RESEARCH ARTICLE

Contamination levels of polychlorinated biphenyls in wildversus cultivated samples of female and male mussels (Mytilussp.) from the Northwest Coast of Iberian Peninsula—newapplication for QuEChERS (Quick, Easy, Cheap, Effective,Rugged, and Safe) methodology

Tânia Vieira Madureira & Cláudia Santos & Susana Velhote &

Catarina Cruzeiro & Eduardo Rocha & Maria João Rocha

Received: 9 April 2013 /Accepted: 16 July 2013# Springer-Verlag Berlin Heidelberg 2013

Abstract A newly analytical method based on QuEChERSextraction followed by gas chromatography with mass spec-trometry (GC-MS) analysis was developed and validated forthe quantification of 18 PCBs in wild (from MatosinhosBeach, Portugal) and cultivated (from Ria de Arousa, Spain)mussel samples, pooled by sex. Wild animals showed higher

PCB levels than cultivated mussels, with males from bothorigins, presenting an upper contamination profile comparingwith females. This fact seems to be correlated with fewbiometric parameters, but other interdependencies, notaddressed herein, such as distinct lipid contents betweensexes, as a consequence of the gametogenic stage, may alsoexplain this data. Overall, data reiterate the importance ofinvestigating the presence of PCBs in marine biological sam-ples, which can act both as bioindicators of environmentalcontamination, either as food quality controls for humanhealth.

Keywords Organic compounds . Chemical pollution .

Mussel watch . QuEChERS . GC-MS

Introduction

In the context of environmental toxicology, the persistentorganic pollutants (POPs) have reached the limelightsome decades ago, but still represent a source of currentconcern due to the potential adverse effects both on humanhealth and on different environmental ecosystems. In particu-lar for polychlorinated biphenyls (PCBs), despite of havingbeen banned from open use in most countries, recent moni-toring studies confirm they are still occurring in diverse envi-ronmental compartments, although in most cases at declininglevels, in comparison with few years ago (Carro et al. 2010;Zhang et al. 2011).

Several studies along the years have linked PCB occur-rences with diseases and/or abnormalities in a large number ofwildlife species, including some fish (Lauriano et al. 2012),mollusks (Höher et al. 2012), and mammals (Vanden Berghe

Responsible editor: Leif Kronberg

Tânia Vieira Madureira and Cláudia Santos are joint first authors.

T. V. Madureira : C. Santos : S. Velhote : C. Cruzeiro : E. Rocha :M. J. RochaICBAS—Institute of Biomedical Sciences Abel Salazar, U.Porto—University of Porto, Rua Jorge Viterbo Ferreira 228,4050-313 Oporto, Portugal

T. V. Madureirae-mail: tvmadureira@icbas.up.pt

C. Santose-mail: claudiasantos2001@gmail.com

S. Velhotee-mail: su_velhote@hotmail.com

C. Cruzeiroe-mail: catarinarcruzeiro@hotmail.com

E. Rochae-mail: erocha@icbas.up.pt

T. V. Madureira : C. Santos : S. Velhote : C. Cruzeiro : E. Rocha :M. J. Rocha (*)CIIMAR/CIMAR—Interdisciplinary Centre of Marine andEnvironmental Research, U. Porto—University of Porto, Rua dosBragas 289, 4050-123 Oporto, Portugale-mail: mjsrocha@netcabo.pt

M. J. RochaISCS-N—Superior Institute of Health Sciences—North, Rua Centralde Gandra, 1317, 4585-116 Gandra, Paredes, Portugal

Environ Sci Pollut ResDOI 10.1007/s11356-013-2017-y

et al. 2013). These evidences of environmental imbalancesounded as serious warnings for human health and stemmingfor such data, the United States Environmental ProtectionAgency and the European Commission soon have recog-nized PCBs as targets for regulation (CEC—Commissionof the European Communities 2001; EPA—EnvironmentalProtection Agency 2000).

Filter feeding organisms, such as mussels (Mytilus sp.), areactually recognized as time integrators, which means theyembed the long-term effects caused by polluting compoundsin a specific study area, and also allow the identification ofpotential sources of contamination (Hunt and Slone 2010).Additionally, as seafood is considered as a major contributorto the total dietary intake of organic contaminants, musselmonitoring studies can provide extra information concerninghuman food safety (Moon and Choi 2009).

For the analysis of POPs in high complexity samples, as theenvironmental ones, there has been a constant demand of newsample preparation methodologies with increasingly specificityand robustness, combined with simple implementation andaffordable costs, which may explain the success ofQuEChERS (Quick, Easy, Cheap, Effective, Rugged andSafe) technique. Extraction of PCBs, using QuEChERS meth-odology, is still very scarce in the literature (Banerjee et al.2012; Norli et al. 2011), and to our knowledge, it has neverbeen used specifically for mussel samples. This application, insuch a complex matrix, may be an excellent alternative extrac-tion technique in relation to others, for future implementationnot only on environmental monitoring campaigns, always verydemanding in terms of number of samples versus the timerequired for processing, either for research/regulatory studies,including quality control within the food industry sector.

In the present work, an adaptation of the original QuEChERSmethod was devised and implemented to extract 18 PCBsfrom mussel samples (Mytilus sp.). Our main scientific aimswere to: (a) identity and quantify the target PCBs in wild andcultivated mussel samples; for that purpose, specimens fromMatosinhos Beach (Portugal) and from the economically im-portant Ria de Arousa (Spain), respectively, were used; (b)analyze potential sex load differences of PCB concentrationsin both types of samples, a still poorly studied issue; and (c)establish an initial starting point concerning the PCB environ-mental contamination profile on these sampling origins, inorder to foster their future temporal monitoring and evaluateenvironmental and consumer health risks.

Materials and methods

Sampling sites

In January of 2012, specimens of Mytilus sp. were obtainedfrom two distinct sampling origins located along the northwest

(NW) coast of Iberian Peninsula, according with Fig. 1. Usingthis strategy, it was possible to have mussel samples from awild population (S1) versus a cultivated population (S2). Thecultivated mussels were commercialized and labeled asMytilus galloprovincialis (production area: GAL—22/02—Polígono Cambados C2, Ria de Arousa).

The Matosinhos Beach—S1 (41º10′16.74″N; 8º41′19.45″W) is located relatively close to the mouth of Douro River,which is considered as one of the longest rivers of the IberianPeninsula, sharing its watershed between Portugal and Spain.The presence of contaminant inputs in Douro estuarine area/NW coast of Portugal have been demonstrated regarding alarge variety of environmental pollutants in water (Madureiraet al. 2010; Rocha et al. 2011a), but also in tissue samples fromaquatic organisms (Reis et al. 2012) and sediments (Rocha et al.2011b). Considering the fact that Matosinhos coastal area isdrained by the Douro river waters, the respective beach isherein included as a potential polluted sampling site, althoughthese coastal bathing waters have been recently classified as“appropriate for baths” (ARH-Norte 2011).

The Ria de Arousa—S2 is located in the coastline of Galicia(NW Spain) and it corresponds to the largest Galician Ria,covering an area of approximately 200 km2. Some POPs, suchas polycyclic aromatic compounds (PAHs), have been quanti-fied at distinct sites along the Galician Rias, especially in theaftermath of the Prestige incident (Viñas et al. 2009). However,in the particular case of Ria de Arousa, this estuary has beenpointed as one of the cleanest ones, when compared with theother Rias (Carro et al. 2010). Overall, the Galician Riasrepresent 95 % of Spanish mussel production, surpassing anannual production of 200,000 ton, which makes them one ofthe world top seafood producers, as a result of their warmthclimate, high amount of nutrients and prime location(Caballero-Miguez et al. 2011).

Mussel (Mytilus sp.) sample collection

The number of mussels collected was established in order toform five pools per sex, each one with a minimum weight of20 g of wet soft tissue. This mass was previously determinedin preliminary assays, considering the inevitable losses thatarise during the preparation and homogenization of sam-ples. At least three individuals were taken to form a poolsample. Thus, 60 wild mussels were picked manually alongMatosinhos Beach (S1), during the ebb tide and at intertidalzone. The mussels were immediately transported to the labora-tory in a refrigerated cooler (±4 °C). Then, the animals wereplaced for 24 h in 30 L tanks with constant salinity (34‰±2.00)and oxygenation, in order to release the sand and other solidparticles that could interfere with the extraction protocol.Afterwards, all mussels were anesthetized in a magnesiumchloride solution (MgCl2) (60 g/L) for approximately 1 h. Foreach animal the length, width and height (cm) were measured

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and the weights (g), total, and soft tissue were registered.Mussel condition index (CI) was calculated using the followingformula: CI=[[wet tissue mass(g)/(shell lenght)3]×100](Dabrowska et al. 2013). In addition, a small fragment ofgonad (approximately 4 mm) was removed from eachmussel specimen for sex differentiation histological anal-yses, in order to established five pools of mussels for each sexand sampling origin. Subsequently, all the collected speci-mens were stored individually in aluminum foil at −80 °Cuntil further procedure.

Regarding the cultivated mussels, 40 animals from the Riade Arousa (S2) were purchased in a local market (Porto,Portugal). These animals were subject to the above men-tioned sampling procedures, with the exception of thecleaning step, since they have already been depurated beforecommercialization.

The cultivated mussels were also used for method valida-tion purposes and prepared as described. However, the ani-mals used for validation were purposely not identified by sexto ensure a robust methodology.

Histological analyses for sex determination

A small fragment of gonad (approximately 4 mm) wasremoved from each mussel specimen, fixed in 10 %(v/v) buffered formaldehyde (J.T. Baker, Netherlands)for 24 h and, subsequently stored in 70 % ethanol,before being processed in an automatic tissue proces-sor (Leica TP1020, Germany), following a 12-h pro-cedure (2 baths of 1 h of 70 % ethanol, 96 % ethanol,99.9 % ethanol, ethanol and xylene 50:50, v/v, xyleneand paraffin). Afterwards, fragments were embedded ina Histosec paraffin wax (Merck, Germany) and sectioned(4 μm thickness) on a fully motorized rotary microtome(Leica RM2155, Germany). Each gonad fragment was ran-domly sliced in order to correctly assess the sex of eachindividual. The slides were stained with hematoxylin solu-tion, modified acc. to Gill II (Merck, Germany), and eosin Y1 % (yellowish; J.T. Baker, Netherlands), cleared in xylene(VWR, France) and mounted with DPX (Sigma-Aldrich,United Kingdom).

Fig. 1 Geographical distribution of mussel sampling origins along thenorthwest (NW) coast of the Iberian Peninsula. IGeneral overview of thelocations in Portugal and Spain. II Location of the wild mussels site

(S1—Matosinhos Beach, Portugal). III Location of the cultivated musselssite (S2—Ria de Arousa, Spain)

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Analytical methods

Reagents and standard solutions

All organic solvents were LC/GC grade and were supplied byRomil (Cambridge, United Kingdom), except acetonitrilewhich was from Sigma-Aldrich (Steinheim, Germany). Theultrapure water was purified through a Milli-Q system(Millipore, Molsheim, France). Anhydrous magnesium sul-fate (MgSO4), sodium chloride (NaCl), and primary second-ary amine (PSA) sorbent were also obtained from Sigma-Aldrich (Steinheim, Germany).

The CEN PCB Congener Mix 1 (10 μg/mL each compo-nent in heptane), includes PCB 18, PCB 28, PCB 31, PCB 44,PCB 52, PCB 138, and PCB 153 and was obtained fromSupelco (Bellefonte, USA). Individual solutions (10,000 mg/L) of PCB 29, PCB 50, PCB 66, PCB 77, PCB 104, PCB 141,PCB 154, PCB 183, and PCB 187 were purchased from Dr.Ehrenstorfer (Augsburg, Germany). The internal standard(IS), 4,4′-difluorobiphenyl, together with PCB 1 and PCB 5were from Sigma-Aldrich (Steinheim, Germany). All refer-ence standards were of >98 % purity.

For IS, an individual stock solution of 5,000 μg/L wasprepared in n-hexane. For all the PCBs included in this work,a 500-μg/L mix solution of each component in n-hexane wasprepared. These solutions were stored in the dark at −20 °C inamber bottles to avoid degradation, which was never observedduring the study period.

For validation purposes, fortified mussel pools (wet softtissue homogenates) with PCB mixture solutions and the ISsolution, at appropriate concentrations, were added to the10 mL of acetonitrile used at the beginning of QuEChERSextraction method. Eight working calibration standards weredone ranging from 0.5 to 50.0 μg/L and three quality control(QC) standard solutions of each PCB were also obtained (lowQC—8.0 μg/L, medium QC—27.0 μg/L, and high QC—44.0 μg/L).

Taking into consideration that PCB levels can be detectedin the cultivated mussels used for validation assays, theobtained chromatograms from non-spiked mussel matrix,which was analyzed on the same day, was automaticallysubtracted (Xcalibur software, version 2.0.7, ThermoScientific) to the chromatograms obtained from the samespiked mussel matrix.

QuEChERS sample preparation

The QuEChERS extraction method performed in this studywas based on the original methodology developed byAnastassiades and co-workers (Anastassiades et al. 2003).

Briefly, once defrosted, the mussel soft tissues of each poolwere first homogenized with an IKAT 10 basic Ultra-Turrax®on ice, to ensure compounds stability, and then treated

individually in order to avoid possible contamination betweensample pools. From the obtained homogenate, 10.0 g wastransferred into a 50 mL Teflon centrifuge tube (Nalgene,Rochester, USA), and 10 mL acetonitrile, previously spikedwith IS (30 μg/L) and/or with adequate PCBmixture solutionsfor validation purposes, was added. Samples were vigorouslyshaken for 1 min using a vortex device (Vortex Mixer VX-200, Labnet International Inc.) at maximum speed, and sub-sequently, they were allowed to stand for 2 min, in order toguaranty an effective interaction between compounds and thebiological matrix. Then, 4 g of anhydrous MgSO4 and 1 gNaCl were added and mixed on the vortex mixer immediatelyfor 1 min. Following this step, samples were centrifuged for5 min at 5,000 rfc, at a constant temperature of 15 °C (Sigma2-16K centrifuge). In the clean-up step, 6 mL of the acetoni-trile layer was transferred into a Teflon tube containing900 mg MgSO4 and 150 mg PSA, capped tightly and shakenusing the vortex mixer for 30 s. Finally, the extracts werecentrifuged for 1 min at 5,000 rfc, and the supernatant wascollected for gas chromatography–mass spectrometry (GC-MS) analysis.

Gas chromatography–mass spectrometry

All analyses were carried out using a gas chromatograph(Trace GC ultra, Thermo Finnigan Electron Corporation),coupled with an ion trap mass spectrometer Thermo ScientificITQ™ 1100 GC-MSn) and an autosampler (Thermo ScientificTriPlus™). A Trace GOLD column (TG-5MS, length—30 m,ID—0.25 mm, film thickness—0.25 μm) was used. The oventemperatures were programmed as follows, based on the liter-ature (Zhang et al. 2007): 40 °C (initial equilibrium time 2 min)to 250 °C at 12 °C/min (hold time 1 min); and finally, from250 °C to 310 °C at 5 °C/min (hold time 1min). The ion sourceand injector temperatures were set at 280 °C and 250 °C,respectively. Helium (99.9999 %) was used as carrier gas at aconstant flow of 1 mL/min. The injected volume was 1 μL(injection needle of 80 mm; splitless straight liner;3×5×105 mm, I.D.×O.D.×L). The mass spectrometer wasrun in Selected Ion Monitoring (SIM). For each PCB thequantification ions selected are presented on Table 1. Eachchromatographic analysis took approximately 33 min.

Method validation

Linearity was evaluated using three independent calibrationcurves, performed in three distinct days, each with eightcalibrators, at the specified concentration range. The curveswere performed in mussel matrix, and all calibrators wereinjected in triplicate. The obtained calibration curves were aplot of the peak area ratio of the compound signal to the ISversus nominal concentration.

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The method detection limits (MDLs) and method quantifi-cation limits (MQLs) were calculated based on the calibrationcurves of each PCB, following the general formulas (ICH1995): MDL=3.3σ/S and MDL=10σ/S, where σ correspondsto the standard deviation of the response and S to the slope ofthe calibration curve.

To assess the precision, accuracy and recoveries of themethod, three pools of mussel matrix were fortified at theconcentrations established for low, medium and high QCs.The extracts were injected in triplicate and the procedure re-peated in three different and consecutive days for each QC.Precision was expressed as the relative standard deviation(RSD) of the replicate measurements and the accuracy of themethod was evaluated as the percentage of agreement betweenthe method results and the nominal amount of compoundadded. Regarding recovery values, the extraction efficiency ofthe method was determined by comparing the peak area ratio ofeach PCB, in spiked mussel matrix, with the peak area ratio ofthe same concentration of the PCB in unextracted solvent.

Finally, stability of PCBs in mussel extracts was evaluatedusing QC samples (matrix spiked at the three concentrationsof QCs) which were analyzed immediately after preparationand after 24 h at −20 °C, which corresponds to the maximumperiod of storage prior to analysis.

Statistical analyses

The statistical analysis was performed with STATISTICAsoftware (version 10.0, StatSoft Inc.). All data were firstexamined for normality and homogeneity of variances usingthe Shapiro–Wilk’sW test and Levene’s test, respectively. Thecomparisons of concentration levels of each PCB betweensexes and sampling sites, as well as the comparisons ofbiometric measurements of specimens (between sexes andsampling sites), were done using the two-way ANOVA withpost-hoc multiple comparisons made by both Tukey’s andNewman–Keuls’ tests. Differences were considered sig-nificant for p<0.05, thus adopting the more convention-al threshold for significance currently seen in the liter-ature. In all cases, the differences were 100 % concor-dant between the two tests mentioned above. Correlationanalysis was done by calculating the Pearson coefficient(r) (p<0.05). All data are presented as the mean of each pool(total number of pools: n=5) in ng/g of wet weight (ww), forevery sampling site and sex, followed by the respective stan-dard deviation (SD).

Results

Biometric characterization of mussel samples

The total population of cultivated mussels acquired from Riade Arousa had an average length of 7.7±0.69 cm, a height of3.9±0.37 cm and a width of 2.5±0.32 cm. The mean weightswere of 21.2±4.81 and 8.7±2.25 g with and without shell,respectively. Males accounted for 43%, and females represent57 % of the total population.

Wild mussels from Matosinhos Beach showed an averagelength of 5.9±0.46 cm, a height of 3.0±0.26 cm and a widthof 2.3±0.20 cm. The mean weights were 15.3±2.90 g withshell and 6.0±1.39 g shelled. Males represented 61 % and thefemales 37 %. In only 2 % of the cases, it was not possible todetermine the sex due to the immature state of the specimens.From the two populations described, several individuals wererandomly selected in order to obtain five pools per sex andsite, as it was previously mentioned. In Table 2, it is presentedthe biometric characterization of the pooled mussels. Overall,it should be noted that cultivated mussels (from Ria deArousa, Spain) of both sexes showed significant greater bio-metric measurements, such as for example length and totalweight, in comparison with the specimens from MatosinhosBeach, Portugal. Nevertheless, wild mussels exhibited thehighest condition indices. Greater condition indices were alsofound for males of both origins; although for the ones fromMatosinhos Beach, it was not possible to prove the signifi-cance of the differences.

Table 1 Conditions of the optimized GC-MS method

Compound tR(min)

Selected ions (m/z) Segmenttimes(min)Target Q1 Q2 Q3

4,4′-difluorobiphenyl(IS)

12.36 190 188 128 10.30–15.00

PCB 1 13.73 151 152 153 188 10.30–15.00

PCB 18 15.74 221 151 152 186 15.00–17.55

PCB 29 16.51 186 258 220 292 15.00–17.55

PCB 31 17.06 186 258 220 292 15.00–17.55

PCB 5 17.32 221 222 151 152 15.00–17.55

PCB 77 17.32 186 258 220 292 15.00–17.55

PCB 50 17.92 220 292 186 258 17.55–18.17

PCB 52 17.92 220 17.55–18.17

PCB 44 18.12 220 222 17.55–18.17

PCB 66 18.26 220 290 292 294 18.17–19.10

PCB 104 18.98 222 328 326 18.17–19.10

PCB 28 19.38 258 256 19.10–20.65

PCB 153 20.24 290 218 145 19.10–20.65

PCB 154 20.48 326 328 324 19.10–20.65

PCB 141 20.93 290 218 20.65–22.60

PCB 138 21.53 290 218 145 20.65–22.60

PCB 187 21.91 324 326 252 162 20.65–22.60

PCB 183 22.00 324 326 252 162 20.65–22.60

tR retention time, Q1, Q2, and Q3 qualifier ions, IS internal standard

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Validation of the analytical methodology—QuEChERS–GC-MS

The selectivity of the method was shown by comparing theretention times and ions presence/intensity of the 18 PCBs, insolvent calibration standards and QC solutions in musselmatrix, which evidenced no significant matrix effects (Fig. 2).

The calibration curves, obtained for all compounds, werelinear for the established calibration ranges. The correlationcoefficients (r>0.98) and the regression equations for the 18PCB are shown in Table 3.

The method detection and quantification limits rangedfrom 0.24 to 1.91 μg/L and from 0.78 to 6.35 μg/L, respec-tively (Table 3).

Intra- and inter-batch precision values were always lowerthan 11.6 % and accuracies were between 81.5% and 119.8%(Table 4).

The data presented in Table 4 also evidences recoveriesranging from 79.1 % to 114.9 %.

The stability assays demonstrated that the storage ofthe QC samples at −20 °C for 24 h had no significanteffect on the quantitative determination of the 18 PCB ana-lyzed (RSD<8.8 %).

PCB levels in mussel samples—patterns between sitesand sexes

Regarding the origin of mussels, the highest total sum ofPCBs was found in specimens collected at MatosinhosBeach (Σ PCBs both sexes 212.5 ng/g of ww), with mean con-centrations ranging from 1.8±0.81 ng/g of ww to22.1±1.71 ng/g of ww. For cultivated mussels from Ria deArousa the PCBmean levels varied between 1.8±0.78 ng/g ofww to 10.4±1.15 ng/g of ww, thereby amounting a total sumof 166.5 ng/g of ww for both sexes (Table 5). Irrespective ofthe sex, the biggest concentrations in Matosinhos musselswere recorded for trichlorobiphenyls, namely PCB 18 (Σ

PCB18 in both sexes 37.3 ng/g of ww) and PCB 29 (Σ PCB29 in

both sexes 21.3 ng/g of ww), which represented approximately17.6 % and 10.0 %, respectively, of the total PCBs quantifiedin these wild mussels. As to mussels from Ria de Arousa, themaximum levels corresponded, once more to PCB 18, whichreached a Σ PCB18 in both sexes of 17.8 ng/g of ww, and onlynarrowly behind the hexachlorobiphenyl PCB 138 (Σ PCB138

in both sexes 15.8 ng/g of ww), corresponding to 9.5 % of thetotal PCBs in cultivated mussels. Overall, low-chlorinatedPCBs (including mono, di, tri, and tetrachlorobiphenyls) and

Table 2 Biometric characterization of mussels selected for the study by location and sex

Sampling site Gender Length (cm) Height (cm) Width (cm) Total weight (g) Soft tissue weight (g) Condition index (Cl)

S1 Male 6.1±0.07A,a 3.1±0.12A,a 2.4±0.04a 16.6±0.14A,a 7.0±0.15A,a 3.1±0.11a

Female 5.8±0.25B,a 3.0±0.09B,a 2.3±0.07 14.8±1.29B,a 5.6±0.68B,a 2.8±0.39a

S2 Male 7.5±0.21b 3.8±0.18b 2.5±0.06b 21.2±1.46b 8.9±1.05b 2.1±0.21A,b

Female 8.0±0.46b 4.0±0.17b 2.5±0.29 22.7±2.71b 9.0±1.04b 1.8±0.15B,b

S1—Matosinhos Beach, Portugal; S2—Ria de Arousa, Spain. Different upper case letters mean significant differences (p<0.05) between sexes at thesame sampling site. Different lower case letters mean significant differences (p<0.05) among a sex at different sampling sites. Data are expressed asmean±SD (n=5 pools per sex and site)

Fig. 2 Chromatograms of a a standard mixture (40 μg/L) of the targetPCBs in n-hexane and b a spiked mussel sample before extraction, at thesame concentration level. 1 4,4′-difluorobiphenyl (internal standard), 2

PCB 1, 3 PCB 18, 4 PCB 29, 5 PCB 31, 6 PCB 5, 7 PCB 77, 8 PCB 50, 9PCB 52, 10 PCB 44, 11 PCB 66, 12 PCB 104, 13 PCB 28, 14 PCB 153,15 PCB 154, 16 PCB 141, 17 PCB 138, 18 PCB 187, and 19 PCB 183

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high-chlorinated ones (including penta, hexa and hepta-chlorobiphenyls) were 67.4 % and 32.6 %, respectively, ofthe total sum of PCBs in mussels from Matosinhos Beach;while in cultivated mussels, the percentages for the two groupsof congeners were more equitable (50.2 % for lower chlorinat-ed and 49.8 % for the higher chlorinated).

As it can be noted in Fig. 3, from the comparative analysisof mussels from Matosinhos Beach and the specimens fromRia de Arousa, for the females five PCBs (PCB 18, PCB 31,PCB 77, PCB 154, and PCB 187) showed significant differ-ences in the concentrations between sites. Meanwhile, thedifferences between sites regarding the concentrations ofPCBs in males were noted for almost all compounds.

When comparing the concentration of PCBs in male versusfemale mussels from the same origin, a clear pattern wasobtained, as shown in Fig. 3. In both sites, male musselspresented higher concentrations of PCBs (Σ PCBs 134.2 ng/gof ww in Matosinhos Beach and 89.1 ng/g of ww in Ria deArousa) than females (Σ PCBs 78.3 ng/g of ww in MatosinhosBeach and 77.4 ng/g of ww in Ria de Arousa). In terms ofmean concentrations, males concentrated PCBs in a rangebetween 2.0±0.81 ng/g of ww and 22.1±1.71 ng/g of ww,taking into account both origins. The female concentrationsvaried from 1.8±0.78 ng/g of ww to 15.2±1.66 ng/g of ww,on both sites.

For cultivated mussels, a statistical analysis of individualPCBs showed significant differences between concentrationsin males and females in only 6 PCBs (PCB 18, PCB 50, PCB104, PCB 141, PCB 138, and PCB 183). In contrast, for thewild mussels, most of the PCB levels were significantlydifferent between sexes (Fig. 3).

Discussion

The knowledge of the high toxicity pattern of PCB for theenvironment, wild and edible specimens and humans, havetriggered the need for eradication as well as the global interestin their periodically and worldwide monitoring. In this context,the development and validation of effective and robust analyticalmethodologies, as the one presented here, using QuEChERS andGC-MS is indeed of crucial interest.

The original method of QuEChERS is adapted to matriceswith high water content (>80 %) and low lipid profile (<4 %;Anastassiades et al. 2007). These requirements are generallymet in mussel samples, thus justifying the successful applica-tion of this method for this specific matrix, without extramodifications. In relation to the method detection and quanti-fication limits, they are very similar, or even lower, to thosedescribed in the literature (Norli et al. 2011), which evidencethe effectiveness of our methodology for the quantification ofPCBs in real samples, either from wild or cultivated origins.

Overall, PCB levels were quantified in almost all musselsamples, irrespective of the sampling origin. This fact was notparticularly surprising, since molecules with approximately alogarithm of octanol–water partition coefficient (Kow) higherthan 6, as PCBs, are highly associated with the organic matterin aquatic environments (including sediments, plankton andsuspended particles), and consequently, can be taken upby filter feeders. Furthermore, bivalves have very lowactivities of enzymes able to metabolize these compounds(Dailianis 2011).

In particular, a selective accumulation of penta andhexachlorinated PCBs in mussels has already been noticedby others (Bellas et al. 2011; Carro et al. 2010), such as forPCB138, which was herein registered as the highest concen-tration in the case of cultivatedmales, probably due to the highpersistence of this compound. Meanwhile, in this study, withthe exception of the above mentioned situation, the mostelevated individual concentrations were found for some low-chlorinated PCBs, such as PCB 18 and 29. High levels of low-chlorinated PCBs were also quantified, for example in mus-sels from Baltic Sea (Lee et al. 1996) and along the coast ofGhana (Dodoo et al. 2013). This last research specificallymentions the predominance of tri-PCBs in the studied bi-valves. The observed data lead us to infer two possible con-comitant scenarios, and so, low-chlorinated PCBs could havebeen simply transported from other locations, or can derive

Table 3 Linearity parameters, detection, and quantification limits of themethod

Compound Linearity parameters MDLs (μg/L) MQLs (μg/L)

Calibration equation r

PCB 1 y=0.109981x 0.98 0.52 1.74

PCB 18 y=0.0984029x 0.98 0.60 1.99

PCB 29 y=0.0999856x 0.98 0.40 1.32

PCB 31 y=0.1274120x 0.98 0.35 1.18

PCB 5 y=0.160409x 0.98 0.57 1.90

PCB 77 y=0.181467x 0.99 0.24 0.78

PCB 50 y=0.0922806x 0.98 0.77 2.58

PCB 52 y=0.102941x 0.98 0.24 0.79

PCB 44 y=0.0905624x 0.98 0.67 2.22

PCB 66 y=0.160371x 0.98 0.65 2.16

PCB 104 y=0.0525188x 0.98 0.42 1.40

PCB 28 y=0.0851368x 0.98 0.60 2.02

PCB 153 y=0.172841x 0.98 0.67 2.24

PCB 154 y=0.0597985x 0.98 0.81 2.71

PCB 141 y=0.048551x 0.98 1.18 3.91

PCB 138 y=0.0472453x 0.98 0.97 3.24

PCB 187 y=0.0158558x 0.98 1.28 4.26

PCB 183 y=0.0116355x 0.98 1.91 6.35

r correlation coefficients, MDLs method detection limits, MQLs methodquantification limits

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Table 4 Recovery, accuracy, intra-, and inter-batch precision

Compound (CQ μg/L) 1st day 2nd day 3rd day

RSD (%) Accuracy (%) Recovery (%) RSD (%) Accuracy (%) Recovery (%) RSD (%) Accuracy (%) Recovery (%)

PCB 1

8.0 5.2 101.8 82.6 11.4 91.8 80.8 6.7 102.0 86.0

27.0 2.8 100.2 88.1 1.9 100.0 88.2 1.7 101.3 89.1

44.0 1.1 100.0 89.0 1.0 100.1 89.3 0.9 100.2 91.8

PCB 18

8.0 10.3 116.7 99.8 10.3 117.7 99.8 6.8 109.8 105.8

27.0 2.0 104.2 107.5 1.8 100.5 102.6 3.6 102.7 107.2

44.0 1.1 99.3 97.2 1.6 101.1 102.3 1.2 100.3 99.9

PCB 29

8.0 8.6 113.0 79.4 10.7 115.4 82.8 8.8 116.6 84.4

27.0 2.2 103.3 86.9 2.0 100.3 84.5 2.8 103.4 85.2

44.0 1.1 99.9 88.8 1.3 100.4 91.5 1.1 99.8 88.3

PCB 31

8.0 11.2 81.5 91.5 11.6 93.1 101.7 9.5 93.2 103.9

27.0 2.4 100.0 99.0 1.3 101.2 101.9 1.6 100.8 101.0

44.0 2.0 99.2 104.7 2.2 100.4 101.1 1.0 99.3 100.8

PCB 77

8.0 5.4 113.1 103.5 8.2 95.6 98.8 5.9 111.1 107.9

27.0 1.8 96.3 99.2 1.5 96.1 98.7 1.2 94.6 98.3

44.0 0.8 98.1 104.7 0.9 99.7 106.6 0.9 97.8 105.9

PCB 5

8.0 8.8 81.7 109.4 11.5 91.0 114.4 6.0 100.9 114.9

27.0 1.9 98.7 106.6 2.1 95.2 104.6 1.2 96.6 107.0

44.0 1.2 100.4 101.7 1.3 99.8 100.2 1.2 100.2 98.3

PCB 52

8.0 7.6 105.5 81.7 7.4 110.0 86.1 6.9 98.8 81.3

27.0 3.0 98.0 88.5 2.4 100.6 91.2 2.2 100.8 93.8

44.0 1.4 98.6 92.3 1.7 99.2 101.3 2.3 99.2 99.1

PCB 50

8.0 6.5 114.1 83.3 7.5 119.8 84.7 5.3 117.3 88.2

27.0 1.9 101.3 88.7 2.2 103.3 89.1 2.7 102.5 89.1

44.0 2.1 102.0 84.4 1.4 99.2 85.9 1.0 100.8 89.8

PCB 44

8.0 7.1 115.9 80.7 7.6 119.3 79.4 8.7 119.6 88.8

27.0 1.4 102.2 85.6 1.8 102.0 83.0 3.3 103.5 89.0

44.0 1.4 102 84.9 1.1 101.3 82.1 0.8 100.8 81.6

PCB 66

8.0 6.9 82.4 85.3 5.9 85.5 79.1 7.2 98.9 83.4

27.0 1.8 108.0 94.9 2.3 101.1 85.7 1.7 100.9 95.1

44.0 1.2 100.3 90.6 1.3 99.3 89.9 1.0 100.3 92.6

PCB 104

8.0 4.3 109.1 106.4 7.2 106.7 106.3 4.9 107.1 101.3

27.0 2.5 102.6 101.5 3.2 104.5 108.2 3.4 103.8 107.0

44.0 2.8 103.1 103.0 2.0 101.7 92.3 1.7 100.0 91.9

PCB 28

8.0 11.2 111.6 84.7 6.2 112.8 88.2 6.0 111.1 81.2

27.0 2.5 100.7 89.9 2.8 101.8 93.1 2.9 102.2 94.2

Environ Sci Pollut Res

from the de-chlorination of high-chlorinated PCBs (Otchere2005). On the contrary, higher-chlorinated PCBs, as PCB 138,present an elevated deposition pattern, which reflects a poten-tial contamination input located closely to the mussels habitat.However, it should be noted that the identification of thesources and clarification concerning the predominance ofcongener profiles are beyond the main goals initially set forthis study.

The total sum of the quantified PCBs included in this studywas significantly higher for the wild mussels (fromMatosinhosBeach, Portugal) comparatively to that calculated to the culti-vated ones (from Ria de Arousa, Spain). When establishing acomparison between the concentrations quantified in musselsfromMatosinhos Beach and those reported on other parts of theworld, approximately similar trends have been found in theEgyptian Red Sea coast (Σ PCBs ranging from 6.75 to 66.44 ng/g of ww; Khaled et al. 2004) and Korean coastal waters (Σ PCBs

ranging from 5.82 to 98.5 ng/g of ww; Khim et al. 2000).Higher levels were detected, for example in the Danish straits

and Southeastern Baltic Sea (maximum 412 ng/g of ww;Mikoszewski and Lubelska 2010), and even exceeding this,the ones from the Scheldt estuary (SE, the Netherlands-Belgium; Σ PCBs ranging from 287 to 1,690 ng/g of ww; VanAel et al. 2012). Although the detected levels in wild musselsare not alarming, probably because the site is an open sea areaand also due to today’s severe control of industrial use andemissions, there are several explanations that can be pointed forthe presence of PCBs along the coast of Matosinhos Beach.These include its location in the metropolitan area of Porto city,where it is located Leixões harbour, with one of the mostintense vessel traffic of the country, and several industrialsettlements comprising a petrochemical complex. Few studiesreported the distribution of PCBs in Portuguese urban soils(Cachada et al. 2012) and the bioaccumulation in distinctfish collected from two watersheds located on the northregion (Antunes et al. 2001) and another closer to the countrycentral area (Baptista et al. 2013). To our knowledge, at oursampling site, PCB levels have never been quantified in any

Table 4 (continued)

Compound (CQ μg/L) 1st day 2nd day 3rd day

RSD (%) Accuracy (%) Recovery (%) RSD (%) Accuracy (%) Recovery (%) RSD (%) Accuracy (%) Recovery (%)

44.0 2.3 103.1 93.6 1.7 101.3 90.8 1.7 99.8 89.6

PCB 153

8.0 6.7 101.6 86.8 2.9 111.0 88.7 6.2 115.2 89.3

27.0 2.9 100.8 96.5 1.7 98.7 91.7 2.2 99.7 95.8

44.0 1.0 99.8 97.0 1.8 100.6 97.8 1.6 99.6 96.3

PCB 154

8.0 7.1 106.0 99.8 4.1 105.8 99.8 8.7 107.3 103.4

27.0 2.0 99.8 97.0 2.4 101.9 97.2 3.0 101.3 99.8

44.0 2.3 100.9 91.1 1.0 99.8 91.0 1.5 99.4 91.0

PCB 141

8.0 8.0 114.7 85.4 4.0 110.3 81.5 6.9 114.4 82.9

27.0 2.4 102.4 84.3 2.1 98.9 82.1 2.7 101.5 88.9

44.0 3.3 102.1 87.3 1.8 100.7 87.1 1.4 100.5 86.2

PCB 138

8.0 6.6 119.7 84.1 7.8 113.8 80.1 5.7 119.7 83.0

27.0 2.4 102.8 79.7 2.8 100.9 79.3 2.9 102.7 82.6

44.0 1.7 99.9 82.4 1.6 99.3 82.0 1.7 99.5 82.0

PCB 187

8.0 10.7 111.8 86.0 10.3 112.7 84.7 6.4 114.7 91.3

27.0 1.8 103.5 82.3 3.5 102.1 81.6 2.0 103.5 85.8

44.0 2.8 100.1 89.1 1.8 99.0 86.9 1.4 100.3 99.4

PCB 183

8.0 7.3 108.5 88.2 5.0 110.7 87.3 8.3 114.3 92.0

27.0 2.5 101.7 82.2 2.5 100.5 89.5 3.5 102.3 90.2

44.0 2.5 101.9 97.3 1.7 99.7 90.8 1.2 99.5 90.8

RSD, accuracy, and recovery were obtained from the repetition of the analytical method in triplicate, on three consecutive days, for the three QCs

QC quality control, RSD relative standard deviation

Environ Sci Pollut Res

environmental matrix; however, PAHs were determined inlocal coastal sediments (Rocha et al. 2011b).

Moving through to the PCB levels in cultivated mus-sels from the NW Spanish coast, it is well known thenotable economic relevance of Galician Rias for shell-fish production, which may explain the intensive monitoringin terms of POPs contamination from few decades ago untilnow, e.g., (Alvarez Piñeiro et al. 1995; Bellas et al. 2011;Carro et al. 2010; Suárez et al. 2013). Recent studies usingmussels purchased in local markets, supermarkets and/or gro-cery stores (Gómara et al. 2005; Perelló et al. 2012) showedsimilar concentrations of PCBs to those referred in this study.

Bivalves reflect their immediate contamination vicinity andlogically their bioaccumulation profile and global health statusis not only dependent on the conditions at the sampling sites,but may also be linked with some endogenous parame-ters of the specimens (i.e., size, sex …). In fact, it hasalready been described a relationship between higher bioaccu-mulation factors with decrease in size, as a direct consequence

of an increased uptake kinetic needs (Gilek et al. 1996). Suchnegative correlation between size and accumulation of com-pounds is evident in the obtained results. Generally, for bothsexes, biometric parameters, such as for instance length andtotal weight were significantly lower in wild mussels (fromMatosinhos Beach) in relation to cultivated mussels (from Riade Arousa), and theΣ PCBs both sexes were 212.5 ng/g of ww and166.5 ng/g of ww, respectively. In particular, the Pearson’snegative correlations found for male mussels fromMatosinhos Beach were r=−0.86 (p=0.06) and r=−0.68(p=0.21), for length and total weight with the PCB sums,respectively. Considering the condition index of mussels,higher values were noted for both males and females fromMatosinhos Beach, comparing to the cultivated ones from Riade Arousa, which may indicate potential differences in theenvironmental conditions on both sites. Despite this inference,it is well known that these cultivated individuals have excep-tional physical and biological conditions in the Galician Rias,and in about 17months, mussels can reach the adequate size forcommercialization (Caballero-Miguez et al. 2011). This canpossibly mean these mussels have probably less time eitherfor growing and to be exposed to possible environmentalcontaminants, than the wild ones. This is reflected in smallercondition indices and lower levels of contamination by PCBs.The obtained data concerning condition index versus the sumof PCBs agrees with the correlation stated elsewhere, whichreports a proportional ratio between tissue concentration andthe condition index, for organic compounds (Andral et al.2004). Another interesting point, comparing musselsfrom both sites is that recorded data are indicative of aslight delay in the gametogenic cycle in wild mussels in relationto the cultured individuals, which can directly influence thelipid content and the PCB concentrations, as evidenced byothers (Suárez et al. 2013).

Several comparative studies have been developed for thedetection of PCBs inmale and female organisms; however, suchdata are not available in the literature for mussels, which makesthe current research as pioneer in this aspect. Herein, for themajority of PCBs under study it was observed significant dif-ferences between the concentrations determined inmalemusselsversus female mussels, with an obvious predominance in males.

Throughout the reproductive cycle, bivalves evidence bio-chemical changes related with stored energy reserves, whichare mainly accumulated in the digestive gland and the mantle(Leonardos and Lucas 2000). In general, females have a higherpercentage of total lipids than males. However, with regardto polar lipids, such as phospholipids, males (mainly inmatured stages) present higher percentages of total dryweight, in the mantle and digestive gland, than females(Martínez-Pita et al. 2012). Moreover, it has been foundthat males have 10 % more reproductive tissue in the mantle

Table 5 Mean concentrations (ng/g of wet weight) and total sums ofPCBs in wild mussels (S1—Matosinhos Beach, Portugal) and cultivatedmussels (S2—Ria de Arousa, Spain) in both sexes

Compound S1 S2

Males Females Males Females

PCB1 3.1±1.32 1.8±0.81 3.1±0.70 2.1±0.76

PCB18 22.1±1.71 15.2±1.66 7.4±1.40 10.4±1.15

PCB29 13.2±1.76 8.1±1.88 6.4±1.01 6.0±1.82

PCB31 9.7±1.12 5.7±1.92 1.0±0.42a 2.5±0.89

PCB5 7.2±1.40 5.9±1.87 5.4±0.92 5.1±1.22

PCB77 5.5±1.89 4.7±1.83 2.0±0.81 1.8±0.78

PCB50 8.0±0.93 4.3±1.04 4.9±1.53 3.3±0.81

PCB52 6.2±1.58 2.9±1.07 3.2±0.86 3.2±1.00

PCB44 5.9±1.79 2.8±0.75 3.8±1.14 3.1±0.57

PCB66 5.0±1.34 2.3±0.74 1.5±0.54a 1.6±0.93a

PCB104 3.6±1.47 2.2±1.08 2.2±0.78 3.3±1.03

PCB28 3.7±0.73 1.9±0.82a 5.2±1.09 4.7±1.33

PCB153 4.4±1.04 3.2±1.23 3.2±0.56 1.8±0.40a

PCB154 7.8±1.25 3.6±0.86 7.3±1.74 6.1±1.83

PCB141 7.1±1.10 4.9±0.77 7.9±1.13 6.0±1.47

PCB138 7.4±1.29 5.8±1.08 9.5±1.45 6.3±0.98

PCB187 7.6±1.38 4.9±1.26 8.4±1.66 6.7±1.29

PCB183 6.7±1.33 3.9±0.67a 9.2±1.52 6.8±1.36bΣ PCBs(sex/site) 134.2 78.3 89.1 77.4bΣ PCBs(both sex/site) 212.5 166.5

S1—Matosinhos Beach, Portugal; S2—Ria de Arousa, Spain. Data areexpressed as mean (ng/g of wet weight)±SD (n=5 pools per sex and site)a Levels below the method quantification limitb Sum of PCBs except those below method quantification limits

Environ Sci Pollut Res

than females (Maloy et al. 2003). These evidences may wellexplain the higher concentrations of PCBs, as lipophilic com-pounds, found in male mussels. Besides, significant differ-ences were observed between males and females with regardto the biometric parameters evaluated, which resulted in great-er condition indices in the case of males.

It has been suggested that the sex differences andPCB levels may be associated with the lower efficiencyof male growth (Madenjian et al. 2010), the loss ofcontaminants through spawning in females (Bodiguelet al. 2009) and to the earlier maturation and at asmaller size in males than in females (Madenjian et al.2009). Given the various hypotheses as to what factorsunderlie the differences found between sexes and com-pound concentrations, and taking into account physio-logical, anatomical and ecological needs of various species,more studies should be done particularly with Mytilus sp., inorder to properly understand the relationship between sexdifferences and the preferred bioaccumulation of PCBs,throughout the reproductive cycle.

Conclusion

An analytical methodology using QuEChERS and GC-MSproved to be successfully applied to the quantification of 18PCBs in both wild and cultivated mussels. Overall, wildmussels from Matosinhos Beach (Portugal) showed higherlevels of PCB contents than cultivated mussels from Ria deArousa (Spain). Additionally, there were observed significantdifferences in the concentrations of PCBs in both sexes, withmales showing higher values in both sites. These differencesmay be related to the type and amount of lipid content and/orto the biometric profile of male and female mussels.

The obtained data contributes to the information available onthe degree of POPs contamination and also represents a crucialbaseline for future comprehensive monitoring, in order to en-sure both human safety and the health of aquatic ecosystems.

Acknowledgments This study was financially supported by CESPUProject ref. 01-GCQF-CICS-10, by the European Regional DevelopmentFund (ERDF) funds through the Competitiveness and Trade Expansion

Fig. 3 Mean concentrations ofPCBs (ng/g of wet weight) infemale and male mussels fromMatosinhos Beach, Portugal—S1and Ria de Arousa, Spain—S2.PCBs marked in bold indicatesignificant differences (p<0.05according to Tukey’s andNewman–Keuls’ tests) betweensexes at the same site. Thesuperscript asterisks meansignificant differences for eachPCB between sites for males(filled diamond) and females(diamond). The filled circlemeans levels were below themethod quantification limit. Dataare expressed as mean±SD (n=5pools per sex and site)

Environ Sci Pollut Res

Program (COMPETE) and by National Funds provided by Fundaçãopara a Ciência e a Tecnologia (FCT) via the research projects PTDC/MAR/70436/2006 and PEst-C/MAR/LA0015/2011. Support wasalso given by the Master of Marine Sciences—Marine Resources(ICBAS-U.Porto).

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