Effects of experimental calcium availability and anthropogenic metalpollution on eggshell characteristics and yolk carotenoid and vitaminlevels in two passerine birds

Silvia Espín a, *, Sandra Ruiz a, 1, Pablo S�anchez-Virosta a, 1, Juha-Pekka Salminen b,Tapio Eeva a

a Section of Ecology, Department of Biology, University of Turku, 20014, Turku, Finlandb Laboratory of Organic Chemistry and Chemical Biology, Department of Chemistry, University of Turku, 20014, Turku, Finland

h i g h l i g h t s

� Metal pollution affected maternal lutein and vitamin D3 allocation to yolk.� Metal pollution did not affect the egg volume, eggshell index and pigmentation.� Both species were capable to obtain sufficient calcium for eggshell formation.� Nestling growth and size was influenced by carotenoids and vitamin D3 in yolk.� The two species differ in their carotenoid and vitamin D3 investment into egg yolks.

a r t i c l e i n f o

Article history:Received 18 November 2015Received in revised form14 February 2016Accepted 16 February 2016Available online xxx

Handling Editor: A. Gies

Keywords:Eggshell pigmentationMaternal effectsLuteinCholecalciferolRetinol

a b s t r a c t

The maternal investment into egg quality depends on the condition of the female, the quality of themate, and the quality of the environment. In that sense, availability of nutrients and exposure to pol-lutants are essential parameters to consider. The main aim of this study is to assess the effects of calcium(Ca) availability and anthropogenic metal pollution on early-stage reproduction in two passerine species,great tits (Parus major) and pied flycatchers (Ficedula hypoleuca), inhabiting a Ca-poor and metal-polluted area in SW Finland. Both species were able to obtain sufficient Ca for eggshell formation, andmetal pollution was below the level of having negative effects in the egg size and eggshell characteristics.However, metal polluted environment negatively affected yolk lutein and vitamin D3 levels in bothspecies, probably because of a lower access to carotenoid-rich diet and higher metal interference withvitamin D3 metabolism. The higher levels of vitamin D3 in yolks in the unpolluted zone could also be dueto upregulated D3 levels as a response to the lower natural Ca availability. Yolk carotenoids and vitaminD3 were positively associated with nestling growth and size, supporting their importance for theappropriate chick development. The interspecific differences in yolk nutrient concentrations possiblyreflect the different growth rate of these species. Pied flycatchers are likely adapted to low Ca availabilitythrough an efficient vitamin D3 metabolism, but their Ca intake could be close to a deficient level.

© 2016 Elsevier Ltd. All rights reserved.

1. Introduction

Early life conditions affect physiology, morphology, health andbehavior in birds (Ahmed et al., 2014; Anisman et al., 1998; Desaiet al., 1995; Zimmer et al., 2013). Females may change prenatalenvironment by transferring varying amounts of essential re-sources into their eggs such as calcium (Ca), vitamins and carot-enoids, and thus influence offspring quality (see e.g. H~orak et al.,2002; Saino et al., 2002). The importance of Ca availability for

* Corresponding author.E-mail addresses: sieslu@utu.fi, silvia.espin@um.es (S. Espín), srruiz@utu.fi

(S. Ruiz), pasanv@utu.fi (P. S�anchez-Virosta), j-p.salminen@utu.fi (J.-P. Salminen),teeva@utu.fi (T. Eeva).

1 Note: Sandra Ruiz and Pablo S�anchez-Virosta have equal contribution to thearticle and appear in alphabetical order (both are considered second author).

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Chemosphere 151 (2016) 189e201

successful breeding in passerine birds has been widely reported(Graveland et al., 1994; Graveland and van der Wal, 1996; Reynolds,2001; Reynolds and Perrins, 2010). This macronutrient is requiredfor shell formation during egg laying since up to 98% of the drymass of eggshell consists of calcium carbonate (Reynolds et al.,2004). The eggshell provides protection and is a source of Ca forthe embryo skeletal growth and calcification (Reynolds et al., 2004;Reynolds and Perrins, 2010). Vitamin D3 (cholecalciferol) isrequired to effectively use Ca (NRC, 1994), and its active metabolite1,25-dihydroxyvitamin D3 (1,25OHD3) is essential for the transportof Ca from the eggshell to the embryo (Elaroussi et al., 1994;Narbaitz, 1987). Thus, both Ca and vitamin D3 levels in femalesand their transfer to the egg during oogenesis are of utmostimportance for an optimum eggshell quality and embryo devel-opment. Furthermore, carotenoids and vitamin A (retinol) areimportant dietary micronutrients transferred from maternal cir-culation and deposited in the egg yolk. Carotenoids function aspigments in feathers, precursors of vitamin A, antioxidants, theyare involved in cell differentiation and proliferation, and playvarious roles in the endocrine and immune systems (Eeva et al.,2008; Møller et al., 2000; Surai et al., 2001a,b). Vitamin A is alsoessential during early stages of embryogenesis for the initiation oforganogenesis (Zile, 2004, 2001). In eggs, carotenoids and vitaminA are involved in regulation of embryonic development by way oftheir antioxidant properties, crucial due to the fast oxidativemetabolism in growing embryos associated with oxidative stress(Ga�al et al., 1995; Hargitai et al., 2006; Surai, 2002).

The maternal investment into egg quality depends on the con-dition of the female at breeding and on the quality of the mate, butalso on the quality of the environment. In that sense, Ca availabilityand metal pollution are essential parameters to consider. Smallpasserine birds cannot store enough Ca in their body for successfulreproduction, so they consume Ca-rich foods such as snail shells inaddition to their normal food (Graveland, 1996; Graveland andDrent, 1997; M€and et al., 2000; Tilgar et al., 1999). Ca provision-ing experiments have shown that tits consumed more Ca duringthe egg-laying and nestling period, and less during incubation(Espín et al., 2016; Graveland and Drent, 1997), which supports thespecial Ca requirement for eggshell formation and skeletal growth(Reynolds et al., 2004; Starck, 1998). Therefore, Ca-poor areaswhere exchangeable Ca in the soil and snail abundance aredepressed, may pose a problem to their reproduction (see Reynoldset al., 2004). Evidence of Ca-limited reproduction in birds has beenobserved mainly as thin-shelled eggs, and reduced egg and clutchsize; thus, egg properties have been preferred as response variablesin Ca-provisioning studies (reviewed in Reynolds et al., 2004).Vitamin D deficiency may also cause decreased egg production andegg weight as well as eggshell thinning, and in growing birds it mayderive in hypocalcemia, which affects skeletal development(Macwhirter, 1994). Another parameter related to Ca limitation isthe eggshell pigmentation. Great tits (Parus major), as some othersmall passerines, lay white eggs speckled with brown protopor-phyrin pigment spots (eggshell maculation) (Kennedy and Vevers,1976; Wegmann et al., 2015). Different authors have evidencedthat the eggshell pigmentation increases with decreasing Caavailability and is related to eggshell thickness (Gosler et al., 2005;Higham and Gosler, 2006). Recent findings support the so-calledstructural-function hypothesis (Hargitai et al., 2013), which ar-gues that protoporphyrin pigments are used to compensate forlocalized shell-thinning, strengthening it when Ca is scarce (Gosleret al., 2005; Solomon, 1997).

On the other hand, metal-polluted environments represent anadditional challenge for passerines, since pollution may affect snailabundance (Eeva et al., 2010b). Along with this, Ca deficiency in thediet is known to increase the absorption and accumulation of

harmful metals, such as lead (Pb) and cadmium (Cd), in birds(Dauwe et al., 2006; Scheuhammer, 1996). Furthermore, thesemetals may alter the homeostasis and function of Ca (Pounds,1984;Suzuki et al., 1985) and interfere with normal vitamin D3 meta-bolism by blocking renal synthesis of 1,25OHD3 (Edelstein et al.,1984; Moon, 1994; Smith et al., 1981). Exposure to metals is alsoknown to cause oxidative stress (Koivula and Eeva, 2010; S�anchez-Virosta et al., 2015). Therefore, metals may interfere with levels ofantioxidants in birds inhabiting polluted areas, as has been foundfor vitamin A and carotenoids in birds naturally or experimentallyexposed to certain contaminants (Fernie et al., 2005; Ortiz-Santaliestra et al., 2015). Moreover, the secondary environmentaleffects of metal pollution on food quality are known to affect levelsof dietary nutrients such as carotenoids in plasma of birds (Eevaet al., 2009, 2005), although the effects on yolk components havebeen scarcely evaluated (Hargitai et al., 2016). Since the concen-trations of vitamins and carotenoids in the egg yolk depend on thematernal intake and transfer of these components (Bortolotti et al.,2003; Ga�al et al., 1995; Griminger, 1966; Hargitai et al., 2006;Karadas et al., 2005; Mattila et al., 2004; Stevens and Blair, 1985),metal exposure and Ca deficiency in laying females could interferein the yolk concentrations of these compounds, which couldcompromise hatching success and chick development.

The main aim of this study is to assess the effects of Ca avail-ability and anthropogenic metal exposure on early-stage repro-duction in two passerine species inhabiting a Ca-poor and metal-polluted area. For this purpose, we experimentally manipulatedthe availability of Ca and evaluated different parameters that couldbe sensitive to Ca deficiency and/or metal effects such as hatchingprobability, egg size and eggshell characteristics (eggshell indexand pigmentation pattern), and vitamin (D3 and A) and carotenoidlevels in yolk. Since bird species may differ in their Ca requirementand metal sensitivity (Eeva and Lehikoinen, 2010), differences be-tween great tits and pied flycatchers (Ficedula hypoleuca) are alsoevaluated. Furthermore, we will explore the effects of yolk nutri-ents (vitamins and carotenoid content) on hatching probability,nestling size and growth, and fledging success.

Since Ca is strongly involved in the first stage of breeding and isclosely related to vitamin D, we expect to find a negative effect ofmetal pollution on eggshell characteristics and yolk vitamin Dcontent, and a positive effect of Ca supplementation on eggshellparameters. Based on previous findings showing higher plasmacarotenoid (lutein) levels and plumage carotenoid chroma in greattit nestlings in the unpolluted area than in the polluted area (Eevaet al., 2009), we hypothesize that eggs of the polluted area willcontain less carotenoids and vitamin A. Finally, since some studieshave shown that yolk micronutrient levels may affect the nestlinggrowth and fledging success (e.g. Marri and Richner, 2014; McGrawet al., 2005), we expect to find an effect of yolk vitamins andcarotenoid content on these nestling traits. Although the impor-tance of evaluating Ca availability in conjunction with environ-mental pollution has been highlighted by different researchers(Graveland, 1995; Poulin and Brigham, 2001; Reynolds, 2001;Scheuhammer, 1991), to the best of our knowledge only twoexperimental studies have been done relating Ca deficiency andmetal pollution in wild passerine populations (Eeva, 1996; Espínet al., 2016).

2. Material and methods

2.1. Ca-provisioning experiment

The feeding experiment with Ca was conducted during thebreeding season 2014 in the surroundings of a copperenickel(CueNi) smelter in Harjavalta (61�200 N, 22�100 E), southwestern

S. Espín et al. / Chemosphere 151 (2016) 189e201190

Finland. Metals (mainly Cu; Ni; Pb; Cd; and arsenic, As) are com-mon contaminants in the vicinity of the smelter (hereafter calledpolluted zone) due to current and long-term deposition (Deromeand Lindroos, 1998; Eeva and Lehikoinen, 1996; Jussila andJormalainen, 1991). Details on metal contamination and Ca avail-ability in the study area are provided by Espín et al. (2016). Theexperiment was carried out on populations of great tits and piedflycatchers using nest boxes (see Lambrechts et al., 2010) situatedalong the pollution gradient, divided into the polluted (0e4 kmfrom the smelter) and the unpolluted zone (4.1e11 km from thesmelter). Nest boxes were emptied before the start of nest buildingand checked in mid-April, and then periodically to monitor thedevelopment of nest building and record the laying date, clutchsize, hatching date, brood size, and number of fledglings. When anew nest in an advanced building stage was found, it was firstidentified for its species and assigned by turns either to the Ca-supplemented or to the control group. In that moment, smallfeeders (plastic cylindrical cups) with 5 g of crushed mussel shells(Versele Laga) for Ca-supplemented nests or empty for controlnests were placed inside the nest boxes. Previous studies confirmedthat tits and pied flycatchers consume snail shell and egg shellfragments supplied in the feeders (Graveland, 1996; Gravelandet al., 1994; Graveland and Drent, 1997; M€and and Tilgar, 2003;Tilgar et al., 2002, 1999). The feeders were regularly checked andrefilled when needed (recording the amount added), so there wasalways a source of Ca material (ad libitum supplementation). Thecrushed shells remains were weighed and replaced at the begin-ning of the incubation period and just after hatching. At the age of12 days in pied flycatchers (hereafter d12) and 14 days in great tits(d14) the feeder was removed and the leftover Ca was weighed. Caconsumption during the laying, incubation and chick rearing pe-riods was calculated with these measurements and considering theextra amount added where appropriate. In total, 10 different siteswith nest boxes were used in this study (5 in the polluted and 5 inthe unpolluted area). We set 36 Ca-supplemented nests (17 inpolluted and 19 in unpolluted zone) and 47 control nests (22 inpolluted and 25 in unpolluted zone) of great tits. Regarding piedflycatchers, we had 42 Ca-supplemented nests (21 in each zone)and 39 control nests (21 in polluted and 18 in unpolluted zone).

2.2. Sampling and measurements

From each clutch we collected the 3rd egg (4th or 5th egg wascollected in few cases) on the day that it was laid. The eggs weremarked with a pencil every day in order to know the exact positionin the laying sequence. In this way, we standardized the samplingto avoid possible differences in egg characteristics and vitamins orcarotenoids content along the laying sequence. In total 83 great titeggs and 81 pied flycatcher eggs were collected. Eggs were trans-ported wrapped in cotton in small bottles to prevent breakage. Inthe day of collection, eggs were weighed and the length and widthwere measured using a digital caliper to the nearest 0.01 mm. Eggvolume was determined using the following equation (from Hoyt,1979): Egg volume (mm3) ¼ 0.51 � Egg length (mm) � Eggwidth2 (mm2). Eggshell pigmentation pattern in great tits wasscored in three categories according to Gosler et al. (2000): pigmentintensity (from 1 for palest spots to 5 for the darkest), distribution(from 1 for > 90% of spots concentrated at one end to 5 for spotsevenly distributed) and spot-size (from 1 for small spots to 3 forlarge spots). The collected eggs were cut into two halves with arazor blade, and yolk and albumen were weighed and split in twodifferent tubes and then stored at �80 �C. The eggshells werecarefully washed with milliQ water, dried to constant mass at roomtemperature and weighed to the nearest 0.1 mg. An eggshell indexwas calculated as: Eggshell mass (g)/(Egg length (mm) � Egg width

(mm)) according to Ratcliffe (1967). Eeva and Lehikoinen (1995)reported that the actual thickness of the egg shell explained 75%and 82% of the variation in the eggshell index of great tits and piedflycatchers, respectively; and according to Ratcliffe (1970) thisparameter can be used as a measure of eggshell thickness. Samplesizes vary for different egg parameters because we were unable tomeasure all components of some eggs.

On d7 after hatching, birds were ringed with individuallynumbered metal rings and combined feces of all nestlings from thesame brood were collected in tubes and conserved at �20 �C formetals analysis. Nestlings were weighed on d7 for both species andd12 (pied flycatcher) or d14 (great tit) post-hatching with a preci-sion of 0.1 g using a Pesola spring balance. Different dates for thesecond measurements were decided based upon pied flycatchersgrowing and fledging faster than great tits (own unpublished dataon wing growth; Cramp and Perrins, 1993). Wing length wasmeasured to the nearest 0.5mmusing a ruler, and tarsus length andtotal head (billþhead) length were measured with a digital caliperto the nearest 0.01 mm. Blood samples were collected for differentanalyses that will be the scope of other publications (for samplingdetails after hatching see Espín et al., 2016).

2.3. Metal analysis

Feces from the 7-day-old nestlings were dried over 72 h at 45 �C.Fecal samples from nestlings of the same brood were combined toassess metal exposure at brood level. Metal concentrations (Ca, As,Pb, Cd, Cu, and Ni) were determined with an inductively coupledplasma optical emission spectrometer (ICP-OES) in which thequantification limit is 1 ppm for Ca and 0.01 ppm for the othermetals (n¼ 67 and 63 for great tit and pied flycatcher, respectively).Total mercury (Hg) was analyzed in a Milestone DMA-80 direct Hganalyzer by atomic absorption spectrophotometry with a detectionlimit of 0.005 ng (n ¼ 65 and 59 for great tit and pied flycatcher,respectively). Details on the analytical process, precision and ac-curacy of the methods and certified reference materials used areprovided in Espín et al. (2016). Metal concentrations were referredto dry mass. The mean percentage for water content in feces was75.7 ± 7.6% in great tits and 79.5 ± 3.7% in pied flycatchers.

2.4. Carotenoid and vitamin analysis

Before vitamin and carotenoid analysis, yolk samples werefreeze-dried at�33 �C during 72 h (freeze-dryer Christ® Beta 2e16)and ground into a fine powder using a tissue lyser (Qiagen,Retsch®). A known amount of yolk powder (c.a. 20e30 mg) wasextracted three times with 100% acetone (600 ml of acetone and 1 hshaking each time). The solvent was evaporated using a vacuumcentrifuge concentrator for c.a. fifty minutes. The extract wasreconstituted with 100 ml of methanol, vortex-mixed for 5 min, andfiltered before analysis. The vitamin and carotenoid composition ofthe extracts was analyzed by ultra-performance liquid chroma-tography coupled with mass spectrometry (UPLC-MS/MS) on theAcquity UPLC system (Waters Corp., Milford, MA, USA), interfacedto a Xevo TQ triple-quadrupole mass spectrometer with electro-spray ionization (ESI) (Waters Corp., Milford, MA, USA). In brief, theUPLC system was equipped with an autosampler, a binary solventmanager, a 100 mm � 2.1 mm i.d., 1.7 mm, an Acquity UPLC BEHPhenyl column (Waters Corp., Wexford, Ireland), and a diode arraydetector. The flow rate was set to 0.5 mL/min, and the mobile phaseconsisted of two solvents: acetonitrile (A) and 0.1% aqueous formicacid (B) with the following gradient profile: 0e1.0 min, 90% A in B(isocratic); 1.0e2.0 min, 90e95% A in B (linear gradient);2.0e2.5 min, 95e98% A in B (linear gradient); 2.5e4.5 min, 98% A inB (isocratic); 4.5e7.0 min, column wash and stabilization. Injection

S. Espín et al. / Chemosphere 151 (2016) 189e201 191

volume was 5 ml. Data collection of both UV and MS occurredcontinuously from 0 to 4.5 min. Positive ESI mode was used, withESI conditions as follows: capillary voltage, 2.4 kV; desolvationtemperature, 550 �C; source temperature, 150 �C; desolvation andcone gas (N2),1000 and 100 L/h, respectively; and argon as collisiongas.

Carotenoids were detected by UPLC-DAD at 450 nm. We iden-tified lutein by co-elution with commercial standards. The otherfour carotenoids that had UV spectra of carotenoids were classifiedas unidentified carotenoids. Lutein and the unidentified caroten-oids were quantified using commercial lutein as a standard(Extrasynthese, France). Vitamins were detected by UPLC-MS/MS.We created multiple reaction monitoring (MRM) methods for themore specific detection of every vitamin. For instance, for vitamin Atype compounds we used the MRMs: 269.2 > 93 with 24 V conevoltage and 22 eV collision energy. Other methods were as follows:Vitamin D3: 385.3 > 367.3, 27 V/14 eV; 25-hydroxy-vitamin D3:383.3 > 109, 20 V/20 eV; 383.3 > 159, 20 V/20 eV; 383.3 > 365, 20 V/10 eV; 1,25-dihydroxy-vitamin D3: 399.30 > 135, 20 V/28 eV;399.30 > 226, 20 V/20 eV; 399.30 > 381, 20 V/12 eV; Vitamin E:431.50 > 165, 35 V/26 eV; Vitamin K2: 445.5 > 187.1, 27 V/23 eV;Vitamin K1: 451.5 > 187, 34 V/25 eV; Vitamin E acetate:473.50 > 165, 20 V/40 eV; 473.5 > 207, 20 V/20 eV. Regarding vi-tamins, retinol was identified and quantified separately as the peakin the samples corresponded to the peak of the commercial stan-dard. Since other peak corresponding to a retinol-like compoundwas detected in the yolk samples, total vitamin A (sum of retinoland retinol-like compound) concentrations are provided. Forvitamin D3, two derivatives of the main compound were detected,and total vitamin D3 concentrations are expressed as the sum ofthese two cholecalciferol-like compounds. Quantification ofvitamin A and D3 was done using retinol (CAS number 68-26-8,Product number 17772, SigmaeAldrich) and cholecalciferol (CASnumber 67-97-0, Product number C0314, Tokyo Chemical In-dustries) standards, respectively. Stock standard solutions (20 mg/ml for each analyte) were prepared by dissolving the appropriateamount of retinol and cholecalciferol in methanol, while luteinstandard was prepared in acetone. Standard dilutions were pre-pared daily. Vitamins K1, K2 and E showed very low signal intensityby MS/MS in the yolk samples and were thus not included in finalquantifications.

2.5. Statistical procedures

Statistical analyses were performed with SAS 9.4 and SPSS 22.0statistical packages. Generalized linear models (GLMs; Glimmixprocedure in SAS) were used to study the effects of the treatment(Ca-supplement/control), zone (polluted/unpolluted) and theirinteraction (selected as explanatory factors) on hatching probabil-ity, egg size and eggshell characteristics (eggshell index, spot in-tensity, spot distribution, spot size), and yolk vitamin (D3 and A)and carotenoid (lutein and unidentified carotenoids) levels in bothgreat tits and pied flycatchers. Normality of variables was checkedfrom the model residuals and some variables were normalizedusing natural log-transformation (i.e. lutein, unidentified caroten-oids, vitamin D3 and vitamin A concentrations). Binomial errordistribution (0 ¼ not hatched, 1 ¼ hatched) was used to model theprobability of hatching in a clutch.

GLMs were also done to explore the effects of metal and Caconcentrations in feces of nestlings (as a proxy of dietary levels) onhatching probability, egg size and eggshell characteristics, andvitamin and carotenoid levels. Although there can be some differ-ences in fecal metal concentrations between adults and nestlings,metal concentrations in nestling excrements are good indicators ofenvironmental metal contamination and metal availability in food

items (Berglund et al., 2011). Since metal concentrations in feces(As, Cd, Ni, Pb, Cu and Hg) were positively correlated(rp ¼ 0.38e0.87, p < 0.01 and rp ¼ 0.29e0.95, p < 0.03 in great titsand pied flycatchers, respectively; metals were log-transformedbefore analysis), we carried out Principal Components Analysis(PCA; Princomp procedure in SAS). The first principal component(PC1, eigenvalue 3.80 and 4.30 in great tits and pied flycatchers,respectively) explained 63% and 72% of the variation in our data ingreat tits and pied flycatchers, respectively, so it was used in themodels as an explanatory variable to describe the general level ofmetal exposure (Eigenvectors: As 0.41, Cd 0.41, Cu 0.48, Ni 0.44, Pb0.28 and Hg 0.40 in great tit and As 0.43, Cd 0.28, Cu 0.46, Ni 0.44,Pb 0.44 and Hg 0.37 in pied flycatchers). Therefore, the first prin-cipal component (PC1) frommetals, Ca in feces, and PC1*Ca in feceswere included as explanatory variables in the model, together withsome possibly confounding variables (laying date and clutch size).Explanatory variables and their interaction terms were retainedwhen statistically significant (p < 0.05). Non-significant variableswere dropped one by one from the model starting from in-teractions, and they were added in the final model one by one andkept if significant. Besides, GLMs with multinomial error distribu-tion were ran to evaluate the relationship between the eggshellindex (explanatory variable in the model) and the distribution,intensity and size of pigment spots (ordinary category dependentvariables) in eggshells of great tits.

Finally, GLMs were used to explore the effects of yolk nutrients(vitamins and carotenoid content) on hatching probability, nestlingsize and growth, and fledging success. Wing, tarsus, head, and bodymass growth rates were calculated for each individual from thehour of the first measurement on d7 to the hour of the last mea-surement on d12/14, and they are expressed as mm/day (wing,tarsus and head growth) or g/day (body mass growth). Body sizeparameters (wing, tarsus, head length and body mass on d7;rs ¼ 0.58e0.85, p < 0.001 and rs ¼ 0.82e0.92, p < 0.001 in great titsand pied flycatchers, respectively) and growth parameters (wing,tarsus, head and body mass growth rates; rs ¼ 0.24e0.64, p < 0.05and rs ¼ 0.27e0.69, p < 0.05 in great tits and pied flycatchers,respectively) of nestlings were positively correlated. Hence, wecalculated the principal components on them, using the PC1 in themodels. The PC1 (eigenvalue 3.38 and 3.64 in great tits and piedflycatchers, respectively) for the size parameters explained 85% and91% of the variation in our data in great tits and pied flycatchers,respectively (Eigenvectors: wing length d7 0.47, body mass d7 0.49,tarsus length d7 0.51 and head length d7 0.53 in great tit and winglength d7 0.49, body mass d7 0.50, tarsus length d7 0.51 and headlength d7 0.50 in pied flycatchers); and the PC1 (eigenvalue 2.61and 2.72 in great tits and pied flycatchers, respectively) for thegrowth parameters explained 65% and 68% of the variation (Ei-genvectors: wing growth 0.50, body mass growth 0.54, tarsusgrowth 0.44 and head growth 0.52 in great tit and wing growth0.47, body mass growth 0.47, tarsus growth 0.49 and head growth0.56 in pied flycatchers). Therefore, yolk nutrients (lutein, un-identified carotenoids, vitamin D3 or vitamin A), treatment, and theinteraction were included as explaining variables in the model,together with laying date and hatchling number. To avoid over-complicate models the effect of each nutrient was tested in aseparate model. All the above mentioned analyses were done at thebrood level, i.e. we used brood means of nestling measurements asindependent units. Pearson (rp) or Spearman's (rs) correlation co-efficient was used to study correlations among response variablesdepending on the normality of data (checked using Kolmogor-oveSmirnov test). Due to the well-known adverse effects of Pb andCd on Ca metabolism (Pounds, 1984; Suzuki et al., 1985), thesemetals were correlated separately with all the variables. The level ofsignificance was set at p < 0.05 in all analyses.

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3. Results

The means for breeding parameters, egg size and eggshellcharacteristics, and carotenoid and vitamin concentrations in yolksamples of great tits and pied flycatchers according to zone(polluted and unpolluted) and treatment (Ca-supplemented andcontrol) are presented in Supplementary Material (Table S1). Cor-relations among variables (Ca, Pb and Cd concentrations in feces,eggshell characteristics and carotenoid and vitamins levels) areshown in Table S2.

3.1. Effects of Ca availability and metal pollution on hatchingprobability, egg size and eggshell characteristics in great tits andpied flycatchers

In general, the Ca treatment and pollution level (zone) had noeffect on hatching probability, egg volume and eggshell character-istics (Table 1). In pied flycatchers, the eggshell index was 2.3%higher in Ca-supplemented nests (0.393 ± 0.019 g/mm2) than incontrol nests (0.384 ± 0.022 g/mm2), but the effect was not sta-tistically significant (Table 1 and Table S1). The Ca consumption offemale pied flycatchers during laying (divided by clutch size) waspositively correlated with the eggshell index (rs ¼ 0.23, p ¼ 0.042,n ¼ 81). The general metal level (PC1) did not explain the eggshellcharacteristics, except for an effect on spot distribution (Table 2,Fig. 1) showing that a higher metal exposure associates with moreevenly distributed spots. Similarly, Pb concentrations were posi-tively correlated with the spot distribution (rs ¼ 0.33, p ¼ 0.006,n ¼ 67; Table S2). In addition, Cd but not Pb concentrations in feceswere negatively correlated with eggshell index (rp ¼ �0.34,p ¼ 0.01, n ¼ 58) in pied flycatchers (Table S2).

The eggshell index had an effect on spot intensity (F ¼ 4.781,78,p ¼ 0.03), showing that eggshells with a lower index were lessintensively colored (Fig. 1). The spot intensity was positivelycorrelated with the spot size (rs ¼ 0.36, p¼ 0.001, n¼ 83; Table S2).

3.2. Effects of Ca availability and metal pollution on carotenoidsand fat-soluble vitamins in yolk samples of great tits and piedflycatchers

Regarding the effects of the Ca treatment on the yolk nutrientscontent, total vitamin A concentrations in yolk of great tits weresignificantly lower in Ca-supplemented nests than in controls

(Table 1 and Table S1, Fig. 2). In pied flycatchers, vitamin D3 levelstended to be higher in yolks from Ca-supplemented nests whencomparing with control nests, the effect of the treatment beingalmost significant (p ¼ 0.054, Table 1 and Table S1, Fig. 2). Inaddition, levels of unidentified carotenoids in yolks from Ca-treatednests of pied flycatchers were significantly higher than levels foundin control yolks in the unpolluted zone, although no effect oftreatment was found in the polluted zone (Table 1, Fig. 2). A similarbut non-significant trend was observed for yolk lutein levels in Ca-supplemented nests of pied flycatchers in the unpolluted zone(Table 1, Fig. 2). A negative association between Ca levels in fecesand vitamin D3 concentrations was found in great tits (Table 2 andTable S2). In pied flycatchers, this relationship was not significantthough the two variables showed marginally significant negativecorrelation, also found for Ca in feces and lutein concentrations inyolk (p ¼ 0.055 in both cases, Table S2).

In regard to the effect of metal pollution on the yolk micro-nutrient content, lutein and vitamin D3 concentrations in yolksfrom the polluted zone were lower (16 and 22% lower in great titsand 25 and 26% lower in pied flycatchers, respectively) than thosefrom the unpolluted zone in both species (Table 1 and Table S1,Fig. 2). Accordingly, fecal metal concentrations (PC1) had a negativeassociation with yolk lutein levels in pied flycatchers and yolkvitamin D3 concentrations in great tits, together with laying dateand clutch size (Table 2). Pb concentrations in feces were negativelycorrelated with yolk lutein levels in both species, and with carot-enoid and vitamin D3 concentrations in yolks of great tits(Table S2).

Pied flycatcher yolks showed significantly higher lutein(F ¼ 100.68, p < 0.001, n ¼ 163), unidentified carotenoid(F ¼ 224.53, p < 0.001, n ¼ 163), vitamin D3 (F ¼ 360.98, p < 0.001,n ¼ 163) and vitamin A (F ¼ 30.75, p < 0.001, n ¼ 163) concen-trations than great tit yolks (Fig. 2, Table S1). Significant positivecorrelations were found between lutein and unidentified caroten-oids for both species (rp ¼ 0.93 and 0.86, p ¼ <0.001, n ¼ 83 and 80in great tits and pied flycatchers, respectively), vitamin D3 andlutein (rp ¼ 0.41, p ¼ <0.001, n ¼ 83) or carotenoids (rp ¼ 0.33,p ¼ 0.002, n ¼ 83) in great tits, and vitamin D3 and vitamin A inpied flycatchers (rp ¼ 0.68, p ¼ <0.001, n ¼ 80) (Table S2).

3.3. Effect of yolk micronutrient levels on nestling size and growth

No significant effects of yolk carotenoid and vitamin levels on

Table 1Generalized linear models for the effects of zone (polluted vs. unpolluted) and Ca treatment on hatching probability, eggshell characteristics, carotenoid and vitamin con-centrations in yolk samples of great tits and pied flycatchers in Harjavalta, Finland. Means and sample sizes are given in the Supplementary material Table S1.

Great tit Pied flycatcher

Zone Treatment Zone � treatment Zone Treatment Zone � treatment

Dependent variable N Fndf,ddf p Fndf,ddf p Fndf,ddf p N Fndf,ddf p Fndf,ddf p Fndf,ddf p

Hatching probabilitya 83 0.021,80 0.89 0.601,81 0.44 0.201,79 0.65 81 1.951,79 0.17 0.031,78 0.87 0.811,77 0.37Egg volume (mm3)b 83 0.381,81 0.54 0.061,80 0.81 0.221,79 0.64 77 0.541,75 0.47 0.121,74 0.73 0.191,73 0.67Eggshell index (g/mm2)b 83 0.051,81 0.83 0.011,80 0.93 0.751,79 0.39 76 0.271,73 0.61 3.571,74 0.063 0.171,72 0.68Spot intensityc 83 0.071,78 0.79 0.001,77 0.95 0.571,76 0.45 e e e e e e e

Spot distributionc 83 0.351,78 0.56 0.031,77 0.87 1.741,76 0.19 e e e e e e e

Spot sizec 83 1.151,80 0.29 0.551,79 0.46 0.141,78 0.71 e e e e e e e

Lutein (mg/g yolk)b,d 83 5.321,81 0.024 0.031,80 0.87 0.141,79 0.71 80 13.261,78 0.0005 3.371,77 0.070 1.931,76 0.17P

Unidentified carotenoids(mg/g yolk)b,d

83 2.431,81 0.12 0.011,80 0.93 0.001,79 0.98 80 3.101,76 0.082 4.671,76 0.034 4.711,76 0.033

PVitamin D3 (mg/g yolk)b,d 83 27.071,81 <0.0001 0.231,80 0.64 0.681,79 0.41 80 5.271,78 0.024 3.841,77 0.054 0.061,76 0.81

PVitamin A (mg/g yolk)b,d 83 0.431,80 0.51 6.201,81 0.015 0.181,79 0.67 80 0.421,77 0.52 1.641,78 0.20 0.401,76 0.53

Note: Terms left in the final model are shown in bold.a GLM with binomial error distribution.b GLM with normal error distribution.c GLM with multinomial distribution.d The variables were log-transformed before analysis.

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Table 2Generalized linear models for variation in hatching probability, egg shell characteristics, and micronutrient concentrations in yolk samples of great tits and pied flycatchers in Harjavalta, Finland.

Response variable Ne Metals (PC1) Ca in feces Metals (PC1) � Ca in feces Laying date Clutch size

Fndf,ddf p Estimate ± SE Fndf,ddf p Estimate ± SE Fndf,ddf p Estimate ± SE Fndf,ddf p Estimate ± SE Fndf,ddf p Estimate ± SE

Great titHatching probabilitya 83 0.601,62 0.44 0.035 ± 0.045 0.371,60 0.55 �0.072 ± 0.12 0.061,59 0.81 �0.019 ± 0.078 0.201,81 0.66 �0.011 ± 0.025 0.761,61 0.39 0.059 ± 0.068Egg volume (mm3)b 83 0.311,60 0.58 �4.15 ± 7.40 0.581,64 0.45 �15.47 ± 20.39 0.101,59 0.75 3.91 ± 12.22 0.081,63 0.78 0.67 ± 2.37 1.371,81 0.25 �9.32 ± 7.96Eggshell index (g/mm2)b 83 0.341,62 0.56 0.00095 ± 0.0016 1.121,65 0.29 0.0044 ± 0.0042 2.951,61 0.091 �0.0044 ± 0.0026 0.311,59 0.58 �0.00029 ± 0.00052 1.081,60 0.30 �0.0025 ± 0.0024Spot intensityc 83 1.051,60 0.31 0.12 ± 0.12 0.531,58 0.47 �0.22 ± 0.31 0.211,57 0.65 0.087 ± 0.19 3.521,78 0.064 0.060 ± 0.032 1.171,59 0.28 0.19 ± 0.17Spot distributionc 83 3.991,60 0.050 ¡0.25 ± 0.12 0.441,59 0.51 �0.21 ± 0.32 0.801,58 0.38 0.16 ± 0.18 0.281,57 0.60 �0.020 ± 0.037 0.071,56 0.80 0.045 ± 0.17Spot sizec 83 0.201,62 0.65 0.054 ± 0.12 0.071,61 0.80 �0.086 ± 0.33 1.381,60 0.24 0.23 ± 0.20 4.781,80 0.032 ¡0.077 ± 0.035 0.161,58 0.69 0.073 ± 0.18Lutein (mg/g yolk)b,d 83 0.811,61 0.37 �0.022 ± 0.025 1.161,64 0.28 �0.068 ± 0.063 0.001,59 0.97 0.0016 ± 0.041 0.311,60 0.58 0.0042 ± 0.0076 0.011,81 0.93 0.0023 ± 0.025P

Unidentified carotenoids(mg/g yolk)b,d

83 0.241,61 0.63 �0.0089 ± 0.018 0.711,64 0.40 �0.039 ± 0.047 0.001,59 0.95 �0.0020 ± 0.030 0.021,60 0.88 0.00086 ± 0.0056 0.121,81 0.73 �0.0065 ± 0.019

PVitamin D3 (mg/g yolk)b,d 83 4.921,60 0.030 ¡0.034 ± 0.015 7.651,60 0.0075 ¡0.11 ± 0.040 0.281,59 0.60 �0.013 ± 0.025 10.461,60 0.0020 0.015 ± 0.0046 4.371,60 0.041 0.047 ± 0.022

PVitamin A (mg/g yolk)b,d 83 1.781,62 0.19 0.035 ± 0.026 0.241,60 0.63 �0.034 ± 0.069 0.171,59 0.68 �0.018 ± 0.043 5.361,81 0.023 ¡0.017 ± 0.0072 2.751,61 0.10 0.063 ± 0.038

Pied flycatcherHatching probabilitya 81 1.971,54 0.17 �0.050 ± 0.036 1.271,59 0.26 0.17 ± 0.15 1.601,53 0.21 0.10 ± 0.082 2.081,78 0.15 0.072 ± 0.050 1.761,79 0.19 0.21 ± 0.16Egg volume (mm3)b 77 0.511,52 0.48 5.40 ± 7.53 1.241,57 0.27 �32.09 ± 28.77 1.381,51 0.25 18.69 ± 15.90 1.531,50 0.22 �5.92 ± 4.79 0.011,49 0.92 �1.83 ± 18.30Eggshell index (g/mm2)b 76 0.151,51 0.70 �0.00051 ± 0.0013 0.241,56 0.63 �0.0024 ± 0.0050 2.121,50 0.15 �0.0041 ± 0.0028 0.061,48 0.81 0.00021 ± 0.00087 0.531,49 0.47 0.0022 ± 0.0030Lutein (mg/g yolk)b,d 80 16.431,55 0.0002 ¡0.080 ± 0.020 1.751,54 0.19 �0.11 ± 0.084 1.401,53 0.24 0.051 ± 0.043 12.271,55 0.0009 0.045 ± 0.013 0.301,52 0.59 0.026 ± 0.048P

Unidentified carotenoids(mg/g yolk)b,d

80 0.071,54 0.79 0.0045 ± 0.017 3.031,59 0.087 �0.12 ± 0.067 2.881,53 0.096 0.060 ± 0.036 20.821,78 <0.0001 0.038 ± 0.0083 3.391,52 0.071 0.071 ± 0.039

PVitamin D3 (mg/g yolk)b,d 80 1.361,56 0.25 �0.046 ± 0.040 0.041,53 0.85 �0.034 ± 0.18 0.041,52 0.85 �0.018 ± 0.093 0.611,54 0.44 0.022 ± 0.028 0.391,55 0.54 0.058 ± 0.093

PVitamin A (mg/g yolk)b,d 80 0.061,55 0.81 �0.0080 ± 0.033 0.001,60 0.95 0.0088 ± 0.13 0.811,54 0.37 �0.064 ± 0.072 0.081,52 0.77 0.0065 ± 0.022 0.051,53 0.83 0.017 ± 0.077

Note:Terms left in the final model are shown in bold.a GLM with binomial error distribution.b GLM with normal error distribution.c GLM with multinomial distribution.d The variables were log-transformed before analysis.e Because of missing values the sample size varies according to the model.

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fledging success were found for any species. The interaction yolklutein*treatment had a significant effect on growth (PC1) in greattits (Fndf,ddf ¼ 4.821,57, p ¼ 0.032, Estimate ± SE ¼ 1.98 ± 0.90)together with laying date (p < 0.001) and hatchling number(p ¼ 0.006, Fig. 3a). Yolk lutein content and growth rate (PC1) werepositively correlated in Ca-supplemented nests (rP ¼ 0.50,p ¼ 0.009, n ¼ 26), but not in control nests (rP ¼ 0.064, p ¼ 0.71,n ¼ 37). Unidentified carotenoid concentration in yolk had a sig-nificant positive association with growth rate (PC1) in great tits(Fndf,ddf ¼ 6.771,59, p ¼ 0.012, Estimate ± SE ¼ 1.69 ± 0.65; Fig. 3b)together with laying date (p < 0.001) and hatchling number(p ¼ 0.019), and almost significantly in pied flycatchers(Fndf,ddf ¼ 3.861,51, p ¼ 0.055, Estimate ± SE ¼ 1.46 ± 0.75).

The vitamin D3 concentrations in yolk showed a significantpositive effect on size at day 7 (PC1) in great tits (Fndf,ddf ¼ 12.341,65,p < 0.001, Estimate ± SE ¼ 2.72 ± 0.77). Finally, the effect of yolkvitamin A level on growth (PC1) in pied flycatchers depended onthe treatment (Fndf,ddf ¼ 5.031,49, p ¼ 0.030,Estimate ± SE ¼ 2.11 ± 0.94), being negative in control nests(rP ¼ �0.46, p ¼ 0.016, n ¼ 27), but not in the Ca supplementednests (rP ¼ 0.15, p ¼ 0.46, n ¼ 26; Fig. 3c).

4. Discussion

4.1. Effects of Ca availability and metal pollution on hatchingprobability, egg size and eggshell characteristics in great tits andpied flycatchers

In general, providing females with supplemental Ca had nosignificant effects on the hatching probability, egg size and eggshellparameters examined, suggesting that egg laying was not con-strained by insufficient Ca availability in great tits and pied fly-catchers (Table 1). Together with a previous study based on this Ca-supplementation experiment (see Espín et al., 2016), the resultssuggest that control females were able to obtain sufficient naturalor anthropogenic sources of Ca for eggshell formation. In pied fly-catchers, the eggshell index was slightly higher in Ca-supplemented nests than in control nests, although the effect wasnot significant (Table 1 and Table S1). However, since we observedlow supplemented Ca consumption in pied flycatchers, especially inthe polluted zone (Espín et al., 2016), we studied the correlationsbetween the amount of Ca consumed by the female during thelaying period and the egg characteristics, and we found a positivecorrelation with the eggshell index. Although we cannot state thatCa availability is constraining the egg laying in this species, theycould take an advantage of the Ca provisioning producing slightlythicker eggs (Reynolds et al., 2004). Thus, Ca intake could be close

to a level that may be deficient to keep a good eggshell quality inthis species.

Hatching probability, egg size and eggshell characteristics werenot affected by pollution zone (Table 1). Different researchers havefound that Cd exposure may decrease eggshell thickness in birds(Kor�enekova et al., 2007; Rahman et al., 2007; Skalick�a et al., 2008),and a negative correlation between Cd concentrations in feces (aproxy for dietary exposure in a territory) and the eggshell indexwas also observed in our study for the pied flycatcher (Table S2).However, general metal exposure seems to be below the level ofproducing negative effects on the eggshell parameters evaluated.As suggested before, pied flycatchers seem to be adapted to low Caavailability and can successfully breed in a polluted environmentwhen metal concentrations are not too high (Espín et al., 2016).However, there may be a delicate balance between Ca levels in theorganism and the tolerance to metals that should be consideredwhen evaluating metal exposure and effects in birds.

The eggshell strength is mainly affected by the shell thickness,closely related to Ca availability. Some authors suggest that onepossible function of protoporphyrin-spotting in speckled eggs is tostrengthen the shell (Gosler et al., 2005; Solomon, 1997), in such away that protoporphyrin acts as a cushion between the calcitecrystals making the shell more resistant (Solomon, 1997). In thepresent study we failed to find an effect of Ca supplementation orzone in eggshell maculation in great tits, nor did we find any effecton eggshell index (Table 1). The lack of treatment effect on spottingpatterns reinforces the idea that female great tits obtain sufficientCa sources for eggshell formation. Likewise, other Ca-provisioningstudies also failed to find an effect of treatment on the pigmentdarkness in blue tits (Cyanistes caeruleus) (García-Navas et al., 2010)and shell pigmentation pattern in great tits in one study year(Hargitai et al., 2013). Contrary to the structural-function hypoth-esis, we found that eggs with increasing eggshell index tend to havedarker spots (Fig. 1). A similar result was found in blue tits by Sanzand García-Navas (2009), and Ca-supplemented female great titslaid eggs with darker spots in other studies, although eggshellthickness was not increased by Ca-supplementation (Hargitai et al.,2013; M€agi et al., 2011). In addition, females exposed to highermetal levels tended to produce eggs with a more homogeneousdistribution of pigment spots over the eggshell surface (Fig. 1), andparticularly Pb showed a positive relationship with the spot dis-tribution (Table S2). This result seems unexpected since metalssuch as Pb and Cd may affect negatively the Ca metabolism andeggshell thickness (Lundholm and Mathson, 1986; Pounds, 1984),and thinner shells are associated with concentrated spots (Hargitaiet al., 2013; Sanz and García-Navas, 2009) while Ca-supplementedfemales laid eggs with a more homogeneous distribution (García-

Fig. 1. Relationship between metals in feces (a proxy of metal intake) and spot distribution (from 1 for > 90% of spots concentrated at one end to 5 for spots evenly distributed), andeffect of eggshell index on spot intensity (from 1 for palest spots to 5 for the darkest) in great tits. PC1 from metals was used to describe the general level of metal exposure. Eggshellindex ¼ Eggshell mass (g)/(Egg length (mm) � Egg width (mm)).

S. Espín et al. / Chemosphere 151 (2016) 189e201 195

Fig. 2. Mean (±95% CIs) carotenoid and vitamin concentrations (mg/g, dry mass) in yolk samples of great tits and pied flycatchers according to zone (polluted and unpolluted) andtreatment (Ca-supplemented and control) in Harjavalta, Finland. The data are predicted and back-transformed values from the GLM models shown in Table 1. Effects of zone andtreatment on the different parameters are shown in Table 1. *Significant differences between zones. **Significant effect of the interaction zone*treatment. Letters in bars denotesignificant differences among treatments (means with different letter are statistically different).

Navas et al., 2010). Other authors have found that eggs with a moreaggregated spotting pattern had higher Cu level in the eggshell(Hargitai et al., 2016). However, in our study metal levels were nothigh enough to cause shell thinning. This positive association couldbe related to Pb effects on the heme synthesis pathway (Flora et al.,2012). This metal may downregulate the enzyme ferrochelatasethat catalyzes the insertion of iron into protoporphyrin to formheme, leading to anemia (Flora et al., 2012; Kelada et al., 2001) andaccumulation of erytrhrocyte protoporphyrin (Flora et al., 2012;Jangid et al., 2012). Then, a Pb-related accumulation of protopor-phyrin in blood and anemia could interfere in the eggshellpigmentation. In this regard, De Coster et al. (2012) found thatanemia in female great tits may affect deposition of protoporphyrinon eggshells.

4.2. Effects of Ca availability and metal pollution on carotenoidsand fat-soluble vitamins in yolk samples of great tits and piedflycatchers

The natural mechanisms of female carotenoid and othermicronutrient allocation to the egg yolk and the potential con-straints are not clearly understood. Whether micronutrient levelssimply reflect maternal intake in the diet or an active regulationmechanism exists is still a matter of study (Bortolotti et al., 2003;Isaksson et al., 2008; Koutsos et al., 2003; Surai et al., 2001a;T€or€ok et al., 2006). The present study shows that lutein andvitamin D3 concentrations in yolks were lower in the polluted thanin the unpolluted zone for both species (Table 1, Fig. 2). Probablyboth indirect and direct effects of metal pollution are altering thelevels of these micronutrients. Previous studies in the same areahave found that metal pollution affects food quality (Eeva et al.,2005) and consequently levels of carotenoids in plasma of birds(Eeva et al., 2009). Therefore, females inhabiting the unpollutedzone probably have better access to lutein-rich food sources, ascompared to the polluted zone, and can invest more of these nu-trients to the yolk, maintaining or restoring their circulating levels.

On the other hand, birds breeding in the polluted zone areexposed to higher metal pollution than their conspecifics in theunpolluted zone (see Espín et al., 2016). Metals (particularly Pb andCd) interfere with normal vitamin D3 metabolism (Edelstein et al.,1984;Moon,1994; Smith et al., 1981). In this sense, PC1 frommetalshad a negative effect on vitamin D3 levels in yolks of great tits(Table 2), and a negative correlation was found between fecal Pbconcentrations in nestlings and yolk vitamin D3 (Table S2). Anotherpossibility that should be mentioned is related to the induction ofoxidative stress by some metals (Pb, Cd, As) (Koivula and Eeva,2010; S�anchez-Virosta et al., 2015). Carotenoids have beenconsidered as part of the antioxidant barriers protecting againstoxidative damage (Møller et al., 2000), though their role as anti-oxidants in birds is not clear (Isaksson and Andersson, 2008). Fe-males in the polluted zone may lay eggs with lower antioxidantlevels due to maternal depletion for the scavenging of free radicals.Lower carotenoid concentrations in yolks of great tits from oxida-tively stressed urban areas compared with rural areas have beenpreviously described (Hargitai et al., 2016; H~orak et al., 2002). Thenegative effect of PC1 frommetals on yolk lutein in pied flycatchers,and the negative correlations between fecal Pb concentrations andyolk lutein in both species (Table 2 and Table S2), could reflect thenecessity of organisms to incorporate these dietary antioxidants tobalance the increased oxidative stress caused by metals. However,lutein seems to have a weak role as antioxidant in birds (Isakssonand Andersson, 2008 and references therein), and previousstudies did not findmuch support for this hypothesis in the studiedarea (see e.g. Eeva et al., 2009; Koivula et al., 2011); thus a lowerlutein concentration due to the poorer food quality in the polluted

zone is the most likely explanation.Supplemental Ca resulted in an increase of unidentified carot-

enoid levels in yolks of pied flycatchers in the unpolluted zone, anda similar trend was observed for lutein (Table 1, Fig. 2). Acquiringsufficient Ca is time-consuming (Graveland and Berends, 1997).Precisely birds in the unpolluted zone tended to consume more ofthe Ca supplements, probably because of the lower Ca availabilityfrom natural and anthropogenic sources in this area (Espín et al.,2016). Thus, supplemental Ca could allow the female to increasethe time for searching for carotenoid-rich food such as lepidop-terans (Eeva et al., 2010a), which might incur in a higher intake andyolk supply of these nutrients.

The lack of differences in yolk vitamin A concentrations betweenzones (Table 1, Fig. 2) could indicate that it is not a limited micro-nutrient to laying females, and probably other molecules are able to

Fig. 3. Effect of lutein (a), unidentified carotenoids (b) and vitamin A (c) in yolk ongrowth (PC1) in great tit (a, b) and pied flycatcher (c) nestlings. The data points (±95%CIs) are predicted and back-transformed values from the GLM models.

S. Espín et al. / Chemosphere 151 (2016) 189e201 197

cope with metal-induced oxidative stress, playing a more impor-tant role in the antioxidant defense than vitamin A. The low con-centrations of this vitamin in yolk samples, as has been proposed byother researchers (Hargitai et al., 2006), is probably due to its pro-oxidant activity and negative effects when the deposition into theyolk is high (Surai et al., 1998b). The significantly higher vitamin Acontent in yolk of pied flycatchers compared with great tits couldbe in part due to the different diet. For example, pied flycatcher hasa higher proportion of Coleoptera in its diet (Eeva et al., 2010a), andthis group may have higher vitamin A content that for examplelepidopterans (Alamu et al., 2013). Moreover, a higher allocation ofthis vitamin due to a higher necessity for embryo development inthis species is also possible.

The lower vitamin D3 concentrations in yolks in the pollutedzone comparedwith the unpolluted zonemay be also related to theCa availability. Vitamin D and its metabolites are the most impor-tant factors regulating intestinal Ca absorption (Bar, 2009). VitaminD3 is absorbed by the intestinal mucosa from food or formed by UVirradiation, and is hydroxylated to 25-hydroxyvitamin D3(25OHD3) in the liver, which is the major circulating form ofvitamin D in the blood stream (Aburto et al., 1998; Rucker et al.,2007; Tian et al., 1994). During eggshell formation, Ca is rapidlymobilized and plasma concentrations rapidly decrease (Peliciaet al., 2009). When there is a lack of Ca, the secretion of the para-thyroid hormone (PTH) is stimulated which in turn stimulates thetransfer and hydroxylation of 25OHD3 in the kidney, originatingthe most active hormonal form 1,25OHD3. This compound willincrease Ca absorption in the intestine, mobilize Ca from the bones,and reduce Ca excretion via the kidney (Bar, 2008; Pelicia et al.,2009). As it was found in the previous study based on this Ca-supplementation experiment (Espín et al., 2016), the Ca supple-ment consumption in both species tended to be higher in the un-polluted zone, likely due to lower availability of Ca sources in theseremote sites. Since maintaining the level of circulating Ca is criticalfor the laying bird, it is likely that the females respond with anenhanced basal metabolism of vitamin D3 to increase intestinal Caabsorption (and reduce its excretion) when faced with low Caavailability, which may result in higher levels of vitamin D3 me-tabolites in yolks in the unpolluted zone. Accordingly, a negativeeffect of Ca levels in feces, indicative of dietary Ca availability, onvitamin D3 concentrations was found in great tits (Table 2 and S2),and a negative correlation between fecal Ca and yolk vitamin D3concentrations was almost significant in pied flycatchers (Table S2).

Vitamin D3 concentrations were much higher in pied flycatcherthan in great tit yolks (4e5 times) (Table S1, Fig. 2). Espín et al.(2016) observed that great tits had significantly higher Ca con-centrations in feces and plasma than pied flycatcher nestlings,suggesting that they need and sustain higher Ca levels and seem tobe more opportunistic than pied flycatchers, consuming more ofthe supplemented Ca, probably saving energy and time. Fecal Calevels in pied flycatcher nestlings were lower in the unpollutedthan in the polluted zone, indicative of the lower Ca availability(Espín et al., 2016) but likely also of the lower Ca renal excretionregulated by 1,25OHD3. Pied flycatchers seem to be adapted to lowCa availability since they can successfully breed at low Ca envi-ronment, showing no significant adverse effect on hatching prob-ability or eggshell characteristics at current metal exposure levels(see previous section). Vitamin D3 may play a key role in thisadaptation. Pied flycatchers may accelerate the vitamin D3 meta-bolism more efficiently than great tits during egg laying, whichcould stimulate Ca absorption in the intestine and reduce Caexcretion. More efficient absorption would also explain why thisspecies consumed less Ca supplements. In addition, they may bemore efficient in vitamin D3 allocation to the egg for an effectiveuse of Ca by the developing embryo. A higher yolk vitamin D3

content may have direct beneficial effects on the embryo but alsomay determine the ability of the nestling to metabolize and effi-ciently use this vitamin later in life, as has been found for othernutrients (Koutsos et al., 2003). Finally, yolks from Ca-supplemented nests showed almost significantly higher vitaminD3 levels than yolks from control nests for this species (Table 1 andTable S1, Fig. 2). It is possible that the easy Ca availability leavesmore time to search other nutritiously important food items, andfemales could maintain Ca levels balanced for their own needs,transferring more vitamin D3 metabolites to their eggs.

4.3. Effect of yolk micronutrient levels on nestling size and growth

It has been proposed that micronutrient levels in yolks mayaffect not only the embryo development, but also the nestlinggrowth and fledging success (Biard et al., 2005; Marri and Richner,2014; McGraw et al., 2005). Although we did not find effects onfledging success, vitamin D3 concentrations in yolk showed a sig-nificant positive effect on size of the nestlings at d7 (PC1) in greattits. This is likely related to the essential function of vitamin D3metabolites in Ca homeostasis and bone mineralization (Elaroussiet al., 1994; Macwhirter, 1994; Narbaitz, 1987). Accordingly,maternal effects by way of yolk Ca on the tarsus length of 8-day-oldgreat tit nestlings were found in a Ca provisioning experiment(Tilgar et al., 2005).

The negative effect of yolk vitamin A on growth found in controlnests, disappeared when female pied flycatchers were Ca-supplemented (Fig. 3c). As stated above, females could benefit ofthe extra Ca for self-maintenance and transfer more vitamin D3(and probably more Ca due to the positive correlations found be-tween female Ca consumption and eggshell index and mass) intoeggs, which is reflected in higher yolk vitamin D3 in Ca-supplemented nests (Table 1, Fig. 2). Vitamin A may interact withvitamin D3 metabolism (Johansson and Melhus, 2001; Rohde et al.,1999), which may affect the Ca mobilization from the eggshell tothe embryo, the Cametabolism in the chick, and in turn the nestlinggrowth in control nests. With a higher availability of Ca and vitaminD3 in Ca-supplemented nests, this effect may be attenuated.Balancing the levels of these three closely-related nutrients seemsessential for a correct embryo and chick development.

Yolk unidentified carotenoid content had a positive effect ongrowth (PC1) in great tits (Fig. 3b), almost significant for pied fly-catchers; and yolk lutein also showed this positive effect whengreat tits were Ca-supplemented (Fig. 3a). These effects may berelated to the role of carotenoids in reducing the oxidative stressassociated to the high metabolic rate during growth, together withits role in cell differentiation and proliferation (Surai et al., 2001b;Surai, 2002). Experimental studies providing carotenoids to pas-serines have found that supplemented females incorporated morecarotenoids into eggs, nestlings had higher plasma and featherlutein concentrations compared to controls, and carotenoid sup-plementation showed positive effects on nestling size, immunesystem development, and hatching and fledging success (Biardet al., 2005; Eeva et al., 2008, 2009; Marri and Richner, 2014;McGraw et al., 2005). It is possible that the quality of prenatalenvironment (higher carotenoid content in yolk) can have directbeneficial effects on embryos but also may determine the ability ofthe nestling to absorb, metabolize and efficiently utilize dietarynutrients later in life (Koutsos et al., 2003).

Pied flycatcher yolks showed 45% higher lutein and unidentifiedcarotenoid levels and 78% higher vitamin D3 concentrations thangreat tit yolks (Fig. 2, Table S1). Females from different species maydiffer in the nutrient investment into egg yolks at the time of laying(Blount, 2004; Hargitai et al., 2006) due to different embryo re-quirements. For example, the growth rates, and hence potentially

S. Espín et al. / Chemosphere 151 (2016) 189e201198

antioxidant requirements, may vary markedly between bird spe-cies. Deeming and Pike (2013) confirmed that antioxidants shouldbe allocated according to the rate of embryonic growth, such thateggs containing embryos that grow faster have higher antioxidantlevels. Although both species have a similar initial egg mass(around 1.7 g, see Table S1), pied flycatchers grow faster than greattits, so they are expected to have higher levels of antioxidant pro-vision. How female pied flycatchers allocate higher carotenoidlevels into yolks compared with great tits is another issue. Piedflycatchers could be more efficient than great tits in the absorption,metabolic transformation and/or transport of nutrients to the egg,as has been observed in other species (Surai et al., 1998a). Alter-natively, there may be a trade-off between time spent searching forCa and for other nutrients. Finally, the species-specific diet (Eevaet al., 2012, 2010a), the variability of natural caterpillar availabil-ity among years, and the fact that pied flycatchers breed about 20days later than great tits and caterpillar availability may vary be-tween the nestling periods of the two species (Eeva et al., 2005),should be considered.

5. Conclusions

In conclusion, metal polluted environment affected maternallutein and vitamin D3 allocation to egg yolk in both species. It islikely that females in the polluted zone have lower access tocarotenoid-rich food items and they may suffer from higher metalinterference with vitamin D3 metabolism, allocating fewer nutri-ents to yolks than their conspecifics in the unpolluted zone. Thehigher levels of vitamin D3 in yolks in the unpolluted zone could berelated to the lower Ca availability in these remote sites. We foundthat nestling growth and size are influenced by carotenoids andvitamin D3, so these micronutrients could limit the appropriatechick development, especially in the polluted zone for great tits,where nestling traits are affected by the lower diet quality (Espínet al., 2016).

Great tits and pied flycatchers were not constrained by Caavailability, but they may employ different strategies duringbreeding. Pied flycatchersmay need less Ca and could be adapted tolow Ca availability. A more efficient vitamin D3 metabolism stim-ulating Ca absorption and reducing Ca excretion could play animportant role in this adaptation. In addition, they may be moreefficient in vitamin D3 allocation to the egg, which may have directbeneficial effects on the nestling. This is evidenced by highervitamin D3 levels in yolks of this species and, apparently, a moresensitive response of vitamin D3 allocation to an extra Ca supply.However, changes in the Ca availability may affect pied flycatchersto a greater extent, since we have seen that Ca provisioning pro-duced eggs with a slightly higher eggshell index, so Ca intake maybe close to a deficient level.

In all, our findings suggest that great tits are more sensitive todiet quality and carotenoids availability, reflected in effects duringthe nestling period; while pied flycatchers are more sensitive tometal-related disturbance of Ca metabolism, which could produceeffects especially during the egg laying stage. A complex endocrinesystem coordinating the metabolism of vitamin D3 and Ca could bethe key of the differences between species in Ca intake and allo-cation into eggs.

Acknowledgments

We thank Jorma Nurmi and Miia Rainio for their help with thefield work, and Anne Koivuniemi and Atte Tuominen for their helpwith the carotenoid and vitamin analyses. Our study was financedby the Academy of Finland (project 265859 to TE) and by Societaspro Fauna et Flora Fennica (to PS-V). The experiment was

conducted under licenses from the Animal Experiment Committeeof the State Provincial Office of Southern Finland (license numberESAVI/1650/04.10.03/2012) and the Centre for Economic Develop-ment, Transport and the Environment, ELY Centre SouthwestFinland (license number VARELY/319/07.01/2014). All applicableinstitutional and/or national guidelines for the care and use ofanimals were followed. The authors declare that they have noconflict of interest. Sandra Ruiz and Pablo S�anchez-Virosta haveequal contribution to the article and appear in alphabetical order.

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.chemosphere.2016.02.074.

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S. Espín et al. / Chemosphere 151 (2016) 189e201 201

1

Supplementary material

Effects of experimental calcium availability and anthropogenic metal pollution on

eggshell characteristics and yolk carotenoid and vitamin levels in two passerine

birds

Silvia Espína*, Sandra Ruiza, 1, Pablo Sánchez-Virostaa, 1, Juha-Pekka Salminenb, Tapio Eevaa.

aSection of Ecology, Department of Biology, University of Turku, 20014 Turku, Finland. sieslu@utu.fi,

srruiz@utu.fi, pasanv@utu.fi, teeva@utu.fi

bLaboratory of Organic Chemistry and Chemical Biology, Department of Chemistry, University of Turku,

20014 Turku, Finland. j-p.salminen@utu.fi

*Corresponding author: Silvia Espín. E-mail address: sieslu@utu.fi / silvia.espin@um.es Telephone: +358

2 333 6006

1Note: Sandra Ruiz and Pablo Sánchez-Virosta have equal contribution to the article and appear in

alphabetical order (both are considered second author)

2

Table S1. Breeding parameters, egg size, eggshell characteristics, carotenoid and vitamin concentrations inyolk samples of great tits and pied flycatchers according to zone (polluted and unpolluted) and treatment (Ca-supplemented and control) in Harjavalta, Finland.

Polluted and Ca-supplemented Polluted and Control Unpolluted and Ca-

supplemented Unpolluted and Control

Parameter n Mean ± SD n Mean ± SD n Mean ± SD n Mean ± SD

Great tit

Laying date (1 = 1st January) 17 126.24 ± 8.11 22 124.32 ± 6.26 19 130.26 ± 4.94 25 127.32 ± 4.96

Hatching date (1 = 1st January) 15 148.67 ± 7.93 17 147.35 ± 5.56 15 151.47 ± 3.27 21 149.62 ± 3.60

Clutch size 17 7.94 ± 1.43 22 7.45 ± 1.84 19 8.74 ± 1.37 25 8.24 ± 1.67

Hatchling number 17 5.24 ± 2.41 22 5.05 ± 3.03 19 5.63 ± 3.17 25 5.52 ± 2.97

Hatching success (%) 17 66.05 ± 28.18 22 63.98 ± 36.70 19 64.33 ± 35.47 25 64.29 ± 31.55

Egg mass (g) 17 1.72 ± 0.11 22 1.70 ± 0.12 19 1.72 ± 0.13 25 1.72 ± 0.15

Length (mm) 17 17.68 ± 0.66 22 17.64 ± 0.65 19 17.74 ± 0.63 25 17.75 ± 0.62

Width (mm) 17 13.45 ± 0.28 22 13.44 ± 0.31 19 13.43 ± 0.40 25 13.51 ± 0.40

Egg volume (mm3) 17 1633.50 ± 108.86 22 1626.56 ± 107.77 19 1633.31 ± 135.3 25 1655.24 ± 127.08

Spot intensity 17 3.88 ± 0.78 22 3.77 ± 0.81 19 3.68 ± 0.95 25 3.72 ± 1.10

Spot distribution 17 3.06 ± 0.75 22 3.41 ± 1.05 19 3.16 ± 0.90 25 3.08 ± 0.81

Spot size 17 2.24 ± 0.66 22 2.09 ± 0.43 19 2.32 ± 0.75 25 2.24 ± 0.72

Yolk mass (g) 17 0.374 ± 0.041 22 0.364 ± 0.030 19 0.360 ± 0.033 25 0.361 ± 0.044

Albumen mass (g) 17 0.912 ± 0.107 22 0.883 ± 0.126 19 0.958 ± 0.116 25 0.900 ± 0.171

Shell mass (g) 17 0.101 ± 0.007 22 0.102 ± 0.007 19 0.103 ± 0.008 25 0.102 ± 0.008

Eggshell index (g/mm2) 17 0.426 ± 0.021 22 0.431 ± 0.024 19 0.430 ± 0.022 25 0.426 ± 0.028

Lutein (µg/g yolk) 17 60.09 ± 24.87 22 57.42 ± 21.54 19 71.94 ± 31.45 25 68.59 ± 20.92

∑Unidentified carotenoids (µg/g yolk) 17 11.51 ± 3.21 22 11.48 ± 3.39 19 13.18 ± 4.87 25 12.34 ± 2.94

∑Vitamin D3 (µg/g yolk) 17 2.62 ± 0.67 22 2.55 ± 0.53 19 3.26 ± 0.88 25 3.36 ± 0.56

Vitamin A (µg/g yolk) 17 0.25 ± 0.07 22 0.27 ± 0.11 19 0.25 ± 0.081 25 0.29 ± 0.09

∑Vitamin A (µg/g yolk) 17 0.77 ± 0.27 22 0.93 ± 0.38 19 0.71 ± 0.30 25 0.91 ± 0.40

Pied flycatcher

Laying date (1 = 1st January) 21 144.14 ± 2.78 21 144.67 ± 3.54 21 147.14 ± 3.93 18 143.94 ± 1.95

Hatching date (1 = 1st January) 18 163.28 ± 2.82 18 164.94 ± 5.35 21 166.29 ± 3.21 16 163.63 ± 1.86

Clutch size 21 5.81 ± 1.03 21 5.81 ± 1.36 21 6.24 ± 0.70 18 6.22 ± 0.94

Hatchling number 21 3.90 ± 2.17 21 4.14 ± 2.03 21 5.00 ± 0.89 18 4.61 ± 1.91

Hatching success (%) 21 65.33 ± 33.85 21 69.98 ± 30.93 21 80.31 ± 12.05 18 74.32 ± 27.77

Egg mass (g) 21 1.670 ± 0.12 21 1.69 ± 0.12 19 1.70 ± 0.12 16 1.69 ± 0.11

Length (mm) 21 17.63 ± 0.58 21 17.48 ± 0.68 19 17.60 ± 0.77 16 17.64 ± 0.62

Width (mm) 21 13.32 ± 0.37 21 13.38 ± 0.38 19 13.45 ± 0.29 16 13.34 ± 0.30

Egg volume (mm3) 21 1595.48 ± 97.47 21 1596.82 ± 121.60 19 1624.55 ± 120.20 16 1603.38 ± 111.97

Yolk mass (g) 21 0.355 ± 0.034 19 0.354 ± 0.025 21 0.347 ± 0.023 18 0.351 ± 0.028

Albumen mass (g) 21 0.934 ± 0.118 19 0.951 ± 0.134 21 0.949 ± 0.111 18 0.954 ± 0.091

Shell mass (g) 21 0.092 ± 0.007 20 0.089 ± 0.006 21 0.093 ± 0.005 18 0.091 ± 0.007

Eggshell index (g/mm2) 21 0.393 ± 0.021 20 0.382 ± 0.021 19 0.393 ± 0.015 16 0.387 ± 0.023

Lutein (µg/g yolk) 21 102.47 ± 36.82 20 98.03 ± 32.18 21 152.18 ± 59.17 18 112.15 ± 23.05

∑Unidentified carotenoids (µg/g yolk) 21 22.05 ± 5.81 20 21.82 ± 4.63 21 28.45 ± 9.86 18 21.06 ± 3.32

∑Vitamin D3 (µg/g yolk) 21 12.90 ± 6.04 20 9.96 ± 5.52 21 16.92 ± 7.96 18 13.84 ± 7.28

Vitamin A (µg/g yolk) 21 0.35 ± 0.19 20 0.34 ± 0.18 21 0.31 ± 0.16 18 0.36 ± 0.09

∑Vitamin A (µg/g yolk) 21 1.44 ± 0.66 20 1.13 ± 0.56 21 1.46 ± 0.76 18 1.29 ± 0.55

3

Note: Hatching success (hatchling number*100/clutch size). Effects of zone and experiment in the different parameters are shown in Table 1. No significanteffects of zone, experiment and their interaction were found for laying date, hatching date, clutch size and hatching number in either species (not shown inTable 1).

Table S2. Matrix of correlations [r (n)] between calcium, lead and cadmium concentrations in feces, eggshellcharacteristics and carotenoid and vitamins levels in A) great tits and B) pied flycatchers in Harjavalta, Finland.A) Great tit

Pb infecesa

Cd infecesa

Spotintensityb

Spotdistributionb

Spotsizeb

Shellmass

Eggshellindex Luteina ∑Unidentified

carotenoidsa∑VitaminD3a

∑VitaminAa

Ca in fecesa -0.022(67)

-0.014(67)

0.070(67) 0.043 (67) 0.020

(67)-0.007(67)

0.121(67)

-0.106(67) -0.103 (67) -0.244

(67)*0.070(67)

Pb in fecesa 0.465(67) ***

-0.034(67)

0.331(67)**

-0.089(67)

0.010(67)

0.040(67)

-0.439(67) *** -0.353 (67)** -0.381

(67) ***-0.028(67)

Cd in fecesa -0.082(67) 0.087 (67) -0.032

(67)0.117(67)

0.094(67)

-0.026(67) 0.005 (67) -0.195

(67)0.182(67)

Spot intensityb 0.150 (83) 0.359(83) ***

0.240(83)*

0.171(83)

-0.086(83) -0.083 (83) -0.176

(83)-0.028(83)

Spot distributionb 0.031(83)

0.051(83)

-0.047(83)

-0.165(83) -0.144 (83) 0.032

(83)-0.147(83)

Spot sizeb 0.105(83)

0.068(83)

0.143(83) 0.227 (83)* 0.051

(83)-0.246(83)*

Shell mass 0.709(83) ***

-0.038(83) -0.039 (83) -0.054

(83)-0.062(83)

Eggshell index -0.074(83) -0.092 (83) -0.069

(83)-0.063(83)

Luteina 0.929 (83) *** 0.407(83) ***

0.095(83)

∑Unidentifiedcarotenoidsa

0.330(83)**

0.148(83)

∑Vitamin D3a 0.180(83)

Significant correlations (p<0.05) are shown in bold *p<0.05, **p<0.01, ***p<0.001. aThe variables were log-transformed before analysis. Pearson’s correlation was

used except when indicated (bSpearman’s correlation).

Table S2. Matrix of correlations [r (n)] between calcium, lead and cadmium concentrations in feces,eggshell characteristics and carotenoid and vitamins levels in A) great tits and B) pied flycatchers inHarjavalta, Finland.B) Pied flycatcher

Pb infecesa Cd in fecesa Shell mass Eggshell

index Luteina ∑Unidentifiedcarotenoidsa

∑VitaminD3a

∑VitaminAa

Ca in fecesa 0.425(63)*** 0.308 (63)* -0.061 (62) -0.084

(58)-0.244(62) -0.147 (62) -0.245

(62) -0.111 (62)

Pb in fecesa 0.516 (63)*** -0.018 (62) -0.090(58)

-0.436(62)*** -0.114 (62) -0.224

(62) -0.023 (62)

Cd in fecesa -0.322 (62)* -0.338(58)**

-0.111(62) 0.044 (62) 0.011

(62) 0.090 (62)

Shell mass 0.673(76)*** 0.004 (79) 0.071 (79) -0.130

(79) 0.059 (79)

Eggshell index 0.012 (75) 0.033 (75) 0.071(75)

0.303(75)**

Luteina 0.859 (80)*** 0.194(80) 0.022 (80)

∑Unidentifiedcarotenoidsa

0.155(80) 0.019 (80)

∑Vitamin D3a 0.684(80)***

Significant correlations (p<0.05) are shown in bold *p<0.05, **p<0.01, ***p<0.001. Pearson’s correlation was used. aThe variables werelog-transformed before analysis.