Science of the Total Environment 408 (2010) 578–585

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Fluctuating wing asymmetry and hepatic concentrations of persistent organicpollutants are associated in European shag (Phalacrocorax aristotelis) chicks

Bjørn Munro Jenssen a,⁎, Jon Birger Aarnes a,1, Kari-Mette Murvoll a, Dorte Herzke b, Torgeir Nygård c

a Department of Biology, Norwegian University of Science and Technology, NO-7491 Trondheim, Norwayb Norwegian Institute for Air Research, The Polar Environmental Centre, NO-9296 Tromsø, Norwayc Norwegian Institute for Nature Research, NO-7485 Trondheim, Norway

⁎ Corresponding author. Tel.: +47 73596267.E-mail address: bjorn.munro.jenssen@bio.ntnu.no (B

1 Present address: Norwegian Pollution Control Authcals and Local Environmental Management, PO Box 810

0048-9697/$ – see front matter © 2009 Elsevier B.V. Adoi:10.1016/j.scitotenv.2009.10.036

a b s t r a c t

a r t i c l e i n f o

Article history:Received 14 May 2009Received in revised form 9 October 2009Accepted 14 October 2009Available online 6 November 2009

Keywords:EcotoxicologyPCBBFRSeabirdsPollutionNorway

In aquatic birds, high body burdens of persistent organic pollutants (POPs) have been associated withdevelopmental effects related to growth, increased fluctuating wing asymmetry, and disruption of thethyroid hormone, vitamin A (retinol) and vitamin E (tocopherol) homeostasis. The aim of the present studywas to examine if morphological variables (body mass, liver mass, wing length, tarsus length and headlength), fluctuating asymmetry of the wings and tarsus, growth rates and endocrine variables (thyroidhormones, retinol and tocopherol) were associated with hepatic levels of POPs (PCBs, OCPs and PBDEs) in21 day old chicks of European shag (Phalacrocorax aristotelis). Partial Least Squares (PLS) analysis showedthat fluctuating asymmetry of wing bone length (FAWBL) was affected by PCB-105, -118, -138, -153, and -180(r2x=0.88, r2y=0.35, q2=0.29). Bivariate correlation confirmed significant positive relationships betweenFAWBL and each of these PCB congeners. In the PLS model no other biological variables were significantlyaffected by any of the POPs. Levels of POPs were much lower in the shag chicks than in eggs and in hatchlingsfrom the same breeding colony, most likely due to growth dilution of the compounds. We suggest that theeffects of the PCBs on FAWBL may be due to effects of these compounds on bone growth and bone structure.FAWBL may have functional effects on the fitness if it persists after fledging.

.M. Jenssen).orities, Department of Chemi-0 Dep, NO-0032 Oslo, Norway.

ll rights reserved.

© 2009 Elsevier B.V. All rights reserved.

1. Introduction

Due to national and international regulations the use ofmany classicpersistent organic pollutants (POPs) such as polychlorinated biphenyls(PCBs) and organochlorine pesticides (OCPs) have gradually declined(Bignert et al., 1993; Henriksen et al., 2001; Kostamo et al., 2002). Manycountries have also introduced restrictions on the use of somebrominated flame retardants, such as the technical penta- and octa-mixtures of polybrominated diethyl ethers (PBDEs). In spite of suchregulations, levels of many of these POPs are still high in the marineenvironment, and especially in fish-eating seabirds and marinemammal top predators (Jenssen, 2006; Law et al., 2006; Letcher et al.,in press).

In seabirds, high body burdens of POPs have been associated withdisruptive effects in all developmental stages, from reduced eggshellthickness (Helander et al., 2002) via effects on the developing embryoand chick (Champoux et al., 2002; Hoffman et al., 1986; van den Berget al., 1994), to fitness related variables in adults (Bustnes et al., 2008).The embryo and foetus, newborns and young individuals undergo large

and rapid structural and functional changes. Developing animals aretherefore particularly vulnerable to the toxic effects of chemicals. POPshave been associated with reduced growth rates of embryos and chicks(Champoux et al., 2002; Hoffman et al., 1986; van den Berg et al., 1994),with reduced bone length and altered bone structure in adult herringgulls (Larus argentatus) (Fox et al., 2008).

Because many endocrine systems are important in regulatinggrowth and development of young animals, POPs that are endocrinedisrupters are of particular concern. In aquatic birds, high body burdensof POPs have particularly been associatedwith disruption of the thyroidhormone and vitamin A (retinol) homeostasis (Champoux et al., 2002,2006; Fox et al., 2007; Greichus and Hannon, 1973; Verreault et al.,2004), and also with the vitamin E (tocopherol) homeostasis (Murvollet al., 2005, 2007). In hatchlings of European shag (Phalacrocoraxaristotelis) increased levels of POPs in the yolk sac have been associatedwith alterations in liver tocopherol and plasma retinol (Murvoll et al.,1999, 2006). Since retinol, tocopherol and thyroid hormones areimportant for the regulation of growth and development, disruptionof the homeostasis of these endocrine variables in hatchlings mayindicate a potential long-term effect of POPs.

Developmental stability during growth is a measure that can beapplied to study developmental stress (Eeva et al., 2000). Developmen-tal stability reflects the organism's ability to buffer its developmentagainst stress (Møller and Swaddle, 1997). Fluctuating asymmetry (FA)

579B.M. Jenssen et al. / Science of the Total Environment 408 (2010) 578–585

is often used as a measure of developmental stability (or instability)(Bechshoft et al., 2008; Zakharov, 1992). FA refers to small deviationsfrom the idealmorphological symmetry and ismeasured as the absolutedifference between traits on the two sides of a bilaterally symmetricalorganism (Møller and Swaddle, 1997; Palmer and Strobeck, 1986;Palmer and Strobeck, 2003). Fluctuating asymmetry of individuals havebeen reported to increase as a response to stress (Leung and Forbes,1996). Thepossible linkbetweenenvironmental stress and FA is that theorganism compensates for stress through increased energy use (Leunget al., 2000), and this may reduce the energy use available formaintaining developmental precision (Sommer, 1996). Thus, anincreased FA indicates increased developmental instability. Since FA isrelated to the quality of the individual, FA is a biologically relevantbioindicator for studying stress caused by environmental pollutantsin situ (Sommer, 1996). Indeed, FA in primarywing feathers of glaucousgulls (L. hyperboreus) have been associated with high levels of severalPOPs (Bustnes et al., 2002). Furthermore, FA of the primary feathers ofgreat tits (Parus major) increased proportionally to the closeness of aheavy metal source (Eeva et al., 2000).

Biomarkers are defined as measurable changes in biochemicalprocesses or (endogen) compounds induced by xenobiotics (Peakall,1992). Thus, variables that have been shown to respond to POPs canbe applied as tools for evaluating exposure and effects of thesecompounds. Retinol, tocopherol and thyroid hormones are importantendocrine variables that are involved in growth and development,and may thus be applied as biomarkers of effects on growth anddevelopment, including FA.

The aim of the present studywas to examine if hepatic levels of POPs(PCBs, OCPs and PBDEs) in 21 day old chicks of European shag(Phalacrocorax aristotelis)were associatedwithmorphological variables(bodymass, liver mass, wing length, tarsus length and head length), FAof the wings and tarsus, growth rates and endocrine variables (thyroidhormones, retinol and tocopherol). Principal ComponentAnalysis (PCA)and Partial Least Squares (PLS) analysis were used to test forassociations between pollutants and biological variables.

2. Materials and methods

The study was conducted in June to July 2004 at the island Sklinnawhich is a protected seabird reserve situated about 50 km west of theNorwegian mainland (65°12′N, 11°00′E). Being uninhabited and veryremote, the pollution input is believed to be long-range transportedfrom multiple sources.

Eighteen chicks were identified as newly hatched (i.e. less than 12 hold), andmeasurements ofmorphological variableswere recorded eachthird day when they were reared by their parents. They were sacrificedat an age of 21 days. Body mass (BM, g) was measured using calibratedspring weights (1 g accuracy between 0–500 g and 5 g accuracy above500 g). Head length (HL, mm), the length of right and left wing bones(metacarpus, basal and terminal phalanx) (WBLR and WBLL, mm) andthe length of right and left tarsi (TLR and TLL, mm)wasmeasuredwith acalibrated electronic calliper (0.1 mm accuracy). WBL (mm) and TL(mm) represent the mean of WBLR and WBLL, and TLR and TLL,respectively. Fluctuating asymmetry of wing bone length (FAWBL, mm),and fluctuating asymmetry of tarsus length (FATL, mm) was calculatedas the absolute differences between WBLR and WBLL, and between TLRand TLL, respectively. Growth rates (GR) of the morphological variablesBMGR (g/day), HLGR (mm/day), WBLGR (mm/day), TLGR (mm/day),were calculated between day 10 and day 20.

When the chickswere 21 days of age, heparinised blood samples (2–4 mL) were taken before they were sacrificed. The blood samples werecentrifuged, and the plasma was transferred to polyethylene vials thatwere covered with aluminium foil and frozen in liquid nitrogen. Allmorphological variables were recorded before the liver was dissectedout, weighted (LM, g), and transferred to polyethylene vials that werecovered with aluminium foil and frozen in liquid nitrogen.

2.1. Analysis of POPs

Tissue samples were extracted and prepared as previously described(Herzke et al., 2002; Vetter et al., 2007). Briefly, liver samples werehomogenized, subsequently dried in a 10 fold amount of dry sodiumsulphate, and extracted with cyclohexane/acetone (50:50 v/v). Theamount of extractable organic material was determined gravimetrically.Lipid removalwasperformedonagel permeationchromatography (GPC)system. An additional fractionationwas carried out on a florisil column. Arecovery standard (octachloronaphthalene, 10 µL of a 1 ng/µL solution inisooctane) was added prior to quantification to all samples. Forquantification of all compounds, reference material was obtained fromCambridge Isotope Laboratories (Woburn,MA,USA). Solvents of pesticidegrade were employed (E. Merck, Darmstadt, Germany). 13C-labelledcompounds were used as internal standards presenting each groupof analytes, containing between 77 and 500 pg/µL of 13C-p,p′-DDE,13C-p,p′-DDT, 13C-labelled PCB-28, -52, -101, -118, -153, and -180, and13C-labelled PBDE-47. The results were not corrected for recovery.

The analyses of the POPswere performed at the Norwegian Institutefor Air Research, Tromsø. The liver samples were analyzed for 12 PCBcongeners (PCB-28, -52, -99, -101, -105, -118, -138, -153, -170, -180,-183, and -187), 8 PBDE-congeners (PBDE-28, -47, -99, -100, -138, -153,-154, and -183), and 18 OCP compounds (p,p′-dichlorodiphenyltri-chloroethane [DDT], o,p′-DDT, p,p′-dichlorodiphenyldichloroethane[DDE], o,p′-DDE, o,p′-dichlorodiphenyldichloroethane [DDD], hexa-chlorobenzene [HCB], α-, β-, γ-hexachlorocyclohexane [HCH], cis-chlordan, trans-chlordan, cis-nonachlor, trans-nonachlor, oxychlordan,heptachlor, heptachloroepoxid [HCE], and the two toxaphene compo-nents Parlar 26 and Parlar 50).

The detection limits were 0.03–0.11 ng/g wet weight (w.w.), 0.03–0.08 ng/gw.w., and 0.002–0.76 ng/gw.w., respectively, for PBDEs, PCBsand OCPs. Recoveries varied between 60 and 125% for labelled PCBs andbetween 97 and 120% for the labelled PBDEs with the exception oflabelled BDE-183, which had elevated recoveries up to 160% in twocases. Standard referencematerial (SMR) cod oil (NIST 1588a)was usedas a control sample. One SMR and one laboratory blank were analyzedalong with every 10th sample. No blank contamination was detected.

2.2. Vitamin analysis

Hepatic concentrations of retinol (RET) andα-tocopherol (TOC)weredetermined by high-performance liquid chromatography (PerkinElmer200 series, USA) at the Department of Biology, Norwegian University ofScience and Technology (NTNU), Trondheim, Norway. The treatment ofthe liver samples and the extraction of the vitamins from the sampleswere conducted in red light to prevent degradation of RET and TOC. Liversampleswere homogenized and the sampleswere sonicated to break thecells and to facilitate the extraction of vitamins. Hexane was used forextraction of vitamins, and the hexane layer which contained thevitamins, was concentrated by evaporation under pure N2 and dissolvedinmethanol (mobilephase). Adetaileddescriptionof theextractionof thevitamins, of the standards and instruments used, and on the calculation ofvitamin concentrations is given in Murvoll et al. (2005).

Each sample was analyzed in duplicate. Four-point standardcurves were used to calculate concentrations of RET and TOC. Therange of detection was 2.73–377.32 μg/L for RET, and 12.44–2046 μg/Lfor TOC. For RET and TOC, the extraction efficiencies using the presentmethod are 86% and 98%, respectively. More details on the qualityassurance during the analysis of the vitamins, including extraction-efficiency and reproducibility, are given in Murvoll et al. (2005).

2.3. Hormone analysis

Concentrations of total and free triiodothyronine (TT3 and FT3),and total and free thyroxin (TT4 and FT4) were determined byradioimmunoassay (Coat-A-Count TT3, FT3, TT4 and FT4, Diagnostic

Table 2Growth rates (from day 10 to day 20 of life) of morphological variables in chicks ofEuropean shags (Phalacrocorax aristotelis).

Mean SD Range n

BM (g/day) 58.4 5.1 47.0–66.1 18Head (mm/day) 3.2 0.3 2.6–3.9 18Tarsus length (mm/day) 2.5 0.2 2.1–3.1 18Wing length (mm/day) 5.6 0.5 4.6–6.2 18

Table 3Liver concentrations of retinol (RET) and tocopherol (TOC), and plasma concentrationsof total and free triiodothyronine (TT3, FT3) and of total and free thryroxine (TT4 andFT4) in 21 day old chicks of European shags (Phalacrocorax aristotelis).

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Products Corporation, Los Angeles, CA, USA) at the Department ofBiology, NTNU. The amount of radioactive labelled antigens wasregistered using a γ-counter (Cobra Auto-Gamma, Packard Instru-ments Company, Downers Grove, IL, USA).

Commercially available SRM (BIO-RAD, Lyphochek® ImmunoassayPlus Control, Levels 1, 2 and 3) were used as controls. As intern-control,plasma from ox (Bos taurus) was analyzed for each 10th sample of shagplasma. The plasma samples were analyzed in duplicate. Standardcurves were 6-point for TT3 and TT4, and 7-point for FT3 and FT4. Thelimit of detection for each of the hormones was estimated by thesoftware in theγ-counter, and thedetection ranges for TT3, FT3, TT4 andFT4were 0.082–9.22 nmol/L, 0.0029–65pmol/L, 0.117–309 nmol/L, and0.0137–128.7 pmol/L for TT3, FT3, TT4 and FT4, respectively.

Mean SD Range n

RET (ng/g) 710 296 214–1316 17TOC (ng/g) 195 669 519–2902 12TT3 (nmol/L) 3.89 1.48 1.80–7.21 16FT3 (pmol/L) 5.07 1.82 2.01–8.40 15TT4 (nmol/L) 5.31 4.06 0.99–11.79 8FT4 (pmol/L) 2.86 1.31 0.98–5.00 15

2.4. Statistical analysis

The data on POPs are presented asmeanvalues±standard deviation(SD), minimum and maximum values, and median values are providedwhere compounds were not detected in all individuals. Since the sum(Σ) of PCB and PBDE congener compounds varies, the number ofcongeners summed is indicated by an x in ΣPCBx and ΣPBDEx. Thestatistical calculation of the datawas conducted using SPSS (Ver 14.0 forWindows, SPSS, Chicago IL, USA) and The Unscrambler (Camo AS, Oslo,Norway). To simplify the models, only lipid-weight (l.w.) concentra-tions of the POPs were used in the multivariate analysis. All POPsincluded in the multivariate analysis were normally distributed.Principal component analysis (PCA) was used to study inter-correla-tions between pollutants and inter-correlations between biologicalvariables. To obtain a simple structure where the factor loadings withineach principal component were as close to one or zero as possible, thecomponents were rotated orthogonally using the varimax method(Quinn and Keough, 2002). The level of significance was set to p<0.05.To test for correlations between pollutants and biological variables, themultivariate regression test Partial Least Squares (PLS) analysis(Esbensen, 2001) was used (Unscrambler software). The PLS modelsboth the x and ymatrices simultaneously to find the latent variables in xthat will give the best prediction of y. The cross-validated correlation(q2) indicates how much of the variance of the particular responsevariable (y) is explained in the multivariate analysis. Thus, each of theresponse variables included in the multivariate analysis is assigned a q2

value, where q2=1.0 indicates that all variance of the y variable isexplained in this model.

3. Results

Morphological variables of the European shags at hatching and at21 days of age are listed in Table 1. Both FAWBL, and FATL differed from0 (t-test, t>6.3, df=17, p<0.001). Growth rates of morphologicalvariables are listed in Table 2, whereas hepatic vitamin and plasmathyroid hormone concentrations are shown in Table 3.

Table 1Morphological variables in European shag (Phalacrocorax aristotelis) chicks at hatching and

Hatchlings

Mean SD Range

Body mass (g) 36 5 25–43Head length (mm) 35.8 2.1 32.4–40.7Wing length (mm) 15.9 0.7 14.9–17.6Tarsus length (mm) 13.2 1.0 12.2–14.9Liver mass (g) – – –

Tarsus asymmetry (mm)Wing asymmetry (mm) – – –

Concentrations of all POPs that were analyzed for are listed inTable 4. With respect to PCBs, PCB-118, -138, -153 and -180 weredetected in all birds, PCB-105 were detected in 14 birds, PCB-187 insix, PCB-99 in five, PCB-28 in four, PCB-183 in two birds, and PCB-170in one of the birds. PCB-52 and -101 were not detected in any of thebirds. Overall, the concentrations of PCB-153, -138, -118, -180, and-105 were highest, representing 43%, 29%, 16%, 9% and 5%,respectively of ΣPCB10. ΣPCB10 represented 45% of the sum of all OCcompounds (ΣOC).

Of the PBDEs, PBDE-47 was detected in 8 birds. PBDE-100 wasdetected only in one specimen, whereas PBDE-28, -99, -138, -153,-154 and -183 were not detected in any of the birds. Theconcentration of ΣPBDE2 was very low, only 1.1% of ΣPCB10.

Regarding OCPs, HCB, HCE, Parlar 26 and Parlar 50 were detectedin all the birds. p,p′-DDE was detected in 17 of the birds, andoxychlordane was detected in 16 of the birds. The rest of the OCPswere detected in only some of the birds (Table 4). Of the OCPs, theconcentrations of HCE, p,p′-DDE, HCB, oxychlordane, Parlar 50 andParlar 26 were highest, and represented 26%, 23%, 15%, 12%, 6% and5%, respectively of ΣOCP. ΣOCP represented 55% of ΣOC.

Relationships between POPs and between the biological variableswere examined using PCA. With respect to POP data, only compoundsthat were detected in >60% of the specimens were included in theanalyses. Thus, PCB-105, -118, -138, -153, -180, p,p′-DDE, HCB, HCE,oxychlordane, Parlar 26, and Parlar 50 were included as contaminantvariables.

PCA showed that all five PCB congeners were highly positively inter-correlated (r>0.764, p<0.002). Furthermore, p,p′-DDE, Parlar 26 andParlar 50 were positively inter-correlated (r>0.569, p<0.027). p,p′-DDEcorrelated inversely with HCE (r=−0.543, p=0.034, n=17), and there

at an age of 21 days.

21 day old chicks

n Mean SD Range n

18 944.0 91.2 800–1089 1818 95.4 3.6 87.5–101.1 1818 103.0 9.1 85.3–114.9 1818 60.1 2.3 55.1–64.5 18– 51.6 5.2 41.8–59.1 18

1.32 0.90 0–1.7 18– 1.09 0.93 1.00–3.6 18

Table 4Hepatic concentrations of persistent organic pollutants (ng/g lipid-weight) in liver of21 day old chicks of European shags (Phalacrocorax aristotelis) from Sklinna, Norway.

Compound Nd/N Mean SD Min Max

PCB-28 4/18 1.2 2.6 nd 8.1PCB-99 5/18 1.6 2.9 nd 9.8PCB-105 14/18 5.8 5.7 nd 13.3PCB-118 18/18 19.3 9.9 10.7 54.3PCB-138 18/18 35.1 15.6 20.2 90.1PCB-153 18/18 43.4 26.5 24.9 142.1PCB-170 1/18 0.9 – nd 16.0PCB-180 18/18 10.7 5.4 6.1 29.6PCB-183 2/18 0.3 – nd 2.8PCB-187 6/18 2.1 0.8 nd 9.0ΣPCB10 18/18 118.3 66.5 66.5 362.9PBDE-47 8/18 1.1 1.3 nd 3.0PBDE-100 1/18 0.2 – nd 4.0ΣPBDE2 9/18 1.3 1.4 nd 4.0o,p′-DDT 1/18 2.3 – nd 41.6o,p′-DDE 2/18 0.9 – nd 10.5p,p′-DDE 17/18 33.8 12.1 nd 54.8o,p′-DDD 1/18 1.2 – nd 21.3ΣDDTs 18/18 38.2 11.2 23.1 60.2HCB 18/18 22.3 2.5 16.8 25.9α-HCH 2/18 1.7 – nd 17.5β-HCH 1/18 0.1 – nd 2.7γ-HCH 4/18 1.0 3.9 nd 16.5ΣHCH 2/18 2.8 9.0 nd 36.7HCE 18/18 38.1 19.6 8.0 74.2Trans-chlordan 1/18 1.2 – nd 20.8Cis-chlordan 1/18 1.2 – nd 25.1Oxychlordan 16/18 17.7 7.7 nd 28.8Trans-nonachlor 6/18 3.0 5.7 nd 21.5Cis-nonachlor 8/18 3.3 6.0 nd 25.1Σchlordan 16/18 26.4 22.6 nd 109.7Parlar 26 18/18 7.0 3.4 3.2 14.9Parlar 50 18/18 8.7 4.7 4.1 21.1Σtoxaphen 18/18 15.7 7.6 9.1 35.6Extractable lipids (%) 18/18 3.99 0.55 3.41 5.77

Nd indicates the number of individuals which the compounds were detected in,whereas N indicates the total number of chicks sampled. The mean and standarddeviation (SD) is calculated from the concentrations in all 18 individuals, and if theconcentrations of the compound were less than the detection limit, a concentration ofzero was assigned.

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was a positive correlation between HCE and HCB (r=0.598, p=0.020,n=18).

PCA showed that the morphological variables were separated intotwo distinct clusters, whereas the physiological variables wereseparated in a third cluster (not shown). BM, WBL, HL, TL, BMGR,WBLGR were positively inter-correlated (Table 5). In addition, FAWBL

was positively correlated with BM, and TL was positively correlatedwith HLGR and TLGR, and FAWBL correlated positively with WBL andwith WBLGR (Table 5).

The variables HLGR, TLGR and FATL were positively inter-correlated(Table 5). FATL was also inversely correlated with FT3 (r=−0.433,p=0.036) and FT4 (r=−0.453, p=0.029).

Some of the physiological variables, RET, TOC, TT4, FT4, TT3 andFT3 were inter-correlated (Table 6). RET correlated positively withTT4 and TOC. Tocopherol correlated positively with TT4, FT4 and RET,and also inversely with TLGR. In addition to RET and TOC, TT4 alsocorrelated positively with FT4. FT4 correlated positivelywith TT4, RET,TOC and also inversely with FATL (r=−0.453, p=0.029). TT3 waspositively correlated with FT3, and in addition FT3 correlatedinversely with FATL (r=−0.422, p=0.036). LM did not correlatewith any of the other biological variables.

To study biological responses to the contaminant variables, separatePLS analyses were conducted for each of the three clusters of biologicalvariables. As in the PCA-analysis, only compounds thatwere detected in>60% of the individuals were included in the analysis. Only one of theclusters gave a significant PLS component. However, this model whichincluded the contaminants and themorphometric and growth variables

(FAWBL, TL, BMGR, WBL, WBLGR, HL, and BM), gave a very low overallprediction coefficient (q2=0.1), and themodel was therefore not good.In thismodel FAWBL had the highest prediction coefficient, andwhen theother variables were removed, PLS analysis showed that FAWBL (y) wasmost influenced by PCB-105, -118, -138, -153, and -180 (r2x=0.88,r2y=0.35, q2=0.29). Themodel, which resulted in only one significantPLS component, thus explained the FAWBL fairly well. Bivariatecorrelation confirmed significant positive relationships between FAWBL

and each of these PCB congeners (Fig. 1) and with ΣPCB5 (PCB-105:r=0.599, p=0.024, n=14; PBB-118: r=0.553, p=0.017, n=18;PCB-138: r=0.543, p=0.020, n=18; PB-153: r=0.585, p=0.011,n=18; PCB-180: r=0.486, p=0.041, n=18). However, it should benoted that the number of samples was relatively low, and that therelationships are driven by the high FAWBL in the bird with the highestliver concentrations of PCBs (Fig. 1).

4. Discussion

4.1. Fluctuating asymmetry

All the PCB congeners included in the PLSwere significantly positivelyassociated with FAWBL (Fig. 1). The observed effect of environmentalpollutants on FA is consistent with several previous studies on birds(Bustnes et al., 2002; Eeva et al., 2000; Maul and Farris, 2005), insects(Chang et al., 2007), fish (Allenbach et al., 1999; Estes et al., 2006) andmammals (Borisov et al., 1997).

In birds it has previously been reported that PCB-99, -118,oxychlordane, DDE and HCB were associated with FA in the length ofthe 3rd primarywing feather of glaucous gulls (L. hyperboreus) (Bustneset al., 2002). Similarly, FA of the length of the 3rd primary of great tits(Parus major) increased proportionally to the closeness to heavy metalsources (Eeva et al., 2000). Furthermore, FA of the mass of the 2ndsecondary wing feather has been reported in breeding common loons(Gavia immer)with high levels ofmercury (Evers et al., 2008). However,in these particular studies, fluctuating wing asymmetry was documen-ted bymeasuring the length ormass ofwing feathers. Herein, the lengthof the outer wing bones (metacarpus, basal and terminal phalanx)represented wing length. Thus, the effect of the PCBs on FAWBL reportedherein is linked to the effects of these compounds on bone growth and/or bone structure.

In free-living aquatic birds, exposures to high concentrations of OCshave previously been linked to skeletal deformities (Hart et al., 1991;Sanderson et al., 1994; Thompson et al., 2006). In Great Lakes herringgulls (L. argentatus), tarsus length of pipping chicks that did not hatchwere shorter at breeding sites thatwere polluted than at cleaner controlsites (Gilbertson and Fox, 1977; Hoffman et al., 1987), and high levels ofOCs, and in particular dioxin-like compounds, affected the biomechan-ical properties of bones of Great Lakes herring gulls (Fox et al., 2008).Furthermore, shorter femur length of Foster's tern (Sterna forsteri)hatchlings has been linked to high levels of OCs (Hoffman et al., 1987).In-ovo exposure to PCBs has also been shown to affect femur growth inchicken (Gallus gallus) (Gould et al., 1997). Interestingly, the low-doseexposure resulted in increased bone length, whereas a higher doseresulted in decreased bone length (Gould et al., 1997). Perinatalexposure of goats (Capra aegagrus hircus) to PCB-153 caused alteredbone composition in their offspring (Lundberg et al., 2006), and inhumans inverse associations between PCB-118 and bone mineraldensity have been documented (Hodgson et al., 2008). Thus, based onthe numerous previous reports in free-living birds, humans andexperimental studies that OCs including PCBs can affect bone growth,bone mineral composition and biomechanical properties of bones, wesuggest that the association between the PCBs and FAWBL found in thepresent European shag hatchlings is caused by effects of PCBs in wingbone growth.

Several mechanisms may be involved in the development of FA inthe wing bones. The most obvious mechanism is that the PCBs can

Table 5Relationships between morphological variables in chicks of European shags (Phalacrocorax aristotelis).

BMGR (g/day) HL (mm) HLGR (mm/day) TL (mm) TLGR (mm/day) FATL WBL (mm) WBLGR (mm/day) FAWBL

r p r p r p r p r p r p r p r p r p

BM (g) 0.857 <0.001 0.818 <0.001 – – 0.714 <0.001 – – – – 0.840 <0.001 0.813 <0.001 0.412 0.044BMGR (g/day) 0.753 <0.001 – – 0.740 <0.001 – – – – 0.702 0.001 0.682 0.001 – –

HL (mm) – – 0.813 <0.001 0.876 <0.001 – – 0.702 0.001 0.682 0.001 – –

HLGR (mm/day) 0.484 0.021 0.876 <0.001 0.434 0.036 – – 0.682 0.001 – –

TL (mm) 0.517 0.014 – – 0.539 0.010 0.555 0.008 – –

TLGR (mm/day) – – – – – – – –

FATL – – – – – –

WBL (mm) 0.966 <0.001 0.440 0.034WBLGR (mm/day) 0.437 0.035

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interfere with the calcium homeostasis (Thompson et al., 2006).Intestinal calcium absorption and bone balance have been shown tobe affected by 1,25-dihydroxyvitamin D3 (1,25(OH)2D), the activeform of vitamin D (Hurwitz, 1996). In grey seals (Haliocherus grypus) aclear association between circulating levels of 1,25(OH)2D andhepatic PCB levels has been reported, and it has been suggested thatbone lesions observed in Baltic grey seals may be associated withvitamin D disruption (Routti et al., 2008).

Thyroid hormones regulate bone development in young birds, bothdirectly and indirectly by enhancing the activity of other endogenoussubstances such as growth hormone and insulin-like growth factors(Merryman and Buckles, 1998). Several studies have shown that OCsincluding PCBs reduce levels of circulating thyroid hormones in free-living birds and mammals (Braathen et al., 2004; Jenssen et al., 2003;Sørmo et al., 2005; Verreault et al., 2004). In the present study, FT4 andFT3were significantly inversely associatedwith FATL, but notwithFAWBL

or WBLGR. Thus, it does not seem that thyroid hormone disruption wasinvolved in FAWBL or was directly associated withWBLGR. However, theinverse associations between FT4 and FT3 and FATL suggest that the roleof thyroid hormones in FA should be studied in more detail.

During their first 21 days of life, the average daily growth rate of thewing bones in European shags from Sklinna is higher than thecorresponding growth rate of the tarsi (Table 2) (Østnes et al., 2001). Inthepresent study, FAWBLwaspositively correlatedwithWBLGR (r=0.437,p=0.035) and WBL (r=0.440, p=0.034). It should also be noted thatFAWBL was positively correlated with BM (r=0.389, p=0.044), but notwith BMGR. The results may indicate that FA is more likely to occur in fastgrowing bones than in slower growing bones. Since growth rates ofdifferent extremities vary between species, it is possible that this factorshould be taken into consideration if FA is to be used as a bioindicator forpollutant exposure and effects in developing animals.

Although there probably are no functional effects of FAWBL in21 day old shag chicks, FAWBL may have functional effects on fitness ifit persists until and after fledging. Wing asymmetry may increase theenergetic costs of the aerodynamic movements of the birds (Thomas,1993), and thus possibly reduce their locomotory and predatoryabilities and/or their abilities to evade predators.

Table 6Relationships between physiological variables in chicks of European shags (Phalacrocorax a

TOC (ng/g) TT4 (nmol/L)

r p r p

RET (ng/g) 0.592 0.005 0.479 0.022TOC (ng/g) 0.624 0.003TT4 (nmol/L)FT4 (pmol/L)TT3 (nmol/L)

4.2. Retinol and tocopherol

In shag hatchlings (less than 12 hof age), plasmaRET levels correlatedinversely to several PCB congeners, ΣPCB and ΣOCPs (Murvoll et al.,2006). Inverse associations between OCs and plasma or liver retinoidlevels have also been reported in other studies on birds (Champoux et al.,2002, 2006; Greichus and Hannon, 1973; Spear and Moon, 1986). It hasbeen suggested that reduced plasma RET levels in shag hatchlings mayindicate that growth and development could be affected because vitaminA is important for these processes in birds (Murvoll et al., 2006). In thepresent study plasma RETwas not analyzed because the sample volumesonly allowed analysis of plasma thyroid hormone concentrations.However, hepatic RET concentrations were not identified as a responsevariable in relation to hepatic POP concentrations. Furthermore, RETconcentrations did not correlatewith any of themorphological or growthvariables, including FAWBL. The hepatic RET concentrations in the 21 dayold chicks were almost 7 times higher than reported in the livers of shaghatchlings (Murvoll et al., 2006). Thismay indicate thatRETcontent in theyolk sac is a limiting factor which may induce retinol-related effectsduring embryonic growth and in hatchlings, but that dietary intake of RETafter hatching is sufficient for normal growth in older shag chicks.

Hepatic TOC concentrations were inversely associated with TLGR(r=−0.403, p=0.048), but not with any of the othermorphological orgrowth variables, including FAWBL. Furthermore, we found no associa-tions between POPs and hepatic TOC concentrations. Previously, aninverse association between PBDE-99 and hepatic TOC concentrationshas been identified in newly hatched ducklings (Anas platyrhynchos)(Murvoll et al., 2005). Furthermore, an inverse associationbetween yolksac concentrations of PBDE-28 and hepatic TOC concentrations wasindicated in shag hatchlings (Murvoll et al., 2006). However, the modelexplaining the effects of PBDE-28 was not good, and thus Murvoll et al.(2006)warned about giving these findings substantial weight. It shouldbe noted that none of these PBDEs were detected in the liver samples ofthe 21 day old shag chicks. Hepatic TOC concentrations have also beenreported to decrease in several species following exposure to PCB-23,-24, and -47 (Palace et al., 1996; Twaroski et al., 2001). Hepatic TOCconcentrations in the 21 day old shag chicks were only 3‰ of that in

ristotelis).

FT4 (pmol/L) TT3 (nmol/L) FT3 (pmol/L)

r p r p r p

0.564 0.007 – – – –

0.504 0.016 – – – –

0.930 <0.001 – – – –

– – – –

0.652 0.002

Fig. 1. Relationshipsbetween log concentrationsofPCBcongeners andfluctuatingasymmetryofwingbone length (FAWBL) in21 dayold chicksof Europeanshags (Phalacrocorax aristotelis)from Sklinna, Norway (see text for statistics).

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shag hatchlings (Murvoll et al., 2006). The lack of relationships betweenhepatic TOC and any of the POPs indicates that the POPs were notresponsible for the reduction of hepatic TOC during the first 21 days oflife in shags. Itmay bemore likely that the TOC reduction is because TOCis used for protection against the oxidation caused by polyunsaturatedfatty acids in the fish they consume (Surai and Sparks, 2000).

4.3. POP levels and patterns

Concentrations of ΣPCB5 (i.e., CB-105, -118, -138, -153, and -180),p,p′-DDE and HCB in the yolk of shag hatchlings from the samebreeding colony were 113, 164 and 55 times higher than in the liversof the 21 dayold chicksherein (Murvoll et al., 2006). Nodata are availablefor HCE in the hatchlings. With respect to PBDEs, only BDE-47 and -100were detected in the livers of 21 day old chicks. In the yolk sac ofhatchlings, BDE-28, -99, -138, -153, -154 and -183were also present, andthe concentration of ΣPBDE was much higher than reported herein

(>100 times) (Murvoll et al., 2006). In shag eggs from the same colony,BDE-28, -47, -99, -100 and -153 were detected (Vetter et al., 2007), andthese concentrations were lower than reported in livers of hatchlings(Murvoll et al., 2006), but higher than in the livers of 21 day old chicksherein. For instance, the averagewet-weight concentrations of BDE-47 ineggs, egg yolk of the hatchlings, and in the liver of the 21 day old chickswere 828 ng/kg (Vetter et al., 2007), 5590 ng/kg (Murvoll et al., 2006),and 43 ng/kg, respectively (assuming a concentration of zero in the ten21 day old chickswhere the compound notwas detected). The reason forthemuchhigher levels of BDE-47 in the yolk sacs of the hatchlings than inthe eggs is because the lipophilic compoundsbecomeenriched in the yolkas the yolk sac is utilized for growth by the embryo. Even though careshould be takenwhen comparing levels of POPs in different tissues, and itshould be noted that the samples were from different years, the dataindicates that contaminant levels in the chicks were diluted duringgrowth. This is presumably mainly because of low contaminant levels intheirdiet. Suchgrowthdilutioneffects of organochlorineshavepreviously

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been reported in double-crested cormorants (Phalacrocorax auritus)(Jones et al., 1994).

With respect to the pattern of the various PCB congeners there wasnomarkeddifference between the shaghatchlings (Murvoll et al., 2006)and the 21 day old chicks in the present study. Thus, the pattern of PCBcongeners (CB-153>-138>-118>-180) was in very good accordancewith the pattern previously reported in European shags from thisbreeding colony at Sklinna (Murvoll et al., 2006, 1999), and in otherPhalacrocoracidae species from Europe (van den Berg et al., 1994).

The liver concentrations of OCs in the present 21 day old shag chickswere much lower than those that were associated with FA in adultglaucous gulls (Bustnes et al., 2002). In contrast, PCB concentrationswere much higher in the egg and in the yolk sac of hatchlings (Vetteret al., 2007; Murvoll et al., 2006). It is therefore possible that the FAWBL

can be explained by contaminants that were present in the egg and yolkof the hatchlings rather than those still present in 21 day old chicks.

4.4. Fluctuating asymmetry as a bioindicator of pollutant stress

Studies related to effects of environmental pollutants in wildlife areoftenbased on correlative associations between tissue concentrations ofcontaminants and response variables (bioindicators and/or biomar-kers). Although associations between contaminants and responses notare proof of cause–effect relationships, statistical associations may stillbe regarded as strong indicators of cause–effect relationships. Also, thenumber of samples in the present study was relatively low. Thus, wecannot conclude firmly on the use of on FAWBL as a bioindicator ofpollution stress. However, when taking into consideration previouslydocumented effects of PCBs on bone growth and structure (Gould et al.,1997; Lind et al., 2003; Lundberg et al., 2006), and that POPs have beenassociatedwith FA (Borisov et al., 1997; Bustnes et al., 2002; Custer et al.,2001), we suggest that FA is a sensitive bioindicator for developmentaleffects of POPs. It is, however, important to note that the FA of the wingbone length found in the present study, is not necessarily related to thefitness of the chicks.

In the present study there were only few associations betweenbiomarker levels and morphological and growth variables. FT4 and FT3correlated inversely with FATL, and TOC correlated inversely with TLGR.Neither of the biomarkers correlated with FAWBL. This indicates that theapplied biomarkers were poor predictors of effects of POPs on FAWBL.

Acknowledgements

The authors thank Jon Aasen and Hanne Nøvik for the assistanceduring the field work, and Grethe Stavik Eggen, Helene Mathisen andJenny Bytingsvik for technical analytical assistance. The collection ofeggs and chickswas conductedbypermission fromtheCounty governorof Nord-Trøndelag and from the Directorate for Nature Management.

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