Metal concentrations and metallothionein levels in Mytilusgalloprovincialis from Elefsis bay (Saronikos gulf, Greece)

Evangelia Strogyloudi & Michael O. Angelidis &

Anastassios Christides & Evangelos Papathanassiou

Received: 11 July 2011 /Accepted: 8 December 2011 /Published online: 3 January 2012# Springer Science+Business Media B.V. 2011

Abstract Spatial and temporal variability of Cd, Cu,Cr, Ni, Zn, Fe and Mn and metallothionein (MT)concentrations were determined in mussels Mytilusgalloprovincialis from Elefsis bay (Saronikos gulf,Greece). Higher concentrations of both metal concen-trations and MTs were recorded in mussels inhabitingindustrial locations (steelworks and shipyard), indicat-ing a markedly higher metal bioavailability. Howeverat these sites, located at the eastern part of the bay,mussel metal concentrations were not always correlatedwith both seawater metal concentrations and MTs pos-sibly due to different time scales of integration of themetal sources into mussels and/or the participation ofother metal regulatory mechanisms except MT induc-tion. The pattern of the temporal variation of musselmetal concentrations and the MT levels was similar

among stations with higher values during the winter–spring season and lower during the summer–autumnperiod. The inverse relationship of flesh condition indexwith mussel metal concentrations was attributed to theinfluence of mussel annual reproductive cycle.

Keywords Mytilus galloprovincialis . Metalbioaccumulation .Metallothionein . Elefsis bay

Introduction

Coastal zone is a complex and dynamic environmentthat receives large amounts of material from the landand may act as a source or sink of contaminants. Tracemetals are of particular concern due to their environ-mental persistence and their potential toxicity. Naturalmetal fluxes from land and atmosphere affect metalconcentrations in seawater while anthropogenic activ-ities increase metal mobilization due to industrial andurban activities.

Mussels of the genus Mytilus used as bioindicatorsin metal biomonitoring studies in coastal areas (Wangand Rainbow 2008). However the use of mussel metalconcentrations as exposure indicator could be complexby the influence of multiple routes of exposure andabiotic environmental factors such as temperature andsalinity as much as they affect metal speciation in thewater column. Moreover the interacting effects ofphysicochemical parameters with mussel physiologycould cause variation in metal bioaccumulation (Wang

Environ Monit Assess (2012) 184:7189–7205DOI 10.1007/s10661-011-2490-z

E. Strogyloudi (*) : E. PapathanassiouHellenic Centre for Marine Research,Institute of Oceanography,P.O. Box 712, Mavro Lithari, Anavissos,Attiki 19013, Greecee-mail: estro@hcmr.gr

M. O. AngelidisDepartment of Environment, University of the Aegean,Lofos Panepistimiou,Mytilene 81100, Greece

A. ChristidesDevelopment Association of Thriassion Plain,Bureau of Pollution Control and Environmental Quality,Perikleous 11,Aspropyrgos 19300, Greece

et al. 1995; Mubiana et al. 2006). Metal concentrationsin bioindicator organisms represent an integration ofall the recent bioavailable metal in the environment(Amiard et al. 1986; da Silva et al. 2005; Ivankovic etal. 2005; Rainbow et al. 2004; Wright and Mason1999).

Suspended particulate matter (SPM) could be con-sidered as a metal vehicle for heavy metals (Burska etal. 2005; Gavriil and Angelidis 2005; Suzumura et al.2004) and consequently to filter feeder organismsthrough food (Turner and Millward 2002). Particulateorganic carbon (POC) including its live phytoplanktonconstituent (Smaal and Haas 1997) can influence thepartitioning of heavy metals between phases (aqueousand dietary) and their potential bioavailability (Fan etal. 2002).

Metallothionein (MT) induction is well documentedas a response to metal exposure and thus MTs have beenproposed as biomarkers of trace metal pollution innumerous species from different zoological groups(Mourgaud et al. 2002; Mouneyrac et al. 2002; Attig etal. 2010). Metallothioneins are low molecular weightcytosolic metalloproteins, sulphydryl-rich, heat stable,having a high heavy metal binding capacity however,with different affinity (Hg2+ > Cu+ > Cd2+ > Zn2+;Waalkes and Perez-Olle 2000). Metallothionein levelsin Mytilus galloprovincialis (Bebianno and Serafim1998; Domouhtsidou et al. 2004; Ivankovic et al.2005) were used as metal exposure biomarker(Cajaraville et al. 2000; Romeo et al. 2003; Fasulo etal. 2008) as well as part of the mussel antioxidantdefence system (Bocchetti and Regoli 2006). Howeverdetoxification processes other than MT induction couldalso restrict the potential toxic effects of metals (Amiardet al. 2006; Marigomez et al. 2002).

MusselsM. galloprovincialis from Elefsis bay (NorthSaronikos gulf) were used as sentinels within the frame-work of the MED-POL (MAP/UNEP National Moni-toring Program) since 1986 (Scoullos et al. 2007) due totheir ability to accumulate and tolerate high concentra-tions of heavy metals. Although, metal bioaccumulationeliminates the problem of daily fluctuation of watermasses, the concentration of contaminants in tissuesalone provides no information on the biological signif-icance and deleterious effects of environmental pollu-tion on biological systems. Biomarkers have beenmeasured in M. galloprovincialis, in Saronikos gulf, inrelation to heavy metals, but results are very limited(Tsangaris et al. 2004; Vlahogianni et al. 2007). This

study intends to achieve a representation of metal con-centrations in the mussel M. galloprovincialis based ona monthly temporal resolution. The objective of thisstudy was to detect general heavy metal concentrationpatterns towards natural variability of environmentalparameters and mussel physiological condition duringan annual cycle. Additionally the validation of MTinduction as a biomarker of metal exposure was inves-tigated in Elefsis bay through spatial and temporal var-iation of MT levels in the digestive gland of the mussel.

Materials and methods

Sampling

Elefsis bay is positioned in the north part of Saronikosgulf near the Athens–Piraeus metropolitan area. It issmall (67 km2), shallow (max. depth 33 m) andconnected to the rest of the Saronikos Gulf by twonarrow and shallow channels: the eastern at Keratsini(12 m depth) and the western at Megara (8 m depth;Fig. 1). The eastern end of Elefsis bay communicates(Keratsini channel) with the wider area of the Piraeusharbour which is the front to the sea of the entireAthens metropolitan area potentially affecting, at least,the eastern part of Elefsis bay due to intense maritimetraffic and mooring activities (Fig. 1). Many decom-missioned ships are scattered not only at the easternpart of Elefsis bay but also up to the front of theLoutropyrgos area (Fig. 1). There are no river dis-charges in the bay and agricultural activities werelimited. The bay receives industrial effluents from amajor industrial area (Thriassio Plain) along its north-ern shore as well as untreated domestic sewage. Themajor industrial plants in the greater industrial zoneaffecting the sampling site include two oil refineries,two cement manufacturing plants, two shipyards andtwo steelworks factories.

Sampling stations were located in the north coastand along the east–west axis of Elefsis bay: stations F(shipyard) and D (steelworks) at the eastern part andstation G (mussel farm) at the western part of the bay(Fig. 1). Monthly, water samplings and in situ meas-urements of salinity and temperature were carried outfrom December 2000 to January 2002. According tosurface seawater temperature, the warm period wasdefined from May (21.2°C) to October (24.6°C). Themaximum annual temperature value was recorded in

7190 Environ Monit Assess (2012) 184:7189–7205

July (27.6°C). Mean temperature from December2000 to March 2001 was 14.6°C while the minimumtemperature was measured in January 2002 (10.2°C).Salinity ranged from 38.38 (March 2001) to 39.20 psu(October 2001). The observed positive correlationwith temperature (Spearman rank correlation, for p<0.05) indicates evaporation as the main factor of salinityannual variability in Elefsis bay.

Mussels M. galloprovincialis were collected fromnatural populations for metal analysis (monthly) andmetallothionein determination (bimonthly). Musselcollection and treatment, followed in the present study,were in accordance to the Guide ‘for the protection ofanimals used for experimental and other scientificpurposes’ (http://www.bioethics.gr) produced by theNational Bioethics Commission in Greece. Althoughformulated specifically for vertebrates and does notaddress in detail aquatic invertebrate, the Guide isestablishes general principles and ethical consider-ations that are also applicable to these species.Samples were immediately transported to the laboratory.Individuals of 4–6 cm shell length were selected as agood representative of the active population average(reproductive mature mussels). The dry weight of themussel whole body soft tissue (freeze dried) and the

shell valves (at 60°C until constant weight) wererecorded.

Analytical methods

Mussels

Metals Mussels were dissected and pooled samples(n0194) from the whole body tissue of 15–20 indi-viduals each were prepared. Nitric acid digestion ofthe dry tissue was performed into Teflon vessels in aCEM MDS 2100 microwave digestion system.Copper, Cr, Ni, Zn, Fe and Mn concentrations weredetermined by a Varian Spectr AA 20 Plus flame AAS.Instrument detection limits were 0.009 mg/l for Cu,0.006 mg/l for Cr, 0.012 mg/l for Ni, 0.004 mg/l forZn, 0.021 mg/l for Fe and 0.007 mg/l for Mn. Cadmiumdetermination was achieved by graphite furnaceAAS (Perkin-Elmer 4100 equipped with a HGA700) with 0.033 μg/l detection limit. The accuracyand precision of metal chemical analysis in organ-isms were verified with the NRC-Dorm-2 referencematerial (Table 1) while intercalibration exerciseswere performed during the study (Quasimeme/Round20 and IAEA/407).

Fig. 1 Sampling sites in Elefsis bay (F shipyard, D steelworks, G mussel farm)

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Metallothioneins Metallothionein content was eval-uated according the spectrophotometric method(Viarengo et al. 1997) in pooled mussel’s digestivegland samples (n099, of 5–6 individuals each). Tissuewas homogenized in three volumes of 20 mM Tris–HClbuffer, pH 8.6 containing 0.5 M sucrose, 0.006 mMleupeptine and 0.5 mM phenylmethylsulfonilfluoride,as antiproteolitic agents, and 0.01% b-mercaptoethanolas a reducing agent. Metallothionein concentration wasquantified using the reduced glutathione as a referencestandard. Intercalibration exercises were performed dur-ing the study (Zorita et al. 2005).

Lipid content Total lipid content was determinedgravimetrically after dry tissue extraction with hexanein a Soxhlet apparatus. The extract was concentratedto dryness in a rotary evaporator (Menchero et al.1994).

Flesh condition index Flesh condition index (FCI) was

calculated according to the equation: FCI ¼ dwbodydwshell �

100 where dwbody and dwshell were the dry weight ofthe whole soft tissue in mg and the shell respectivelyin gr.

Seawater

For the SPM and chlorophyll α (Chl α) determination,seawater samples (3–4 l) were filtered through poly-carbonate membrane filters (pore size 0.45 μm). Chlo-rophyll α recovery was achieved by filters extractionin 90% acetone for 24 h and Chl α was determined by

fluorimetry (UNESCO/SCOR 1966). Seawater sam-ples (1–2 l) were filtered through pre-combusted(450°C) GF/F filters with nominal pore size 0.7 μm.After drying at 60°C, the filters were acidified withHCl to remove inorganic carbon (Cutter and Radford-Knoery 1991; Verardo et al. 1990). POC was deter-mined using an EA 1108 CHN Fisons Instrumentsanalyzer, calibrated with acetanilide or atropine whereasPOC concentrations were corrected according to ‘blankfilter’ measurements.

Metals Seawater samples for the dissolved metal con-centrations determination (Cd, Cu, Cr, Ni, Zn, Fe, Mn)were passed through Chelex-100 columns (mesh 200–400). The metals were eluted with a mixture of 2NHNO3/1N HCl (3:1 v/v) suprapur acids. Metal concen-trations were determined in the eluates by a Perkin-Elmer(Αanalyst100) atomic absorption spectrophotometer.The procedure of standard additions in synthetic seawa-ter was applied to verify the accuracy of the method.

Filters, after SPM determination, were digested witha mixture of HNO3/HCl/HF (3:1.5:2.5 v/v) suprapuracids into Teflon vessels in a CEM MDS 2100 micro-wave digestion system. Particulate Cd, Cu, Cr and Niconcentrations were determined by graphite furnaceAAS, using a Perkin-Elmer 4100 equipped with aHGA 700 and Zn, Fe and Mn by a Varian Spectr AA20 Plus flame AAS. Instrument detection limits for theflameless AAS were 0.033 μg/l for Cd, 0.097 μg/l forCu, 0.66 μg/l for Cr and 0.509 μg/l for Ni. The marinesediment MESS-3, provided by NRC, was used asreference material to test the accuracy and precision ofseawater particulate metal analysis as well as blankfilters treatment and digestion (Table 1).

Statistical analysis

Results were reported as average (avg)±standard devi-ation (std) while median and lower and upper quartileswere presented. Data were logarithmically transformed(log10). Differences in investigated variables amongsampling stations and efforts were evaluated by theanalysis of variance (Kruskal–Wallis or ANOVAdepending on data distribution). Post hoc comparisonsassessed by the Tukey HSD test at the 95% confidencelevel. Spearman rank correlation was applied in order toinvestigate the possible correlation between variablesfor p<0.05. Outlying values detected by the Box-and-Whisker Plot. Tests were performed using the statistical

Table 1 Measured and assigned metal concentrations (in μgg−1 dw) of the certified reference materials Dorm-2 (dogfishmuscle tissue) and MESS-3 (marine sediment) provided byNRC (National Research council Canada)

n06 Dorm-2 MESS-3

Measured Assigned Measured Assigned

Cd 0.037±0.002 0.043±0.008 0.22±0.02 0.24±0.01

Cu 2.14±0.31 2.34±0.16 30.4±1.6 33.9±1.6

Cr 35.6±5.0 34.7±5.5 99±7 105±4

Ni 20.3±0.6 19.4±3.1 48.0±5.8 46.9±2.2

Zn 22.8±0.7 25.6±2.3

Fe 145±4 142±10

Mn 3.54±0.26 3.66±0.34

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packages Statgraphics for Windows (version 3.1) andWINKS SDA (6th Edition). For the presentation of themussel metal data periodicity, the MATLAB programwas used (version 6.1).

Results

Mussel physiological condition

Flesh condition index was used as a biometric variablerelated to mussels physiological status. Higher FCIwere recorded in mussels collected at station G–mussel farm (western Elefsis bay). The lower physio-logical condition was observed in the mussel popula-tion growing in the shipyard (station F—easternElefsis bay; Fig. 2). Mean lipid content of the mussel

body was 7.5% during winter (December and January),11.5% in July, while during spring (April) and autumn(September), the mean lipid content was intermediate(9.4%). Similar seasonal pattern of FCI and lipid contentwas observed: elevated values during the warm periodof the year followed by an additional peak in autumn tominimum levels in January and a subsequent gradualincrease through spring. Mussels from all populationspresented their best physiological condition from Aprilto July (Fig. 2).

Surface concentrations of SPM, POC and Chl αweremeasured as indicators of both quantity and quality ofmussel food. Mean SPM and POC concentrations were1.40 mg/l (±0.85) and 181 μg/l (±81), respectively,while total Chl α varied between 0.09 and 1.21 μg/l (Table 2). No spatial gradient was observed for theabove parameters among sampling stations (Kruskal–

station D

DecJan1

MarchAprilMayJuneJulyAugSeptOctNov

Jan2

1,2 1,3 1,4 1,5 1,6 1,7 1,8 1,9 2

station F

DecJan1

MarchAprilMayJuneJulyAugSeptOctNov

Jan2

1,2 1,3 1,4 1,5 1,6 1,7 1,8 1,9 2

FCI

station GDec

Jan1March

AprilMayJuneJulyAugSeptOctNov

Jan2

1,4 1,5 1,6 1,7 1,8 1,9 2

Fig. 2 Seasonal variation ofFCI during an annual cyclein mussels from Elefsis bay(D steelworks, F shipyard,G mussel farm). Box andWhiskers plots of log10transformed values (analysisof variance Kruskal–Wallis,95% Tukey HSD intervals)

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Wallis analysis of variance, 95% Tukey HSD intervals).Maximum Chl α concentrations were measured duringwinter (March and January) and autumn (October). Thehigh POC/Chl α ratio (Table 2) showed that particulatematter in Elefsis bay was more enhanced in POC than inChl α.

Metal concentrations

Mussel metal concentrations are summarized inTable 3, where standard deviation accounts fortemporal variability. The lowest median metal values

were determined at station G (mussel farm) for allmetals except Ni. According to statistically significantdifferences per metal and station (Kruskal–Wallisanalysis of variance, 95% Tukey HSD intervals), aspatial ‘pseudo-gradient’ was presented due to thegeneral consistency of the higher ranking of thesites with industrial activity (Table 4) although thepresence of only three sites reduces the power ofthe rank comparison. Additionally to spatial differences,from July to September, Cu, Zn, Fe and Mn musselconcentrations displayed their lower values regardlessof station while outlying metal concentrations weremeasured mainly during the cold period of the year.Seasonal variation was limited for Cr and Ni (Fig. 3).Fluctuations of mussel metal concentrations in Elefsisbay could be described by cosine functions of time fittedin the field data (Table 5). Fitting of the field data wasstronger for Cu, Zn, Fe andMn (p>0.80,Wilcoxon ranktest). Inconsistency of Cr, Ni and Cd to follow the cosinefunction could be, partly, attributed to the highpercentages of outlying mussel metal concentrationsobserved for those metals: Cd at station D (steelworks),Cr at station F (shipyard) and Ni at station G (musselfarm).

Dissolved and particulate metal concentrations insurface seawater are summarized (median, range) in

Table 2 SPM, POC and Chl α concentrations (mean values±standard deviation and range) in surface seawater from threesites in the Elefsis Bay (D: steelworks, F: shipyard, G: musselfarm)

n012 D F G

SPM (mg/l) 1.16±0.47 1.27±0.76 1.76±1.15

0.57–2.16 0.15–3.22 0.67–4.98

Chl α (μg/l) 0.50±0.27 0.55±0.26 0.39±0.31

0.09–1.21 0.28–1.10 0.09–1.08

POC (μg/l) 155±50 201±106 186±78

87–225 110–500 107–340

Table 3 Summary statistics of metal concentrations in mussels whole soft tissue (μg g−1 dw) from Elefsis bay (D: steelworks, F:shipyard, G: mussel farm)

Cd Cu Cr Ni Zn Fe Mn

Station D (n071)

Avg±std 1.34±1.25 20.0±12.2 3.7±1.2 4.9±2.0 269±78 593±388 24.5±12.6

Range 0.14–5.36 5.1–53.1 1.7–8.1 3.0–16.0 154–570 82–1,686 7.2–60.7

Median 0.85 14.9 3.4 4.5 257 568 21.7

L.Q.–U.Q. 0.58–1.37 10.1–29.5 3.0–3.8 4.0–5.5 215–323 230–878 14.7–33.3

Station F (n054)

Avg±std 0.98±0.77 31.2±23.6 9.1±10.5 6.1±3.6 330±117 599±468 18.7±9.8

Range 0.11–3.30 7.1–120 1.4–59.9 3.1–27.5 179–709 81–2,040 7.0–53.9

Median 0.69 23.4 4.0 4.9 289 482 15.2

L.Q.–U.Q. 0.50–1.10 11.5–50.3 3.2–11.2 4.4–7.0 242–394 241–873 12.1–22.9

Station G (n069)

Avg±std 0.54±0.40 6.6±2.7 3.7±1.1 7.4±2.9 164±43 431±296 11.5±6.1

Range 0.02–1.85 2.4–11.0 2.3–7.4 3.3–18.7 106–301 72–1,053 3.4–25.2

Median 0.42 6.4 3.2 6.7 159 355 9.5

L.Q.–U.Q. 0.28–0.75 4.9–9.0 3.0–4.2 5.2–9.8 136–176 119–696 6.6–13.4

The range indicates minimum–maximum values; median refers to median value

n number of samples, avg±std average±standard deviation, L.Q–U.Q. lower and upper quartile

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Table 6. A spatial gradient along the east–west axis ofElefsis bay was observed with higher particulate metalconcentrations at station D–steelworks and dissolved atstation F–shipyard (Kruskal–Wallis analysis of vari-ance, 95% Tukey HSD intervals). However some ele-vated metal concentrations in seawater were measuredat the western station (G—mussel farm; dissolved Crand particulate Cu). Negative correlations were ob-served between particulate metal concentrations andSPM (Fe excluded) while correspond correlations withPOC and Chl α were positive although limited (Cu, Znand Mn positively correlated with POC and Cu, Fe andMn positively correlated with Chl α; Spearman rankcorrelation, for p<0.05).

Cadmium, Cu, Cr and Zn concentrations in musselsand the particulate phase were positively correlated atthe mussel farm station (G). Corresponding correla-tions were limited at station D–steelworks and totallyabsent at station F–shipyard (Table 7). Soluble Cd, Cu,Zn and Mn were positively correlated to mussel metalconcentrations at station G (mussel farm). Metal bio-availability from the dissolved phase was evident onlyfor Zn at stations with industrial activity (D—steel-works and F—shipyard; Table 7).

The effect of the food components (SPM, POC andChlα) onmussel metal concentrations was differentiatedamong stations (Table 7) although their distribution(SPM, POC and Chl α) was homogenous in Elefsis

bay. POC was negatively correlated to mussel metalconcentrations at station G (mussel farm) while thecorresponding relationship with Chl α concentrationwas positive at station F (shipyard). When correlationbetween SPM and mussel metal concentration was sta-tistically significant, was observed to be negative regard-less of station. The relationship between mussel metalconcentrations and FCI was statistically significant neg-ative (Spearman rank correlation for p<0.05). Correla-tion was stronger at station G–mussel farm (Table 7).

Metallothionein induction

Maximum MT concentrations were determined at sta-tions F (shipyard) and D (steelworks) while minimumvalues were similar among stations (Table 8). MeanMT levels were statistically significant lower in mus-sels from station G–mussel farm (ANOVA, 95%Tukey HSD intervals). Lower MT levels were ob-served during August and October sampling effortsat stations F–shipyard and G–mussel farm while noseasonal variation was presented at station D–steel-works (January 2002 was excluded; Fig. 4). In anycase, seasonal variation of MT induction did not con-ceal spatial differences among sampling sites sincemean MT levels at stations D and F (steelworks andshipyard respectively) were almost twofold higher themean level at station G–mussel farm (Table 8).

Metallothionein levels were positively correlatedwith mussel Cu and Zn concentrations at station G–mussel farm and with Cd and Cu at station D–steel-works (Table 9). Correlations with metal concentrationsin seawater were phase depended at stations with indus-trial activity: positive with the soluble and negative(station F—shipyard) or absent (station D—steelworks)with the particulate phase. Only at station G (musselfarm), MT concentrations were negatively related withFCI (r0−0.39 for p<0.05, Spearman rank correlation)

Discussion

Mussel food and mussel physiological condition

Mean SPM concentrations (lower than 2 mg/l) werecomparable with corresponding values reported forenclosed or semi-enclosed gulfs while minimum con-centrations were comparable to values found in theopen waters of the Eastern Mediterranean (Gavriil and

Table 4 Significant differences between pairs of rank means, ofmussel metal concentrations, for each station in Elefsis baybased on Tukey HSD test (p<0.05) after non-parametric analy-sis of variance Kruskal–Wallis (D: steelworks, F: shipyard, G:mussel farm)

Stations D F G

Cd 1 1, 2 3

Cu 1 1 2

Cr 2 1 1, 2

Ni 2 2 1

Zn 1 1 2

Fe 1 1, 2 2

Mn 1 1, 2 3

final score 1 2 3

Stations were indicated by the same number when no significantdifference of mussel metal concentrations was observed amongstations (decreasing order of concentrations is 1>2>3). Finalscore was calculated for each station as the sum of the individualscores per metal

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0

2

4

6

Cd

(µg/

g dw

)

0

20

40

60

80

100

Cu

(µg/

g dw

)

0

10

20

30

40

50

Cr

(µg/

g dw

)

0

6

12

18

24

30

Ni (

µg/g

dw

)

DecJan1

March

AprilM

ayJune

JulyAug

SeptOct

NovJan2

station D station F station G

Fig. 3 Seasonal variation of metal concentrations in the whole soft tissue (μg g−1 dw) of mussels from Elefsis bay (D steelworks, F shipyard,G mussel farm)

7196 Environ Monit Assess (2012) 184:7189–7205

Angelidis 2005; Karageorgis et al. 2008). Anthropo-genic activities at the extended industrial zone ofElefsis bay are validated as major point sources ofSPM and pollutants (Karavoltsos et al. 1999; Mavrakiset al. 2004) while the absence of central sewage outfallor effluents treatment plant in the area could alsocontribute to the surcharge of the bay (Krasakopoulouand Karageorgis 2005; Zeri et al. 2009). The trophicstatus of Elefsis bay was ranged from oligotrophic tolower mesotrophic according to the eutrophication scaledesigned for the Greek seas. Only Chl α annual maxi-mum concentrations were defined within the highermesotrophic interval (0.6–2.21 μg/l; Moncheva et

al. 2001). Minimum POC concentrations measuredin Elefsis bay were comparable to values obtained fromthe oligotrophic eastern Mediterranean (Karageorgis etal. 2008) while mean POC concentrations were notelevated (Price et al. 2005). Mussel pseudofaeces pro-duction threshold of SPM was never exceeded (5 mg/l for SPM) while Chl α concentrations were near thelower limit for mussel feeding activity inhibition(0.5 μg/l for Chl α; Dolmer 2000). Mussels have theability to compensate efficiently seston quality fluctua-tions by maintaining an effective preingestive mecha-nism of selection for organic particulate matter(Hawkins et al. 1996) in order to avoid overload filtering

0

200

400

600

800

Zn

(µg/

g dw

)

0

500

1000

1500

2000

Fe

(µg/

g dw

)

0

20

40

60

80

Mn

(µg/

g dw

)

DecJan1

March

AprilM

ayJune

JulyAug

SeptOct

NovJan2

station D station F station G

Fig. 3 (continued)

Environ Monit Assess (2012) 184:7189–7205 7197

mechanisms, dilution of the nutrition value of the foodand metabolic energy waste (Bayne 2001). Sara et al.(1998) showed that mussels could use the non-livingportion of the POM as a supplementary food source inorder to retain part of their energy for maintenance.

There was a significant decline in the physiologicalcondition of the mussels from east to west along Elefsisbay suggesting lower available energy for somaticgrowth at the eastern stations (stations F—shipyard andD—steelworks) despite the same nutritional value of thefood among stations. It has been observed that understressful environmental conditions and/or exposure topollutants, the physiological condition of the musselscould be reduced (Blanck 2002; Mauri and Baraldi2003; Widdows et al. 1997). The sharp reduction of theFCI during winter–early spring in Elefsis bay was attrib-uted to the release of ripe gametes in the environment aswas confirmed by zooplankton studies in the area (Dr. S.Zervoudaki, personal communication). Both observed

spawning periods (primary during early spring and sec-ondary during early autumn) were sustained by theconcurrently high Chl α concentrations. The periodfrom April to July was corresponded to the resting stageof mussel reproductive cycle with the highest lipidcontent in their body in Elefsis bay. The same seasonalvariation in the biochemical composition and conditionindex was observed by Okumus and Stirling (1998) inMytilus edulis and was in agreement with the reproduc-tive cycle of the Mediterranean mussel as was de-scribed by Bocchetti and Regoli (2006), Pampanin etal. (2005) and Regoli and Orlando (1994).

Metal concentrations and metallothionein induction

Metal concentrations

Mean and median mussel Cu and Cr concentrations inElefsis bay were found to be higher in the mussel

Table 5 Mussel Cu, Zn, Fe and Mn concentration periodicity in Elefsis bay as cosine function of time fitted in the field data

Cu log10ðCuÞ ¼ 0:33 cos½ 2p400 ðt � 45Þ� þ 1:11 Fe log10ðFeÞ ¼ 0:41 cos½ 2p395 ðt � 80Þ� þ 2:61

Zn log10ðZnÞ ¼ 0:11 cos½ 2p399 ðt � 72Þ� þ 2:37 Mn log10ðMnÞ ¼ 0:24 cos½ 2p415 ðt � 23Þ� þ 1:19

y ¼ A cos½2pT ðt þ wÞ� þ B where A0amplitude, B0baseline, Τ0period, ω0phase shift and t0time in days

Table 6 Particulate (A) and dissolved (B) Cd, Cu, Cr, Ni, Zn, Fe and Mn concentrations (median values and range) in seawater fromthree sites in the Elefsis Bay (D: steelworks, F: shipyard, G: mussel farm)

A. Particulate B. Dissolved

(n036) D F G (n012) D F G

Cd Median μg g−1 13.0 2.2 1.4 μg l−1 2.6 2.9 1.8

Range 1.2–41.3 0.7–23.3 0.5–12.1 1.7–2.8 2.4–3.3 1.0–2.2

Cu Median 464 265 113 2.5 3.9 2.0

Range 130–1,208 124–4,365 28–7,448 2.3–3.6 3.4–5.1 1.8–3.7

Cr Median 799 813 443 1.3 1.3 4.5

Range 340–1,310 315–2,663 129–987 0.9–1.9 1.1–1.7 0.5–4.6

Ni Median 212 201 139 10.5 12.8 8.3

Range 65–3,897 54–3,537 32–3,545 6.2–12.6 9.9–15.8 7.9–9.8

Zn Median mg g−1 2.60 1.73 0.88 12.3 17.4 9.0

Range 0.49–6.38 0.18–13.1 0.05–2.61 8.1–14.2 15.3–21.0 6.7–10.9

Fe Median 34.9 21.8 19.5 10.6 9.5 12.0

Range 16.9–56.5 9.0–174 9.6–34.2 8.5–11.2 8.3–10.8 7.9–14.3

Mn Median 1.98 1.03 0.55 5.9 4.2 2.9

Range 0.95–4.40 0.53–8.59 0.18–3.91 4.1–13.0 3.3–5.5 2.8–3.0

Median indicates median value; range means minimum–maximum values

n number of samples

7198 Environ Monit Assess (2012) 184:7189–7205

samples collected at station D (steelworks) and F(shipyard) in Elefsis bay, than those reported for mus-sels from the Montenegrin (Joksimovic et al. 2011),the Croatian (Kljakovic-Gaspic et al. 2006) and theItalian Ionian coast (Storelli et al. 2000; Cardellicchio

et al. 2008). The Zn and Fe levels within Elefsis baywere on average higher (Topcuoglu et al. 2004;Storelli et al. 2000; Kljakovic Gaspic et al. 2002;Romeo et al. 2005) or similar (Topcuoglu et al.2002) than those reported in the literature for theeastern Mediterranean and the Black Seas. At the sametime, the levels of Cu, Cr, Ni and Zn in mussel fromthe present study were lower than those reported forcaged mussels M. galloprovincialis at an offshore gasplatform in the central Adriatic Sea (Gorbi et al. 2008).Mean and maximum Ni concentrations from this studywere similar or higher than those reported from theMarmara (Topcuoglu et al. 2004) and the Turkishcoast of the Black Sea (Topcuoglu et al. 2002).

Seawater soluble and particulate metal concentra-tions were elevated in Elefsis bay although compara-ble with other gulfs (Gavriil and Angelidis 2005;Herut et al. 1999; Price et al. 2005; Ladakis et al.2007). Soluble and particulate Cd, Cu, Cr, Zn andMn concentrations measured by Scoullos et al.

Table 7 Correlation coefficients (Spearman rank correlation) ofmetal concentration in mussel soft tissue with condition index(FCI), metal concentrations in the seawater dissolved (solub)

and in the particulate phase (part), with SPM, POC and Chl αconcentrations in Elefsis bay (D: steelworks, F: shipyard, G:mussel farm)

Cd Cu Cr Ni Zn Fe Mn

Station D

FCI −0.36* −0.30* −0.17 −0.14 −0.40* 0.06 −0.30*solub 0.34 −0.20 −0.02 −0.62* 0.78* −0.47* −0.45*part 0.36* −0.22 −0.22 0.39* 0.20 −0.27* −0.34*SPM −0.38* −0.18 −0.30* 0.11 −0.20 −0.13 0.10

POC −0.33* −0.42* −0.20 −0.02 −0.23 −0.44* −0.63*Chl α 0.09 0.21 0.05 0.24* 0.32* 0.16 0.16

Station F

FCI −0.43* −0.48* −0.66* −0.39* −0.52* −0.42* −0.60*solub 0.38 −0.23 −0.24 −0.76* 0.72* −0.03 0.30

part −0.02 0.11 −0.25 −0.18 0.13 0.24 −0.12SPM 0.07 0.12 0.06 −0.15 −0.36* −0.52* −0.01POC 0.37* −0.02 0.10 −0.34* −0.28* −0.05 −0.01Chl α 0.55* 0.30* 0.39* 0.15 0.13 0.37* 0.52*

Station G

FCI −0.62* −0.77* −0.56* −0.61* −0.49* −0.65* −0.74*solub 0.58* 0.83* −0.85* 0.38 0.74* −0.53* 0.56*

part 0.32* 0.31* 0.31* −0.29* 0.44* 0.08 0.10

SPM −0.44* −0.34* −0.05 −0.06 −0.18 −0.26* −0.28*POC −0.51* −0.65* −0.46* −0.47* −0.61* −0.52* −0.62*Chl α 0.27* 0.004 0.03 −0.12 −0.11 −0.21* 0.06

* p<0.05, statistically significant correlation

Table 8 Summary statistics of metalothionein concentrations inmussel digestive gland (μg g−1 ww) from Elefsis bay (D: steel-works, F: shipyard, G: mussel farm)

D F Gn038 n030 n031

Avg±std 195±83 221±83 126±33

Range 99–349 94–358 73–201

Median 163 228 121

L.Q.–U.Q. 117–256 146–293 73–201

Median indicates median value; range means minimum–maxi-mum values

n number of samples, avg±std average±standard deviation, L.Q.–U.Q. lower and upper quartile

Environ Monit Assess (2012) 184:7189–7205 7199

(2007) in Saronikos gulf, including Elefsis bay, werelower than those measured in the present study. Theimpact of the land based industrial activities at stationsD and F (steelworks and shipyard, respectively)was confirmed by the spatial gradient of watermetal concentrations. In a previous study in Elefsisbay, concerning point sources of pollution, Cu andZn concentrations in both seawater phases werehigher than in the present study (Karavoltsos etal. 1999). Additionally to the industrial activity,the coastal populated urban zone at the easternpart of the bay and local sewage outfalls could probablycontribute to elevated seawater metal levels.

Elevated percentages of outlying Cd, Cu and Crconcentrations in mussels and higher seawater andmussel metal concentrations from the shipyard (station

F) and the steelworks (station D) highlighting theimportance of the industrial activities as a source ofbioavailable metals in Elefsis bay. However, statisticallysignificant correlations between metal concentrations inseawater (dissolved-aqueous and particulate-dietaryphase) and mussel were either absent or negative atstations with elevated metal levels in both seawaterand organisms (D—steelworks and F—shipyard).Possibly the lack of positive correlations at the moremetal polluted sites could be due to metal regulation anddetoxification processes. Invertebrates’ tolerance hasbeen developed through their ability to accumulateexcess metal ions in non-toxic forms (Marigomez et al.2002; Viarengo and Nott 1993).

In Elefsis bay mussel metal concentrations wereclosely linked to the mussel’s life cycle in all samplingpopulations. The strength of the negative correlationsbetween mussel metal levels and FCI was higher atstation G (mussel farm) where mussel metal concen-trations were lower and FCI higher. Maximum musselmetal concentrations in Elefsis bay were measuredduring the stage of sexual maturity including spawn-ing. Growth rate is included as a separate additionalfactor in the biokinetic model affecting inversely metalconcentrations in mussels (Wang and Rainbow 2008).Andral et al. (2004) proposed a linear model explain-ing the relationship of FCI and metal concentrations inmussel whole soft tissue: tissue concentrations areinversely proportional to the condition index althoughstations located in contaminated areas did not obeythis rule due to their consistently higher results.

Seasonal variability of mussel metal concentrationswas similar among sampling stations in Elefsis bayranging between maximum and minimum concentra-tions (during the cold and the warm period respectively),specific for each station and metal. The observed sea-sonal pattern of metal bioaccumulation in Elefsis baywas similar with that presented in other studies following

MTs (µg/g ww)

station D

Dec

Jan

1

Apr

il

June

Aug Oct

Jan

2

1,8

2

2,2

2,4

2,6

2,8station F

Dec

Jan

1

Apr

il

June

Aug Oct

Jan

2

station G

Dec

Jan

1

Apr

il

June

Aug Oct

Jan

2

Fig. 4 Seasonal variation ofmetalothionein concentra-tions in the digestive gland ofmussels from Elefsis bay(D steelworks, F shipyard, Gmussel farm). Mean valuesand standard errors of log10transformed values(ANOVA, 95% Tukey HSDintervals)

Table 9 Correlation coefficients (Spearman rank correlation) ofmetallothionein concentrations with metal concentrations in mus-sels (tissue) and seawater (dissolved—solub and particulate—partphase) in Elefsis bay (stationD: steelworks, F: shipyard, G: musselfarm)

Cd Cu Zn

Station D

tissue 0.30* 0.53* 0.15

solub 0.67* −0.11 0.67*

Part 0.14 −0.23 −0.21Station F

tissue −0.46* −0.30* 0.29

solub −0.59* 0.95* 0.59*

Part −0.21 −0.65* −0.53*Station G

tissue 0.09 0.60* 0.68*

solub 0.61* 0.61* 0.61*

Part 0.25 0.44* 0.37*

*p<0.05, statistically significant correlation

7200 Environ Monit Assess (2012) 184:7189–7205

the same general trend: higher metal concentrationswhen mussel body weight is low and vice versa (Gorbiet al. 2008; Ivankovic et al. 2005; Viarengo et al. 1997).

In Elefsis bay, phytoplankton blooms in winterand early spring coincided with maximum musselmetal concentrations, suggesting that phytoplanktonblooms represent periods of enhanced metal exposure.Phytoplankton cells, due to their ability to absorbdissolved metals from seawater (Luoma and Rainbow2005), could act as regular carriers of both organiccarbon and metals to the mussels (Davies et al. 1997;Luoma et al. 1998). It has been observed that the additionof microalgae to complex natural particle assemblagescould improve the bioavailability of associated metals tobivalves (Lee and Luoma 1998).

Additionally, higher trace metal levels during wintermight be also related to the type of fuel used for heatingat that season. As reported in the study of Nguyen et al.(2005) burn brown coal for house-heating purposes andfor industrial activities, adding considerable amounts tothe pollutant-load in the atmosphere increasingtrace metal levels in precipitation. Within the greaterAthens metropolitan area, during the cold season (fromNovember to March) domestic heating becomes aneffective source of particles enriched in metals com-pared with the warm period (ranging between May toSeptember) although differences are not statisticallysignificant (Manalis et al. 2005). This fact is of particu-lar importance for the industrial area of Elefsis (21 kmfrom the centre of Athens) where large-scale industrialoperations taking place in the area due to the combus-tion of heavy fuel oil powering-up heavy industrialplants.

Metallothionein induction

Diverse methodologies used to isolate and quantifyMTs and different units to express MT concentrationscomplicate the interpretation and the comparison ofthe results obtained from different studies (Bodin et al.2004; Ivankovic et al. 2005; Geffard et al. 2005; Zoritaet al. 2005; Bebianno and Serafim 1998; Hamza-Chaffai et al. 1999). According to the spectrophoto-metric method, MT levels in Elefsis bay were notconsidered elevated except some maximum concen-trations at stations with industrial activity (Viarengo etal. 1997; Ivankovic et al. 2005).

At the mussel farm site (station G) mussels aremore vigorous with lower metal and MT levels in their

body. At that site MTs were positively correlated withmetal bioaccumulation (Cu, Zn) and metal exposure inthe soluble (Cd, Cu, Zn) and particulate (Cu, Zn)phase. Although positive correlations between MTsand mussel metal content have been demonstrated inother studies (Bebianno and Serafim 1998), negativeor no correlation was observed between mussel metalconcentrations and MTs at stations F and D (shipyardand steelworks, respectively) in Elefsis bay. Howeverthe same has been observed in other metal pollutedareas (Amiard et al. 2006; Geffard et al. 2005; Fernándezet al. 2010). The biological half-life of cadmium boundto be inducedMTs is 70 days, whereas the half-life of theMT protein itself is only 4 or 20 days for the oysterCrassostrea virginica and 300 and 25 days, respectivelyinM. edulis.Therefore metals released during the proteinbreakdown have the potential to bind to newly synthe-sized MT, so extending the turnover time of the metal inrelation to the protein (Mouneyrac et al. 2002). Addi-tionally, it is possible, other regulatory mechanismsexcept MT induction to be activated in order to de-toxify high metal levels in the mussels from the nat-ural population in the shipyard (station F; Marigomezet al. 2002). It has shown that high molecular weightproteins, important for metal binding and depurationin mussels, can act as a temporary pool for storingmetals, besides the low molecular weight metallothio-nein (Ng and Wang 2005).

Seasonal variations in MT content in the mussel farmsite (station G) and the shipyard (station F) were ob-served to be similar with metal bioaccumulation tempo-ral pattern (higher levels during the cold period of theyear). These seasonal trends of MTs and trace metalconcentrations could be mainly related to the reproduc-tive cycle and the growth activity of the mussel in Elefsisbay at least in station G (mussel farm) as indicated by theopposite seasonal trend of the mussel FCI as was alsoobserved by Mourgaud et al. (2002) and Andral et al.(2004). Several studies dealing with either MT or tracemetal concentrations in bivalves have also evidenced asimilar seasonal fluctuation (Regoli and Orlando 1994;Ivankovic et al. 2005) while environmental variables areable to influence the cellular concentration of MTs too(Viarengo et al. 1999; Pellerin and Amiard 2009;Pytharopoulou et al. 2008), inducing part of the vari-ability of the data. No seasonality at station D could beattributed to thermal pollution, although the steelworksfactory at that station has the infrastructure to limit theemission of hot water in the marine environment.

Environ Monit Assess (2012) 184:7189–7205 7201

Part of the seasonal MT variation in Elefsis baycould be due to the antioxidant defence system of themussels. It has been observed that during summer,mussels’ antioxidant enzymes activity is high(Cavaletto et al. 2002; Regoli 1998; Sheehan andPower 1999) and it is possible that during the warmperiod of the year in Elefsis bay there is nonecessity of additional MT induction. Moreoverthe phytoplankton peak during winter in Elefsisbay might cause oxidative changes as a consequenceof intense food consumption (Bocchetti and Regoli2006) which could be faced by additionalMT induction.Findings of Bordin et al. (1997) on Macoma balthicashow that the clams are more sensitive to metals inwinter, taking more Cd and Cu and inducing moreMTs than in warmer months.

Conclusions

In summary, the present study has shown that con-centrations of Cd, Cu, Zn and Mn in the body ofthe mussel M. galloprovincialis from the coastalmarine environment of Elefsis bay follow a spatialgradient according to land-based anthropogenic ac-tivities. However seawater metal concentrationswere not correlated with mussel metal concentra-tions at stations with industrial activity. Possiblecauses would include the different time scales ofintegration of the metal sources into mussels inrelation to water. Correspond inconsistency betweenmussel metal concentrations and metallothionein in-duction in Elefsis bay could be attributed to thedifferent biological half-life of metals and MTs and/orthe participation of other regulatory mechanisms exceptMTs in mussel metal detoxification especially at themost contaminated stations.

The seasonal pattern of mussel Cu, Zn, Fe and Mnconcentrations was similar among stations: lower sepa-rately grouped values during the warm period of theyear and elevated concentrations during the cold period.Additionally, mussel metal concentrations and fleshcondition index were inversely correlated especially atstations G (mussel farm) and F (shipyard). The upperand lower mussel metal concentrations during an annualcycle were station depended (according to metal expo-sure) whereas their temporal pattern was specified bythe annual periodicity of the flesh condition index(according to the mussel reproductive cycle).

Acknowledgements We thank Mr. V. Skarakis, the ProductionManager of Halyvourgiki Inc. (station D) and Mr. S. Sarvanidisfrom the Hellenic Shipyards S.A. (station F) for their collaborationand practical help during the sampling. The owner of the musselfarm ‘Poseidon’ in Nea Peramos (station G), Mr. N. Kastanis, wasthanked for his valuable help during the fieldwork. Dr. G.Assimakopoulou and Mr. Th. Zoulias were thanked for Chlα analysis and Dr. E. Krasakopoulou and Mrs. A. Andronifor POC analysis. Financial support was provided by theHellenic Centre for Marine Research (HCMR).

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