gaggiet al., 1996). The vast majority of THg in hair, therefore,is considered MeHg (National Research Council, 2000).Once MeHg is incorporated into the hair, it does notdemethylate and represents a continuous record of thedietary intake of this trace element's concentration in thebody for many months (Subramanian, 1991). MeHgalso can be transferred to seal fetuses via the placentaand to a lesser degree via the milk of the mother (Joneset al., 1976; Reijnders, 1980; Wagemann et al., 1988).Some researchers have documented that MeHg crossesthe placenta (Jernelov, 1986) and accumulates in thedeveloping fetus; therefore, the fetus acts as a mercury“trap” or “sink” by sequestering the toxicant from thematernal system (Chang and Reuhl, 1983).

Adverse health effects of MeHg occur in the repro-ductive (Koller, 1979), immune (Koller, 1979), andcentral nervous systems (Scheuhammer, 1991; Clarkson,1994). Some harmful effects of Hg can be counteractedby the presence of selenium, Se, an essential element(Parizek and Ostadalova, 1967; Martoja and Berry,1980). It potentially binds to Hg in equimolar ratios in

the liver of marine mammals (Koeman et al., 1975;Smith andArmstrong, 1978); however, some researchershave determined molar ratios deviating from 1 (Wage-mann et al., 1988; Himeno et al., 1989; Woshner et al.,2001b; Dehn et al., 2005). The 1:1 molar relationship,thus equimolar binding of Hg and Se, may serve as aprotective mechanism against toxic effects of Hg bybiotransformation of ingested MeHg into a less toxicchemical form. Deviations from the 1:1 molar ratio,however, may indicate that the mechanism for Hgdetoxification is overwhelmed or is not completely Se-dependent (Woshner et al., 2001b).

Accumulation of trace elements (Hg and Se) in marinemammal tissuemay be dependent on age and trophic level(Anas, 1974; Drescher et al., 1977; Himeno et al., 1989;Miles et al., 1992; Woshner et al., 2001b; Dehn et al.,2005). Gender based differences in Hg also may occur invarious tissues of phocids, especially in adults (Reijnders,1980; Wagemann et al., 1988; Skaare et al., 1994). Eachtissue type incorporates and excretes Hg during varioustimeframes. THg concentrations in blood represent Hgexposure during previous days and are dependent ontypes and amounts of prey consumed (National ResearchCouncil, 2000). THg concentrations in hair represent Hgthat was available to the growing pile via blood duringtheir annual moult (Berlin, 1979), which lasts 6–8 weeks(Montagna and Harrison, 1957). Trace metals tend toaccumulate in liver because Hg binds to certaincomponents of the cytoplasm of hepatocytes and stellatemacrophages and is retained (Woshner et al., 2002). THgconcentrations in liver may represent more closely alifetime exposure because HgSe complexes can be storedindefinitely in liver of marine mammals (Nigro andLeonzio, 1996). Relationships should exist between Hgconcentrations in the various tissue types; these relation-ships, however, may be confounded by age, demethyla-tion efficiency, health of the animal, and form of Hgingested (Dietz et al., 1989; Himeno et al., 1989; Mileset al., 1992; National Research Council, 2000).

The objectives of this study were (i) to determineconcentrations of various trace elements in blood, hair,and liver in harbor seal throughout central and northernCalifornia, (ii) to determine if these trace elements differwith age, sex, location, tissue type, and trophic level,and (iii) to evaluate various elemental interactions.

2. Materials and methods

2.1. Sampling of live harbor seals

Harbor seals (n=186) were captured near theirhaulout sites in Monterey Bay, San Francisco Bay, Pt.

Fig. 1. Locations of live harbor seals sampled in central and northern California, USA, from 2003 to 2005.

678 T.J. Brookens et al. / Science of the Total Environment 372 (2007) 676–692

Reyes, and Humboldt, CA, from March 2003 to January2005 (Fig. 1), using methods described by Jeffries et al.(1993). Briefly, two-outboard powered boats were usedto set a net, approximately 120 m in length and 8 m indepth, in waters adjacent to haulout sites. Each end ofthe net was pulled ashore, encircling, and capturingharbor seals. Seals then were removed from the net,placed headfirst into hoop nets, and physically re-strained. Once secured, standard length (±1 cm), girth(±1 cm), mass (±1 kg), sex, and age class were deter-mined. Age classes were defined by criteria from Bigg(1981) and based on size and pelage.

Approximately 10 mL of blood was collected from theextradural intervertebral sinus of each live harbor seal(n=175) in royal blue top Vacutainers® containingsodium heparin (Hansen, 1991). Blood then was storedin 20 mL or 40 mL trace metal clean I-Chem™ Teflon™-coated vials in coolers until transported to Moss LandingMarine Laboratories (MLML) and placed in a −20 °Cfreezer. An approximate 15 cm×15 cm patch of hair,weighing 3 g, was shaved from the dorsal midline region,just anterior of the tail (n=186). A battery operatedshaver, Oster® PowerPro, with a 1/10 mm cryotechstainless steel blade was used to remove the hair. After


hair was removed, it was placed in a polyethylene bag andtransported to a −20 °C freezer. All animals were sampledunder a permit to Dr. James T. Harvey, MLML (NationalMarine Fisheries Service, NMFS, No. 555-1565-01), andSan Jose State University Internal Animal Care and UseCommittee (IACUC No. 835).

2.2. Sampling of dead harbor seals

Dead, beachcast harbor seals (n=53) were sampledalong the central California coast from March 2003 toSeptember 2004 (Fig. 2). Harbor seals were measured,

Fig. 2. Locations of dead, beachcast harbor seals sampled in ce

sex determined, and examined externally. Full post-mortem examinations followed when appropriate sam-ples were taken for histology, pathology, parasitology,and cytology. Hair was shaved using a 1 mm stainlesssteel blade following previously stated methods. Livers(n=40) were removed with stainless steel instrumentsand placed either whole or subsampled in a separatepolyethylene bag. Approximately equal proportions ofeach lobe of the liver were subsampled. Two uppercanines were extracted and placed in polyethylene bagsfor age estimations. All tissue samples were stored in a−20 °C freezer. Animals were sampled under permits to

ntral and northern California, USA, from 2003 to 2005.


Dr. James T. Harvey, MLML (California Department ofFish and Game No. 801135-05, Letter of Authorizationfrom NMFS, Marine Mammal Protection Act/Endan-gered Species Act Permit No. 932-1489-08, and San JoseState University's IACUC No. 835).

2.2.1. Age estimationOne canine tooth per animal was sectioned and

prepared by Matson's Laboratories, Milltown, Montana.Teeth were decalcified, thin sectioned across the midlinein 14 μm sections using a rotary microtome, stained withGiesma® histological stain, and mounted. The mountedsections were examined under transmitted light througha compound microscope at high magnification (100×).Growth layer groups, GLGs, represent one translucentand one opaque layer; and one GLG is deposited eachyear in the cementum of harbor seal canines (Dietz et al.,1989). Age was estimated at least three times each bytwo independent, blind readers at MLML and Univer-sity of Alaska Fairbanks (UAF). Accuracy and precisionof age estimations for both blind readers were com-parable based on average percent error (APE=5.6% and5.2%; Beamish and Fournier, 1981) and index ofprecision (D=1.8% and 0.7%; Chang, 1982).

2.3. THg analysis

Hair, 0.5 to 2.0 g, from beachcast and live harborseals was washed in deionized (D.I.) water with a serialracking method and dried at 60 °C in a drying oven for48 h before analysis. Six cleaning blanks and sixcrossover contamination blanks also were analyzed toensure minimal or undetectable Hg contaminationoccurred during the washing process. Cleaning blankswere D.I. water samples that were washed and dried;whereas, crossover blanks were D.I. water samples thatwere used to rinse out the beakers in between washing ofhair samples to ensure no hair was transferred, poten-tially contaminating the following sample. Both types ofblanks were below the method detection limit (MDL) of0.011 μg/g wet weight (ww). Hair samples were con-sidered to have 0% moisture because the moistureattached to the hair was not inherent in the hair but anartifact of environmental conditions.

THg was analyzed at Marine Pollutions StudiesLaboratories in cooperation with California Departmentof Fish and Game (CDFG) at MLML following theprocedure established by Hatch and Ott (1968) withslight modifications. Approximately 1.0 g of blood and0.5 g of hair were digested with 10 mL of 70:30 v/vHNO3/H2SO4 solution and heated to 125 °C for 2 h.Samples were allowed to cool and diluted to 40 mL with

a 5% v/v solution of 0.2N BrCl in MilliQ® ultra purewater. THg samples were analyzed by Atomic Absorp-tion Spectroscopy (AAS) using a Perkin–Elmer® FlowInjection Mercury System (FIMS-100) with the softwareapplication AAWinLab. A peristaltic pump, in conjunc-tion with an auto-sampler (Perkin–Elmer®AS-90), drewan aliquot of the sample solution into the mixing blockwhere the reducing agent (1.1% SnCl2 in 3% HCl v/v)was pumped simultaneously, reducing ionic mercury toelemental mercury with argon as the carrier gas. MDLwas 0.011 μg/g ww tissue. Concentrations wereexpressed as microgram per gram wet weight.

2.4. Trace element analyses

Livers from dead, beachcast harbor seals weresubsampled, approximately the same proportionalvolume grossly extracted from each lobe, and thenhomogenized using a trace metal clean Brinkman®Polytron PT-10/35 homogenizer. Approximately 0.5 gliver were digested in Teflon™ vessels with 6 mL ofconcentrated double distilled HNO3 in a CEM®MARS5 pressurized microwave. Digestates then weretransferred into 30 mL precleaned, preweighed poly-ethylene bottles and brought up to a mass of 20 g byadding MilliQ® ultra pure water (method 3052; USEnvironmental Protection Agency, 1996). High Reso-lution Inductive Coupled Plasma-Mass Spectroscopy,HR-ICPMS, was used to determine Se and lead (Pb)concentrations in liver tissue. ICPMS method followedcriteria of US Environmental Protection Agency method200.8 (US Environmental Protection Agency, 1994) andwas performed at MLML, CA, using a ThermoFinni-gan® Element2 HR-ICPMS. The MDL was 0.012 μg/gww for Se and 0.007 μg/g ww for Pb. Following themicrowave digestion, liver also was analyzed for THgusing previously mentioned methods with a MDL of0.011 μg/g ww. All trace element concentrations wereexpressed as microgram per gram wet weight.

2.5. MeHg analysis in liver

MeHg was analyzed at CDFG-MLML using theprocedure established by Bloom (1989). Approximately0.5 g of liver homogenate was digested in 25% KOHin methanol. A 1% NaB(C2H5)4 solution in 2% KOHactivated the aqueous phase ethylation. MeHg wasdetermined by isothermal gas chromatography separationof ethyl analogs and cold vapor atomic fluores-cence spectrometer (Tekran®Model-2500 CVAFS mercu-ry detector). TheMDLwas 0.002μg/gww.Concentrationswere expressed as microgram per gram wet weight.


2.6. Quality control

All trace element analyses were run based on qualitycontrol (QC) criteria from CALFED Quality AssuranceProgram Plan (QAPP; Puckett and van Buuren, 2000).Reference materials (DOLT-2 and DOLT-3) wereobtained from the National Research Council ofCanada; whereas, marine mammal reference material(liver of beluga whale; QC97LH2-NIST) was providedby the National Institute of Standards and Technology.Reference materials, instrument standards, methodblanks, instrument blanks, method spikes and dupli-cates of samples were analyzed with each batch of 20samples. All QC fell within criteria limits, and all methodblanks were less than MDL except two Se blanks andone Pb blank. The absolute values of each of these threeblanks (0.013, 0.014, and 0.010 μg/g ww) were wellwithin two times the MDL (0.024 and 0.014 μg/g ww);therefore, samples were not blank subtracted.

2.7. Stable isotope analyses

Liver samples were analyzed for δ13C and δ18N stableisotopes using Elemental Analysis-Isotope Ratio MassSpectrometry (EA-IRMS) at the Alaska Stable IsotopeFacility at UAF. Liver samples were freeze-dried for 48 h;then approximately 0.30 mg were aliquoted into tincapsules, placed in a Costech® EA (ESC 4010)autosampler, and combusted. The CO2 and N2 combus-tion gases were separated chromatographically andtransferred to Finnigan® MAT Conflo III interface withaDelta+XP®Mass Spectrometer, where the isotopesweremeasured (e.g. Dehn et al., 2005). International isotopestandards used were Pee Dee Belemnite limestone forcarbon and atmospheric N2 for nitrogen. QC includedanalyzing tin capsule blanks, laboratory working stan-dards, and isotope standards. δ13C and δ18N values wereon a per mil basis or parts per thousand (‰).

2.8. Statistical analyses

Todetermine homogeneity among variances, Cochran'stest of equal variances was used. If variances wereunequal, data were log-transformed to achieve homoge-neity. Normal distributions then were evaluated usingKolmogorov–Smirnov test. Once all assumptions weremet, ANOVAs were used with interaction terms. To testfor differences in THg concentrations in hair ofbeachcast and live harbor seals, a Model I, two-wayANOVA was used with dead or live and age class asfactors. If factor of dead or live indicated no effect on thesamples, all of the THg concentrations in hair were

pooled. To test for differences in THg concentrations inblood and hair, Model I ANOVAs with Tukey's post-hoctest were used based on age class, sex, and/or location.Monterey Bay, San Francisco Bay, Pt. Reyes, andHumboldt were locations for all analyses. Age classes,unless stated otherwise, were defined as pups (SLb100 cm), juveniles, and adults (males weighingN50 kg; females weighing N45 kg) using criteria ofBigg (1981) and field characteristics.

To test for differences in mean THg, MeHg, Se, Pbconcentrations, %MeHg, and δ18N and δ13C signaturesin livers, a Model I, two-way ANOVAwas used with sexand age (pups and non-pups) as factors. To furtherdetermine if differences in mean concentrations of THgin livers were based on age class (pups and non-pups),sex, and trophic position, a Model III General LinearModel (GLM) was used. All interaction terms were notsignificant according to type III sums of squares, andthe model was reduced to:

log THg concentrations in liver

¼ l þ age class þ sex

þ d15N þ age class ⁎ sex þ e

where μ is a constant and ε is the random errorcomponent for model.

Simple linear regressions also were used to determinerelationships between the following: THg concentrationsin blood and standard length, THg concentrations in hairand standard length, THg concentrations in hair andblood, THg concentrations in hair and blood of adultfemales, THg concentrations in hair and blood of adultmales, THg concentrations in liver and age, Se concen-trations in liver and age, molar THg and molar Seconcentrations in liver, and %MeHg and δ15N liverconcentrations. To determine if the linear regression slopefor THg concentrations in blood and hair of adult femaleswas different from adult males, a one-sample t-test wasused. Additionally, one-sample t-tests were used todetermine if the linear regression slope for molar THgand molar Se concentrations in liver and the ratio ofmolar Se to molar THg were equal to one. Non-linearregressions were used to determine the relationshipsbetween the following: MeHg concentrations in liver andage, %MeHg in liver and age, MeHg and THgconcentrations in liver, THg concentrations in hair andTHg concentrations in liver, THg concentrations in hairand MeHg concentrations in liver, and THg and δ15Nconcentrations in liver.

To determine if harbor seals with hepatic lesions hadgreater trace element concentrations in their livers,Fisher's exact test was used. THg liver concentrations

Table 1Mean (±SE) trace element (μg/g wet weight, ww) and stable isotope (‰) concentrations, ranges, and sample sizes in tissues of harbor seals in centraland northern California, USA, from 2003 to 2005 based on age class

THg blood THg hair THg liver MeHg liver Se liver Pb liver δ13C δ15N

Pups 0.093±0.023 8.200±0.611 1.414±0.254 0.449±0.071 0.751±0.049 0.029±0.008 −17.65±0.29 17.01±0.310.015–0.555 0.409–22.303 0.151–7.003 0.068–1.845 0.400–1.293 b0.007–0.174 −21.89– –14.37 12.37–20.18n=23 n=63 n=28 n=28 n=28 n=28 n=28 n=28

Juveniles 0.284±0.026 9.869±0.7320.061–0.981 1.520–30.733n=46 n=70

Adults 0.302±0.023 15.111±1.168 63.673±15.068 1.211±0.179 24.060±5.343 0.027±0.007⁎ −17.41±0.52 18.20±0.380.052–1.398 2.378–92.782 13.576–162.817 0.506–2.852 4.658–57.969 b0.007–62.057 −21.42– –15.61 16.81–21.70n=106 n=106 n=12 n=12 n=12 n=12 n=12 n=12

⁎Denotes mean±SE calculated without 62.057 μg/g ww outlier.


and %MeHg were partitioned into ≥median andbmedian, and histological data from post-mortemswere used to score livers as with (+) or without (−)lesions. Livers were scored + lesion if there wasmoderate to severe histological findings described bycertified veterinary pathologists at University of Cali-fornia Davis. All statistical tests were calculated usingSystat® version 10.0 (SPSS Inc., 2000) with α= 0.05.

3. Results

Mean concentrations of trace elements (THg, MeHg,Se, and Pb) and stable isotope signatures (δ15N andδ13C) in blood, hair, and liver of harbor seals fromcentral and northern California were partitioned basedon age class (Table 1). Harbor seals ranged in age fromfetus to 21 years of age. Approximately 5% (9 values

Fig. 3. THg concentrations (μg/g ww; mean±SE) in blood of harbor seals inCalifornia (Humboldt), USA, from 2003 to 2005 partitioned amongst age clasadults (A). Sample sizes are noted in parentheses above each SE bar.

out of 175) of THg blood concentrations were less thanthe lowest standard of the instrument but greater than theMDL; whereas, 25% (10 values out of 40) of Pb liverconcentrations were less than the MDL.

THg concentrations in blood differed significantlyamong age classes ( pb0.001) but not location (Fig. 3).There were no significant interactions ( pN0.05) in thisANOVA or any of the subsequent ANOVAs. Mean THgconcentration in blood of pups was significantly lessthan juveniles and adults ( pb0.001; Table 1). There wasno significant difference in mean concentration of THgin hair between dead and live animals ( p=0.907).However, there was an effect of age; THg concentrationin hair of adults was significantly greater than pups( pb0.001) and juveniles ( p=0.002; Table 1).

The assumptions of the Model I, three-way ANOVAwere met after concentrations of THg in hair were log-

central (Monterey Bay, San Francisco Bay, and Pt. Reyes) and northerns and location. Age classes were defined as pups (P), juveniles (J), and

Fig. 4. THg concentrations (μg/g ww; mean±SE) in hair of harbor seals in central (Monterey Bay, San Francisco Bay, and Pt. Reyes) and northernCalifornia (Humboldt), USA, from 2003 to 2005 partitioned amongst age class, sex (males=M and females=F), and location. Age classes weredefined as pups and non-pups because only one juvenile male was sampled in San Francisco Bay. Sample sizes are noted in parentheses above eachSE bar.


transformed. There were no significant differences inmean THg concentrations in hair based on sex orlocation (Fig. 4), but mean (±SE) THg concentration inhair for pups (7.289±0.635 μg/g ww) was significantlyless than non-pups (113.086±0.797 μg/g ww; pb0.001). Gender differences were assessed by analyzinglog-transformed THg concentrations in hair of adultswith pooled data of live and dead individuals andlocation. Adult male harbor seals (18.025±2.148 μg/gww; mean±SE) had significantly greater mean concen-

Fig. 5. THg concentrations (μg/g ww) in blood (○) and hair (▪) in relation toCalifornia, USA, 2003–2005. Linear regressions fitted to both data sets incl

tration of THg in hair than adult female harbor seals(13.041±1.240 μg/g ww; pb0.003).

Significant linear relationships were determined forTHg concentrations in blood and hair based onstandard length (Fig. 5). Standard length increaseswith animal age and is the closest approximationfor age that can be assessed in live harbor seals, withoutcollecting a tooth. There also was a significant rela-tionship between concentrations of THg in hair andin blood (y=27.604x+5.414; r2 =0.324; pb0.001).

age based on standard length of harbor seals from central and northernuding a log-based y-axis for graphical purposes.

Fig. 6. Concentrations (μg/g ww) of THg (▴), Se (○), and Pb (▪) in liver in relation to age based on canine cementum analysis of harbor seals fromcentral and northern California, USA, 2003–2004. A 5-year-old female harbor seal (□) that died of acute lead toxicity was excluded from regressionanalysis.


The relationship between concentrations of THg inhair and blood in adult females (y=21.785x+6.789;r2 =0.180) was significantly less than adult males(y=40.428x+3.492; r2=0.616; pb0.001; t-test).

Assumptions of Model I, two-way ANOVAs weremet after THg and Se liver data were log-transformed.THg (pb0.001), MeHg (p=0.004), and Se concentra-tions (pb0.001), and %MeHg (pb0.001) in liver dif-

Fig. 7. MeHg concentrations (μg/g ww;□) and %MeHg (▴) in liver in relatioand northern California, USA, 2003–2004. Power-based regression was fittewas fitted to %MeHg data.

fered significantly based on age class but not sex. Non-pups had significantly greater THg, MeHg, and Seconcentrations in their livers than pups (Table 1);whereas, pups had significantly greater %MeHg thannon-pups. In all of these instances, most non-pups weremature adults. No significant differences were deter-mined for Pb concentrations, δ13C, or δ15N in liverbased on age or sex (pN0.05). Pb statistical analyses

n to age based on canine cementum analysis of harbor seals from centrald to MeHg concentration data; whereas, a power-based decay function

Fig. 8. THg concentrations (μg/g ww) in relation to δ15N (‰) in liver of harbor seal pups (○) and adults (▴) from central and northern California,USA, 2003–2004. Power-based exponential regression analysis fitted to pup data with a log-scaled y-axis for graphical purposes. Two outliers (▪), aharbor seal one trophic level higher than the mean and a harbor seal one trophic level lower than the mean, were removed from regression analysis.


were calculated without a 62.057 μg/g ww outlier, aharbor seal that died of acute Pb toxicity.

There were significant linear relationships betweenTHg and Se concentrations in liver and age (Fig. 6).Concentrations of MeHg in liver, however, increasedexponentially to approximately 5 years of age reachingan asymptote around 1.3 μg/g ww (Fig. 7). As harborseals aged, %MeHg in the liver reached a minimum afterapproximately 4 years of age (Fig. 7). MeHg concentra-tions in the liver (y=0.316x0.365; r2 =0.673; pb0.001)also increased exponentially with THg concentrationsreaching an asymptote around 2 μg/g ww of MeHg.There was a significant linear relationship betweenmolar THg and molar Se concentrations in liver(y=1.071x−5.175; r2 =0.992; pb0.001), with a slopeapproaching one (β=1.071) but not equal to one (t-test;pb0.001). The molar ratio of Se to THg was 1.88 andwas significantly greater than one (t-test; pb0.001).Concentrations of THg in hair increased exponentiallywith concentrations of THg in liver reaching anasymptote at approximately 18 μg/g ww of THg(y=7.343x0.179; r2 =0.267; pb0.001) and with concen-trations of MeHg in liver reaching an asymptote atapproximately 23 μg/g ww of MeHg (y=13.065x0.518;r2 =0.481; pb0.001).

Most harbor seals sampled had δ13C and δ15N valuesnear mean values of each isotope (Table 1); however,two outliers existed. One harbor seal was feeding onetrophic level less than the mean (δ13C=−20.34‰ andδ15N=12.37‰); whereas, the other harbor seal was

feeding one trophic level greater than the mean(δ13C=−19.06‰ and δ15N=21.70‰). THg concen-trations in liver increased significantly with δ15N inpups (Fig. 8), but minimal data were available todetermine this relationship in adults. Liver concentra-tions of THg were dependent on trophic position asdetermined by δ15N (pb0.007) and age (GLM;pb0.001). Increased δ15N values were related toincreased concentrations of THg in liver. Additionally,δ15N decreased significantly (y=−6.995x+150.49;r2 =0.145; pb0.001) with increasing %MeHg inliver. Neither THg concentrations nor %MeHg wererelated to the presence or absence of lesions in theliver (p=0.167 and p=0.283), but a lack of signifi-cance may have been an artifact of minimal samplesize and low power.

4. Discussion

4.1. Trace element concentrations in various tissuetypes

THg concentrations in blood are not routinely mea-sured in wild marine mammals. Mean concentrations ofTHg in blood of harbor seals in this study were greaterthan values previously published (Kopec and Harvey,1995; Moser, 1996). Trace metal concentrations inbenthic and pelagic schooling fish that comprise themajority of harbor seal diet have yet to be investigatedfrom this area; therefore, it would be impossible to


determine if greater concentrations of THg in bloodfrom the present study represents an overall increase inMeHg in the prey over time. These apparent differencesalso may be caused by differences in various biologicalvariables, sampling design, and overall sample size.More samples were collected during a shorter timeframein a more confined area in the present study than in twoprevious studies. Mean THg concentrations in hair wereless than previously published values (Wenzel et al.,1993; Moser, 1996); maximum concentration of THg inhair, however, was greater than that reported by others(Sergeant and Armstrong, 1973; Freeman and Horne,1973; Wenzel et al., 1993; Moser, 1996).

Hg concentrations in the liver of harbor seals have beenmeasured extensively. Mean THg concentrations in liverof harbor seals from central California are within the midrange of previously published values (Sergeant andArmstrong, 1973; Anas, 1974; Drescher et al., 1977;Reijnders, 1980; Law et al., 1991). Mean Se concentra-tions in liver of harbor seals from central California,however, were greater than most previous studies(Himeno et al., 1989; Frank et al., 1992; Skaare et al.,1994;Moser, 1996) except for harbor seals inWadden Sea(Koeman et al., 1975; Reijnders, 1980).MeanTHg and Seconcentrations in livers of harbor seals in this study alsowere greater than concentrations associated with toxicityin terrestrial animals (N50 μg/g ww for THg and N7 μg/gww for Se; Puls, 1994). It is interesting to note, that meanconcentrations of THg (62.9±15.1 μg/g ww; mean±SE)and Se (23.9±5.4μg/gww) in livers of harbor seals in thisstudy were greater than a prior study completed in thesame sampling locales (43.7±17.7 and 3.9±2.1 μg/gww;Moser, 1996). Fewer researchers have investigatedMeHgconcentrations in harbor seals; however, mean MeHgconcentrations in the present study were generally greaterthan those published for harbor seals on the east coast ofNorth America (Gaskin et al., 1973) and less than thosepublished for harbor seals in the Wadden Sea (Reijnders,1980). Harbor seals in the present study had lesserbaseline Pb concentrations than previously reported(Roberts et al., 1976; Drescher et al., 1977; Law et al.,1991; Moser, 1996). One harbor seal, though, wasremoved from all statistical analyses because it had thegreatest Pb liver concentration reported and subsequentlydied of acute Pb toxicity (Zabka et al., 2006).

4.2. Influence of age

Age-related differences in concentrations of THg inblood and hair corroborated past work that indicatedolder harbor seals had greater concentrations of THg inliver and kidney than younger harbor seals (Anas, 1974;

Roberts et al., 1976; Reijnders, 1980; Himeno et al.,1989; Miles et al., 1992; Skaare et al., 1994). Newlyweaned pups are not as efficient at foraging as adults, andpups tend to consume more crustaceans than older,mature harbor seals (Oates, 2005). THg concentrationsin crustaceans should be less than in fish in our study areabecause fish are at a higher trophic level than crustaceans(Cappon and Smith, 1982; Horn and Ferry-Graham,2006). It follows that adult harbor seals had greaterconcentrations of THg in blood and hair than pups.Juveniles, on the other hand, had similar concentrationsof THg in blood as adults and similar concentrations ofTHg in hair as pups. Juveniles, consisting of yearlingsand subadults, probably forage on similar prey species asadults and feed on more prey types than newly weanedpups (Oates, 2005), which would explain similarconcentrations of THg in blood of juveniles and adults.Greater THg concentrations in hair, however, would beexpected in adults compared with pups or juveniles.Because THg bioaccumulates through time andMeHg isslowly excreted from the body (Berglund and Berlin,1969), pups and juveniles have a lesser overall burden ofTHg because they have been exposed to MeHg for ashorter timeframe than mature adults. Pups and juve-niles, therefore, should have lesser concentrations ofTHg incorporated into their hair than adults.

Numerous researchers have determined a positiverelationship between concentrations of THg, MeHg,and Se in liver and age in harbor seals (Anas, 1974;Koeman et al., 1975; Roberts et al., 1976; Reijnders,1980; Himeno et al., 1989; Skaare et al., 1994). Thecontinuous uptake of THg (i.e. MeHg) via prey, slowelimination, storage, and relatively long half-life of THg(Wagemann et al., 2000) explain this positive relation-ship. Although the half-life of whole body MeHg isapproximately 500 days in ringed seal (Tillander et al.,1972),MeHg concentrations become asymptotic in olderindividuals, which indicate that animals reach anequilibrium when enhanced detoxification capabilitiesbalance new pollutant intake (Aguilar et al., 1999).Because MeHg can be demethylated, %MeHg in theliver is greater in pups, decreases to a minimum of lessthan 10% at 4 years of age, and then remains constantwith increasing age. In the livers of younger animals,greater %MeHg indicated that the demethylation processwas not yet well developed (Caurant et al., 1996);whereas, various researchers of pinnipeds have deter-mined comparable %MeHg levels between 5 and 15% inadults (Gaskin et al., 1973; Roberts et al., 1976;Reijnders, 1980; Woshner et al., 2001b; Dehn et al.,2005). These lesser values of %MeHg in adults indicatedietary uptake of MeHg in harbor seals remaining in


equilibrium with physiological detoxification processes(Dehn et al., 2005).

Mammalian fetuses, however, are exposed to MeHgthrough direct placental transfer from the mother(Berglund and Berlin, 1969; Jones et al., 1976; Jernelov,1986; Wagemann et al., 1988; Ask et al., 2002). Twofetuses (4-month old and full-term) were opportunisti-cally examined in this study. A 4-month old fetus hadminimal amounts of THg in its liver, but %MeHg wasapproximately 100%. In the liver of the full-termfetus, THg concentration was 60 times that of the liverof the 4-month old fetus, but %MeHg (65.3%) was lessthan in the liver of the 4-month old fetus. Fetuses inearly gestational stages do not have fully matured andfunctioning organ systems, including the liver (Beath,2003; Teitelbaum, 2003). Detoxifying and demethylatingmechanismswithin the fetus, therefore, probablywere notoperating at early gestational stages, but were beginningin the full-term fetus. IHg accumulates in the placenta butis not necessarily transferred in large concentrations intothe umbilical blood; whereas, MeHg readily crosses theplacental barrier and accumulates in the developing fetus(Ask et al., 2002). During these critical periods ofdevelopment, MeHg suppresses immune responses inthe embryo and neonate of mice (Koller, 1979), whichmay have detrimental long-term effects on the animal.

4.3. Influence of sex

No significant differences were determined betweensexes in THg concentrations of blood. Harbor seals lack amarked sexual dimorphism and do not fast during lactation(Bigg, 1981); therefore, animals at any specific age ofeither gender should be ingesting the same relativeamounts of prey, i.e. MeHg. Significant differences basedon gender in THg concentrations of hair for mature, adultharbor seals were expected and determined when locationswere pooled. Adult male harbor seals were expected tohave greater concentrations of THg (i.e. MeHg) in theirhair than adult females, because females transfer MeHg totheir pups via gestation and minimally via lactation (Joneset al., 1976; Reijnders, 1980;Wagemann et al., 1988). Bothsexes slowly eliminate Hg in urine and feces, butHg in bilecan be reabsorbed in the intestines (Klaassen and Rozman,1991). A significant source of removal of THg for malesthen would be sequestering it in growing hair follicles.Because there were more females (n=133) than males(n=85) sampled in this study, testing gender differenceswas more difficult especially when only a portion weremature adults. Given a more equal sample size of adultfemales and males, gender differences would likely bedetermined for the entire dataset.

THg, MeHg, and Se concentrations in the liver didnot differ based on gender. Several studies have indi-cated differences in Hg concentrations between sexesbecause mature, females excreted THg, mainly in theform of MeHg, across the placenta into the developingfetus (Berglund and Berlin, 1969; Chang and Reuhl,1983; Wagemann et al., 1988; Hansen, 1991; Aguilaret al., 1999). There also were no differences in %MeHgbased on gender in this study. There were no differencesbetween sexes in trace element concentrations in liverprobably because sample size of livers from dead ani-mals was minimal for mature adults (n=12), especiallyadult males (n=2).

4.4. Influence of location

Significant site-based differences in THg concentra-tions of blood and hair were not found. This is surprisingbecause THg concentrations in sediment and other faunalspecies differ among these four sites (Downing et al.,1998; Hunt et al., 1998; Jacobi et al., 1998). Concentra-tions of THg in blood and hair of harbor seals of a specificage class at a fixed trophic level, therefore, were expectedto differ among sites. Fish, oysters, and birds hadincreased concentrations of Hg in San Francisco andTomales Bays compared with other bays and estuaries(Martin et al., 1984; Ohlendorf et al., 1988; Davis et al.,2002). San Francisco and Tomales Bays have aconsiderable amount of Hg cycling in the ecosystemsdue to runoff from rivers in close proximity to abandonedcinnabar mines, specifically, the presence of the NewAlmaden mine draining into South San Francisco Bay(Bradley, 1918) and the Gambonini mine draining intoWalker Creek and Tomales Bay in Pt. Reyes (Bradley,1918; Hornberger et al., 1999). The Humboldt area,conversely, has or had pulp mills, petroleum plants, fossilfuel and nuclear power plants, and coal and oilgasification plants that contribute Hg pollution (Jacobiet al., 1998). There, conversely, is only one Hg mine atNew Idria (Bradley, 1918) possibly contaminating thePajaro and Salinas Rivers that flow into Monterey Bay.

With an increase in sample size per location andequal sampling of age and sexes (blood n=102 andeffect size=0.165; hair n=145 and effect size=0.138),power of statistical tests would have increased to 80%and significant site differences may have been deter-mined. To determine location differences in THg con-centrations in blood, at least 102 blood samples will beneeded at each location partitioned equally amongst ageclasses without taking into account gender becauseanimals of the same age should be ingesting the sametype and relative amount of prey no matter their sex. To


determine location differences in THg concentrations inhair, however, at least 145 hair samples will be neededfor each gender partitioned equally amongst age classesat each location.

Some of the southerly sites were within 50 km of oneanother. The distance between the two extremesampling sites, Monterey Bay and Humboldt, alsowere relatively short, approximately 560 km. There wasa possibility of movement of harbor seals among allareas sampled. Radio-tag data from these individualsduring the same sampling timeframe has indicated thatharbor seals from Monterey Bay and Humboldt re-mained in their respective areas; whereas, some harborseals from San Francisco Bay and Pt. Reyes traveledgreater distances (Harvey and Goley, 2005). A fewharbor seals (n=7) tagged in Tomales Bay traveledsouth to the Marin Headlands and Año Nuevo Island;whereas, a few seals (n=4) tagged in San Francisco Baytraveled north to Pt. Reyes and south to Half Moon Bay(Harvey and Goley, 2005). Harbor seal prey also havethe ability to migrate great distances. Pelagic schoolingfish, which are at a lower trophic level than benthic fish(Horn and Ferry-Graham, 2006), may have constitutedmore of the overall seal diet during this sampling periodthan more sedentary benthic fish. Because benthic fishare sedentary, they reflect Hg contamination of a distinctlocation; whereas, pelagic schooling fish are moremobile and would have Hg contamination based onvarious locales. Location differences, therefore, may notbe easily determined because either the seals or prey aremoving among sampling locations.

4.5. Tissue comparisons

There was a significant positive relationship betweenTHg concentrations in blood and hair. It is important tonote, however, THg concentrations in these hair sampleswere indicative of the bioavailable Hg during theirmoult, summer of 2003, not 2004 when most animalswere sampled. Harbor seals shed their old hair and growa new pelage in 6–8 weeks during their annual moult(Montagna and Harrison, 1957). The amount of THgsequestered in the hair is dependent on the amount of Hgcycling in the ecosystem, bioaccumulated in preyspecies, consumed as prey, retained in blood, andsequestered into the hair of harbor seals through thegrowing follicle during moult. Hair functions as anexcretory tissue for harbor seals because considerableamounts of toxic substances can be removed from theblood and retained in hair (Wenzel et al., 1993). Bloodsupplies amino acids, which may be bound to Hg, to thekeratinized structure for protein growth while the pile

grows over various weeks. There was a significantpositive relationship between concentrations of THg inhair and concentrations of THg and MeHg in liver. THgin hair was comprised primarily of MeHg and was morerelated to MeHg concentrations in liver than THgconcentrations in liver. It had been proposed, however,that THg concentrations in seal fur could be used as ageneral indicator of the extent of Hg contamination inthe animal (Freeman and Horne, 1973). The indicatorfunction for metal pollution in hair alone, however,could be determined by comparing seal hair betweentwo moults (Wenzel et al., 1993), which did not occur inthis study because of the lack of sample size for a moultother than summer of 2003. A more significantrelationship between THg concentrations in hair andblood or other internal tissues likely could be deter-mined if sampled shortly after the old hair has moulted,and the new hair has formed (Watanabe et al., 1996).

4.6. Influence of trophic level

13C indicates sources of primary productivity; where-as, 15N enriches with increasing trophic levels and thusreflects trophic position (DeNiro and Epstein, 1981;Minagawa and Wada, 1984). The majority of harborseals from this study had comparable δ13C and δ15Nand subsequently were feeding at the same trophic level,only two outliers were found. One animal was probablyfeeding a trophic level higher than the mean, becausegross necropsy and histological results indicated thatthis animal died of invasive adenosquamous genitalcarcinoma with 95% of the liver infiltrated with cancer.Pathways by which proteins are taken up and digested inlysosomes are non-selective and inducible, being activeprimarily under circumstances of nutrient deprivation orstress (Dice and Chiang, 1989). Because the major por-tion of the liver was neoplastic, the harbor seal waslikely catabolizing its own liver as opposed to feedingone true trophic level higher than the mean. Acomparable stepwise increase in δ15N of 3.7‰ wasfound in Arctic char that were practicing cannibalism(Hobson and Welch, 1995). Another harbor seal hadlesser values of δ13C and δ15N and was feeding atrophic level lower than the mean. This animal wasbrought into rehabilitation as a neonate and was fedformula for an entire month before it was euthanized dueto complications from pylonephritis and possible aspira-tion pneumonia. The formula was primarily a zoologicmilk matrix, which was probably either corn- or soy-based, and supplemented with salmon oil (TMMC pers.comm.). Because trophic positioning of organisms bystable N analyses is based on assimilated food of many


meals (Tieszen et al., 1983) and liver has a great proteinturnover rate on the order of days to weeks (Welle,1999), this animal's δ13C and δ15N were probably anartifact of rehabilitation not of typical prey or lactation.

THg concentrations in liver of pups increased withδ15N or trophic level. The plot was biphasic indicatingthat differences also existed based on age class ofindividual harbor seals. As pups increased in trophiclevel, consuming more fish and squid and lesscrustaceans, THg in their livers increased. Biomagnifi-cation of Hg with trophic level was reported by someresearchers (Jarman et al., 1996; Atwell et al., 1998) butnot by other researchers (Wagemann and Muir, 1984;Atwell et al., 1998; Dehn et al., 2005). Thesediscrepancies seem to be dependent on the type of preythe animal consumes and type of tissue sampled. Someresearchers have sampled muscle for stable isotope andHg determinations. THg concentrations in the liver,however, were approximately two orders of magnitudegreater than muscle (Sergeant and Armstrong, 1973) andare dependent on the demethylating efficiency and healthstatus of the animal (Himeno et al., 1989; Aguilar et al.,1999; National Research Council, 2000). Relationshipbetween susceptibility to disease and high pollutantconcentrations is highly contested and may be explainedby depressed immunocompetence caused by pollutants,mobilization of pollutants stored in reserve tissues inindividuals thinned by disease (Joiris et al., 1991), oralterations in physiological functions leading to in-creased concentrations (Aguilar et al., 1999).

4.7. Interaction amongst trace elements

Concentrations of MeHg in liver increased withrespect to THg concentrations to a maximum around2 μg/g ww. MeHg concentrations were asymptotic, with%MeHg remaining constant with increasing THgconcentrations; thereby, an equilibrium has occurredbetween dietary intake of MeHg from prey anddetoxification processes of MeHg. Hg also forms stablecomplexes with Se associated with glutathione andcysteine, which are thiol compounds that act asantioxidants and radical scavengers. HgSe complexthus inhibits these enzymes and lessens the level ofavailable antioxidants to the cells (Fishbein, 1987). Se,moreover, may providemarinemammals with protectionagainst toxicity caused by Hg (Martoja and Berry, 1980;Wagemann and Muir, 1984), because insoluble tiemma-nite (HgSe complex) can be stored indefinitely in theliver of marine mammals (Nigro and Leonzio, 1996).

Se was first reported to counteract acute mercuricchloride toxicity by Parizek and Ostadalova (1967).

Numerous researchers have discussed the protectiveeffect of Se on Hg toxicosis in marine mammals andsubsequent association of the two trace elements,namely in a molar ratio of 1:1 for THg:Se (Koemanet al., 1975; Smith and Armstrong, 1978; Frank et al.,1992; Dietz et al., 2000). Other researchers, however,have determined a molar ratio deviating from unity(Wagemann and Stewart, 1994; Woshner et al., 2001b;Dehn et al., 2005). Mean molar Se:THg ratio did deviatefrom one in the present study, but if molar ratios werepartitioned age-wise, adults (0.99:1) had a more unifiedmolar Se:THg ratio than pups (2.3:1). It has beenproposed that the typical 1:1 molar ratio is only found inadults with greater concentrations of Hg (Dietz et al.,2000; Wagemann et al., 2000; Dehn et al., 2005) withpups having a greater molar ratio of Se:THg (Wagemannet al., 1988; Frank et al., 1992).

These elements also may occur in consistent pro-portions only when a physiologic threshold has beenreached or when adherence to this ratio is not phys-iologically necessary (Woshner et al., 2001b). It has beensuggested that a threshold concentration of Se isequivalent to what is physiologically necessary, plus anadditional reservoir for which Hg has a far greater bindingcapacity (Krone et al., 1999;Woshner et al., 2001a). OnceHg concentrations exceed baseline Se concentrationsaround 3 μg/g ww, Se is likely to increase in parallel withHg concentrations. Coaccumulation may be a result ofcompensation by the organism for the depletion of thephysiologically essential concentrations of Se as Hg isaccumulated and linked to Se present, a normal homeo-static regulation (Beijer and Jernelov, 1978). Below thisbaseline Se level, molar Se:THg deviate from one (Kroneet al., 1999). This continued uptake and subsequentbinding of THg to Se was observed in adult harbor seals,with deviations from the 1:1 molar ratio only in pups,when overall Se concentrations were suspected belownormal physiological concentrations.

4.8. Summary and conclusion

Hg accumulated in harbor seals in central andnorthern California via various prey species. THg con-centrations in blood and hair of harbor seals increasedwith age but did not differ amongst locations in centraland northern California. Gender differences were onlypresent in hair of adult harbor seals; males sequesteredmore THg, hence MeHg, in hair than females. Hair mayhave been a key source of Hg excretion in males;whereas, females excreted Hg via hair, placenta,neonate, and possibly milk. THg and MeHg increasedin livers with age and increased with greater δ15N or


trophic level. MeHg can be demethylated into IHg;therefore, %MeHg was at minimum levels in olderharbor seals although they had greater THg concentra-tions than younger harbor seals. Se in the liver alsoincreased with age and was in an equimolar ratio withTHg in adults. Molar ratio of Se:THg in pups did deviatefrom a 1:1 ratio because THg did not bind with all Sefound below baseline physiological concentrations.Harbor seals from central and northern California hadsignificantly greater concentrations of Hg and Se thanterrestrial mammals, in some instances these concentra-tions were considered toxic to terrestrial mammals.

THg concentrations in blood are useful for deter-mining present day contamination associated with aspecific locale; whereas, THg concentrations in hair arebeneficial for comparing differences during variousmoults over approximately the same timeframe eachyear, which could be valuable in monitoring changes inHg concentrations through time. THg, MeHg, and Seconcentrations in liver represent long-term exposure andretention with additional insight into proportions andpotential binding of these trace elements. Trophic levelassessments are advantageous in terms of comparisonsamong individuals. Finally, the appropriate sample sizeis needed to investigate and determine age, sex, andlocation effects on trace element concentrations.

Acknowledgements

We graciously thank T. Voss, M. Gordon, and A.Byington for their analytical expertise at Moss LandingMarine Laboratories. A special thanks to The MarineMammal Center (D. Greig and F. Gulland) and LongMarine Laboratories-UCSC (E. Wheeler) for theirunparalleled commitment to necropsing and samplinga myriad of animals for this study. This study could nothave been completed without the help of L. Boden-steiner, C. Gibble, T. Goldstein, D. Greig, S. Hansen, S.Hayes, M. Lander, B. Long, H. Nevins, T. Norris, E.Phillips, M. Rutishauser, T. Sigler, S. Simmons, J.Sweeney, K. Thomas, B. Watts, L. Wertz, E. Wheeler, E.Woolery, and many others in the field. We thank K.Knott, S. Moses, and B. Meyer at University of AlaskaFairbanks for assisting in stable isotope analyses andage estimations. A special thanks to S. Allen at theNational Park Service for aiding in original brainstorm-ing aspects of the study and facilitating portions of thestudy at Pt. Reyes National Park. Our appreciation alsois extended to M. Graham and G. Cailliet (MLML), whogave much needed statistical advice, and to K. Thomasfor making site maps. We would like to thank K. Coalefor reviewing the thesis and two anonymous reviewers

whose thoughtful comments improved the manuscript.Lastly, we are grateful for the generous support providedby the following funding sources: American CetaceanSociety–Monterey and San Francisco Chapters, Dr. EarlH. Myers and Ethel M. Myers Oceanographic andMarine Biology Trust, National Park Service, PackardFoundation, and San Francisco Estuary Institute.

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trace element concentrations in the pacific harbor seal (phoca vitulina richardii) in central and...

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