gy, Part C 145 (2007) 588–594www.elsevier.com/locate/cbpc

Comparative Biochemistry and Physiolo

Variability in biochemical components of the mussel(Mytilus galloprovincialis) cultured after Prestige oil spill

Laura G. Peteiro, Uxío Labarta, María José Fernández-Reiriz ⁎

Instituto de Investigaciones Marinas CSIC, Eduardo Cabello 6, 36208 Vigo, Spain

Received 5 October 2006; received in revised form 5 December 2006; accepted 4 February 2007Available online 12 February 2007

Abstract

The biochemical composition (proteins, carbohydrates, glycogen, total lipids and lipid classes) of the mussel Mytilus galloprovincialis wasinvestigated during an experimental culture using mussel seed from areas with different degree of exposure to the Prestige oil spill. The aim of thestudy was to identify alterations in the biochemical composition of mussel seed from natural populations commonly used in Galicia for mussel raftculture that might be linked to previous oil exposure. We have selected three mussel seed populations from Pindo, Miranda and Redes, that werecharacterised in a previous study according to the oil exposure three months after the spill. These populations were transplanted to a raft culturesystem in the Ría de Ares-Betanzos where our experimental culture followed standard commercial techniques from March 2003 to February 2004.Mussels from Pindo (characterised as the most affected area by the oil spill) showed marked differences in lipid composition with regard to otherpopulations in the content of triacylglycerols, (Pb0.001), free fatty acids (Pb0.001) and phospholipids (Pb0.05) at the onset of the culture.Although these differences in lipid composition might reflect their previous exposition to hydrocarbons, this pattern did not last longer most likelydue to depuration of hydrocarbons stored in the tissues or by the development of certain tolerance to PAHs. These significant differences were notdetected between Miranda (designed as hardly affected area) and Redes (designed as reference area) which may reflect that Miranda mussels werenot affected or only hardly affected by the spill. With the exception of the onset of the culture, biochemical composition showed similar patterns inall mussel populations. Then, the fact of being cultured in a common environment seemed to be more responsible for the long-term variability inthe energetic reserve than the origin of the populations or their previous biochemical status.© 2007 Elsevier Inc. All rights reserved.

Keywords: Biochemical composition; Lipid classes; Mytilus galloprovincialis; Prestige oil spill; Raft culture; Sublethal effect

1. Introduction

After the Prestige oil tanker sank near the Finisterre Cape inNovember 2002, successive black waves affected a wide area ofthe Galician Coastline (NW Spain). Although significantamounts of spilled oil did not enter the inner part of theestuaries in which commercial raft culture of mussels is carriedout, there was a significant effect on an extensive area wheremussel seed is gathered for culture purposes.

Bivalves, particularly mussels, are commonly used as sen-tinel organisms that may help to identify the status and trends ofchemical exposure and to establish pollution gradients in avariety of marine coastal environments. The latter fact occursbecause mussels are sessile, filter feeders, widely distributed

⁎ Corresponding author. Tel.: +34 986 231930; fax: +34 986 292762.E-mail address: mjreiriz@iim.csic.es (M.J. Fernández-Reiriz).

1532-0456/$ - see front matter © 2007 Elsevier Inc. All rights reserved.doi:10.1016/j.cbpc.2007.02.006

and may react to changes in the environment throughphysiological responses that are easily measurable (Salazarand Salazar, 1995; Dame, 1996). Ecological and physiologicaldisturbances have been reported in different species of molluscsdue to their exposure to hydrocarbons (Widdows et al., 1982;Strömgren et al., 1986; Larratxea and Pérez Camacho, 1996;Modassir and Ansari, 2000; Porte et al., 2000; De Luca-Abbot,2001; Le Floch et al., 2003; Amiard et al., 2004; Duquesneet al., 2004; Olsson et al., 2004; Pérez-Cadahía et al., 2004).These disturbances might be manifested at different levels ofbiological organization, although biochemical and cellularlevels are the first in which the effects are perceptible(Livingston, 1985; Smolders et al., 2004). Alterations in thebiochemical composition and specifically in those fractions thatinvolve changes in the cycle of accumulation and use ofenergetic reserve are commonly used to identify pollutionexposure (Bayne et al., 1978; Patel and Eapen, 1989; Leavitt

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et al., 1990; Capuzzo, 1996; Modassir and Ansari, 2000;Smolders et al., 2004). Several surveys have reported a decreasein the glycogen concentration (Patel and Eapen, 1989; De Luca-Abbot, 2001; Duquesne et al., 2004; Smolders et al., 2004) thatis considered one of the most important energetic reserves inbivalves, although alterations in total carbohydrates or proteinshave also been documented (Patel and Eapen, 1989; Modassirand Ansari, 2000; Olsson et al., 2004; Smolders et al., 2004).Nevertheless, studies about lipophilic organic contaminantsexposure such as PAHs or PCBs are primarily focused in theimpairment of lipid metabolism, mainly in the distribution andconcentration of polar and neutral lipids (Capuzzo and Leavitt,1988; Lowe, 1988; Leavitt et al., 1990; Capuzzo, 1996; Ferreiraand Vale, 1998; McDowell et al., 1999; Bergen et al., 2001; Chuet al., 2003; Petrovic et al., 2004).

The aim of the present study was to observe the seasonaltrend of the biochemical composition (proteins (prot), totalcarbohydrates (CH), glycogen, total lipids and lipid classes) ofmussel seed that were exposed to the spill in their early life stage(settlers of late summer or early autumn).

2. Materials and methods

2.1. Experimental design

In March 2003, mussel seed Mytilus galloprovincialis wasgathered from three locations along the Galician Coastline:Pindo, Miranda and Redes (Fig. 1). All sources of mussel seedcorresponded to the first collected after the Prestige oil spill for

Fig. 1. Map of Galicia (NW Spain) showing the three mussel seed origins (in squaPrestige oil tanker sank, and the area designed by Labarta et al. (2005) as the most

commercial cultivation. We have selected three mussel seedpopulations Miranda, Pindo and Redes, firstly because thesecorresponded to locations commonly used for gathering musselseed in Ría de Ares-Betanzos and secondly, because thesepopulations were characterised according to their exposure tohydrocarbons three months after the spill by Labarta et al.(2005) according to several biochemical and physiologicalstress indicators. Pindo is located in the central area of the spill(Labarta et al., 2005; González et al., 2006), Miranda is locatedfar from the most exposed area (Labarta et al., 2005; Gonzálezet al., 2006) and Redes is the location that we have consideredas reference because it is far from the central area of the spill andis inside the Ría de Ares-Betanzos where oil did not enter(Labarta et al., 2005; Laffon et al., 2006). These musselpopulations were transplanted from their original locations tothe Lorbé area in the Ría de Ares-Betanzos. A number of tworopes were used for each mussel population, with a density ofapproximately 15 kg of mussel seed per rope, an average den-sity commonly used in the Galician raft culture (Pérez Camachoand Labarta, 2004). Experimental culture was carried out untilFebruary 2004 using a raft system (500 m2) usually employedfor the mussel culture in Galicia with commercial protocols andtechniques described by Peteiro et al. (2006).

2.2. Mussel sampling

Individuals of M. galloprovincialis were collected monthlyfrom each rope at 3–4 m depth, using two replicates of 200–300individuals. Individual mussel lengths were recorded as the

res), the location of the experimental culture (underlined), the place where theaffected by the oil spill (between Finisterre Cape and Ría de Pontevedra).

Fig. 2. Temporal trends of gross biochemical components (average values(n=6)±SD) for the three populations during the experimental culture in Ría deAres-Betanzos (March, 2003/February, 2004). A) Protein contents B)carbohydrate contents C) glycogen contents D) total lipid contents.

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maximum measure along the anterior–posterior axis to thenearest 0.1 mm using callipers (Mitutoyo ®), and each samplewas divided into 1 mm length classes. Adjusted shell length wascalculated by the formula: L=∑(CL ·F) ·N−1 (Box et al.,1989), where L is the shell length average value, CL is theindividual length class, F is the frequency, and N is the totalnumber of individuals considered. From each sample, three sub-samples were selected: one from the adjusted length class, onefrom five length classes below and one from five length classesabove. The number of mussels collected in each sub-sample toobtain flesh tissues for the biochemical analysis varied duringthe culture according to the increase of the adjusted length inorder to obtain approximately 100 mg of dry tissue weight.After dissecting, tissues were frozen, freeze-dried and stored at−70 °C. Prior to the different biochemical analyses the tissueswere pulverised in a pottery mortar and homogenised with andultrasonic Branson Sonifier (250/450 USA).

2.3. Biochemical composition

Proteins were determined using the method described byLowry et al. (1951) after alkaline hydrolysis with 0.5 N NaOHat 30 °C. Carbohydrates were quantified as glucose by thephenolsulphuric acid method (Strickland and Parsons, 1968).Glycogen was also quantified as carbohydrates after precipita-tion with 100% ethanol. Lipids were extracted according to amodified Bligh and Dyer (1959) method (Fernández-Reirizet al., 1989) and gravimetrically determined.

The lipid classes were determined by thin layer chromatog-raphy (TLC). The chromatographic plates were developedfollowing the method of Freeman and West (1966). The plateswere developed with a 10% CuSO4 and 0.85% H3PO4 solutionheated to 180 °C (Bitman and Wood, 1982). Qualitative stan-dards employed for the quantitative analysis of the sterol estersand waxes, sterols, free fatty acids (FFA) and triacylglycerols(TAG), were cholesterol palmitate, cholesterol, palmitic acidand tripalmitin (Sigma), respectively. The standard for thequantitative analysis of phospholipid content was obtained frommussels (M. galloprovincialis).

2.4. Data analysis

Gross biochemical data were analysed on the basis of μg ofbiochemical fraction per mg of total organic matter (OM) andlipid classes data on the basis of μg of lipid class per mg of totallipids.

In order to verify that sub-samples from different lengthclasses but from the same sample could be treated likereplicates, we used an ANOVA between sub-samples for eachbiochemical fraction. When Levene's test showed no homoge-neity of variances, Kruskal–Wallis test was used. None of thesamples showed significant differences between their sub-samples (PN0.05) for any biochemical fraction, then all wereconsidered replicates.

Differences in concentrations of gross biochemical compo-nents and lipid classes among locations during the experimentalculture were tested by ANOVA and Tukey's tests as post-hoc

test. When Levene's test showed no homogeneity of variances,Kruskal–Wallis test followed by the U Mann–Whitman pair-wise contrast were used. We applied the Bonferroni correction(Zar, 1984) each time a set of pair-wise contrasts were used. Alldata analysis was performed following the statistical packageStatistica 6.0.

3. Results

3.1. Gross biochemical composition

Although with punctual differences, all populations showedsimilar patterns in gross biochemical components during theculture either in the proportion in which proteins, carbohy-drates and lipids are present or in their seasonal distribution(Fig. 2A–D).

Fig. 3. Temporal trends in lipid classes (average values (n=6)±SD) for the threepopulations during the experimental culture in Ría de Ares-Betanzos (March,2003/February, 2004). A) Phospholipid contents B) triacylglycerol contents C)free fatty acid contents D) sterol contents.

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Proteins were the main biochemical component during mostof the culture time (Fig. 2A). Maximum concentrations ofprotein were recorded in early spring (March, 2003), whenindividuals from Redes and Miranda reached values of 739.28and 736.59 μg prot/mg OM, respectively which weresignificantly higher than values of Pindo mussels (724.14 μgprot/mg OM; F2,15=5.07 Pb0.05). During spring and summer,protein contents gradually decreased in all populations, untilminimum values were obtained in early autumn (September,2003) when mussels from Redes showed significant lowervalues than the rest of populations (315.01, 353.93 and355.21 μg prot/mg OM for Redes, Miranda and Pindopopulations, respectively; F2,15=5.27 Pb0.05). Protein con-tents increased gradually during autumn–winter until valueswere similar to those of the onset of the culture (March, 2003;Fig. 2A) for all populations.

Carbohydrates showed a seasonal trend opposite to that ofproteins (Fig. 2B) with minimum values at the onset of theexperimental culture (103.85, 97.58 and 89.28 μg CH/mg OMfor Pindo, Redes and Miranda populations, respectively inMarch, 2003; F2,15=5.10 Pb0.05 between Pindo and Miranda)and maximum values in early autumn (551.28, 522.48 and518.20 μg CH/mg OM for Redes, Miranda and Pindo popu-lations, respectively in September, 2003; F2,15=2.61 P=0.107)when carbohydrates were the main component for all thepopulations. Carbohydrate contents decreased in all populationsprogressively during autumn–winter until values similar tothose in spring were reached.

With regard to glycogen contents, all populations showedsimilar trends to those of total carbohydrates (Fig. 2C).Glycogen values represented between 25 and 76% of totalcarbohydrates depending on the season of the year. Months withlowest glycogen contribution to total carbohydrates corre-sponded to spring period, with percentages between 25.5% forRedes population (March, 2003) and 27.9% for Mirandamussels (May, 2003; Fig. 2C). During the early autumn,glycogen represented about 70% of total carbohydrates forRedes and Pindo populations (340.69 and 345.90 μg glycogen/mg OM for Redes and Pindo populations, respectively) whereasfor the case of Miranda population, glycogen only representedabout 55% of total carbohydrates, which is a significantly lowermaximum (254.76 μg glycogen/mg OM) in September 2003(F2,15=9.64 Pb0.01; Fig. 2C).

Lipid contents were more stable during the experimentalculture for all populations with regard to the other components.Despite lipid stability, all populations showed a peak in May2003 and a recovery of the spring values in late winter(Fig. 2D). These fluctuations were less marked than in proteinsvariability which duplicated their concentration as well as thatof carbohydrates that increased their minimum concentration 5–6 times at certain moments of the year (Fig. 2A–B). During thespring peak, Redes population reached the greatest lipid con-centrations (170.34, 182.68 and 227.80 μg total lipids/mgOM; Pindo, Miranda and Redes populations, respectively;F2,15=39.29 Pb0.001). During the late winter peak, bothRedes and Miranda mussels showed higher lipid concentrationsthan Pindo mussels (142.64, 175.64 and 176.60 μg total lipids/

mg OM for Pindo, Redes and Miranda populations, respectivelyin February, 2004; F2,15=4.75 Pb0.05).

3.2. Lipid classes

At the onset of the experimental culture (March, 2003),Pindo mussels showed triacylglycerol concentrations 3–4 timeshigher than the values registered in the other populations (61.59,80.35 and 253.77 μg TAG/mg total lipids; Miranda, Redes andPindo populations, respectively; F2,15 = 50.52 Pb0.001;Fig. 3B). Moreover, Pindo population showed lower concentra-tions in free fatty acids (76.41, 135.22 and 155.17 μg FFA/mgtotal lipids; Pindo, Redes and Miranda populations, respective-ly; F2,15=19.81 Pb0.001; Fig. 3C) and phospholipids (538.64,625.32 and 718.86 μg phospholipids/mg total lipids; Pindo,

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Redes and Miranda populations, respectively; F2,15=18.21Pb0.005; Fig. 3A) than the rest of populations, also at the onsetof the culture.

Excluding the onset of the culture, lipid composition keptsimilar trends in all populations during the experimental culture(Fig. 3A–D).

Phospholipids were the main lipid class during the wholeculture in all populations (Fig. 3A). Although sharp decreaseswere registered in May and September 2003, and in late winter(February, 2004 for the population of Miranda and January,2004 for Pindo and Redes populations), phospholipid contentskept almost stable and with similar ranges for all the populationsthat represented similar annual average values for the threepopulations (684.61, 687.92 and 688.84 μg phospholipids/mgtotal lipids for Pindo, Redes and Miranda populations, re-spectively; F2,199=0.015 P=0.985; Fig. 3A).

Minimum phospholipid concentrations coincided in timewith peaks in triacylglycerol contents for all populations(Fig. 3A–B). Triacylglycerol peaks recorded in spring (May,2003) were the most important for all populations (344.70,381.26 and 432.20 μg TAG/mg total lipids; Miranda, Pindoand Redes populations, respectively), although with differentmagnitude between the three mussel origins (F2,15=30.68Pb0.005). In the autumn peak (September, 2003), Pindo mus-sels showed significantly higher triacylglycerol contents thanthe rest of populations (274.25, 291.24 and 361.69 μg TAG/mgtotal lipids for Redes, Miranda and Pindo populations, re-spectively; F2,15=12.62 Pb0.005), but in the late winter peak,Redes showed, as in the spring, the highest values (191.81,285.44 and 318.10 μg TAG/mg total lipids for Pindo, Mirandaand Redes populations, respectively; F2,15=10.35 Pb0.005;Fig. 3B). Maximums described here were followed by sharpdecreases in triacylglycerol contents for all populations.

With regard to sterol contents, maximums were recorded atthe onset of the culture for all populations (93.78, 97.57 and110.92 μg sterols/mg total lipids for Redes, Pindo and Mirandapopulations, respectively in March, 2003; Fig. 3D). During thefollowing months, sterol contents showed a sharp decrease andthese low values were kept until early autumn when concen-tration increased again and kept stable until the end of theculture (Fig. 3D). Although Miranda and Pindo populationsshowed an increase of sterol concentrations during summer, thisincrease was followed by a quick decline that equalized sterolcontents in the three populations at early autumn (30.5, 34.9 and38.3 μg sterols/mg total lipids for Pindo, Redes and Mirandapopulations, respectively in September, 2003; F2,15=1.09P=0.363).

Sterol esters and waxes only appeared as traces during themost of the culture (March, 2003/February, 2004) for all popu-lations. Only in early spring (March, 2003) Pindo and Redesmussels showed quantifiable concentrations of this compoundin their tissues (33.6 and 65.3 μg sterol ester+waxes/mg totallipids, respectively; F2,15=303.9 Pb0.001).

Free fatty acids showed two peaks during the experimentalculture (Fig. 3C). The first peak was recorded in early spring(March, 2003) by Redes and Miranda populations (135.22 and155.17 μg FFA/mg total lipid, respectively) and the second

during the summer for all populations (164.8 μg FFA/mg totallipid for Redes population in June, 2003; 141.6 and 201.7 μgFFA/mg total lipid for Miranda and Pindo populations,respectively in July, 2003). In the second maximum, Pindomussels showed significantly higher free fatty acid values thanthe rest (F2,15=12.7 Pb0.001). Peaks in free fatty acid contentswas followed by sharp decreases, and the minimum concentra-tions were recorded for all populations during late summer(22.56 and 23.94 μg FFA/mg total lipids for Redes and Mirandapopulations, respectively in August, 2003) and early autumn(25.77 μg FFA/mg total lipids for Pindo population inSeptember, 2003). During autumn–winter a progressive in-crease in free fatty acid values was recorded for all populations(Fig. 3C).

4. Discussion

Despite punctual differences, all populations showed similarpatterns in their biochemical composition during the experi-mental culture. Only at the onset of the culture (March, 2003),Pindo mussels showed marked differences in their lipidcomposition with regard to the other populations in triacylgly-cerol, free fatty acid and phospholipid content. These differ-ences are in agreement with values reported by Labarta et al.(2005) for the same populations in February, 2003. Labarta et al.(2005) characterised several mussel seed populations after thePrestige oil spill and detected for Pindo population disturbancesin their lipid fraction that implied high triacylglycerol levels andlow values in phospholipid contents. These alterations in thelipid composition as well as other physiological and biochem-ical stress indicators and hydrocarbon concentrations in theirtissues allowed the latter authors to classify Pindo mussels asindividuals from the area most affected by the spill. Gonzálezet al. (2006) agree in indicating a central area of the spillaccording to PAH concentrations in water that includes Pindolocation. Capuzzo and Leavitt (1988), when studying the lipidcomposition of the digestive gland of Mytilus edulis along apollution gradient, observed a direct relationship between totallipid contents and pollution level. With regard to the lipidcomposition, Capuzzo and Leavitt (1988) reported an incrementin triacylglycerol concentrations and a reduction in free fattyacid and phospholipid contents as pollution level increased.

Alterations in lipid metabolism as sublethal effect of organiccontaminants exposure such as PAHs or PCBs are extensivelydocumented (Capuzzo and Leavitt, 1988; Lowe, 1988; Leavittet al., 1990; Ferreira and Vale, 1998; Bergen et al., 2001; Chuet al., 2003; Petrovic et al., 2004; Smolders et al., 2004; Orbeaet al., 2006). Most of the surveys pointed out impairments in thedistribution and concentration of polar and neutral lipids, whichare commonly reflected by increases in triacylglycerol contents(Capuzzo and Leavitt, 1988; Leavitt et al., 1990; Bergen et al.,2001; Chu et al., 2003; Petrovic et al., 2004). Lowe (1988)suggested that many structural alterations in the digestiveepithelium of M. edulis were due to increased lipid accumula-tions associated with abnormalities in secondary lysosomes.Moreover, Capuzzo and Leavitt (1988) proposed a disruption inthe mobilisation of triacylglycerols into phospholipids pools, so

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decreases in polar lipids could affect the biosynthesis andintegrity of cell membranes.

Although differences in lipid composition in Pindo musselsat the onset of the experimental culture could reflect theirprevious exposure to hydrocarbons, this pattern did not lastduring the culture. In one hand, this could be due to a depurationphenomenon, since individuals were kept in an area not affectedby the spill and far away from the focal point of pollution.Larratxea and Pérez Camacho (1996) have reported a reductionon hydrocarbon concentration in M. galloprovincialis, affectedby the Aegen Sea oil spill in 1992, to levels of controlindividuals after keeping them for two months in a clean area.Moreover, Laffon et al. (2006) detected not only decreases inhydrocarbon content in mussel exposed to Prestige oil spill butalso signs of a certain DNA repair after a recovery time of sevendays in laboratory with clean seawater. Lack of long-termalterations in the biochemical composition, could be also aresult of a certain adaptation of individuals to the hydrocarbonsstored in their tissues. There are surveys that suggest certaintolerance of individuals to chronic hydrocarbons exposure(Thomas et al., 1999) and this adaptation might indicate a lackof sensitiveness of biochemical composition as long-term stressindicator in our case.

The hypothesis on lack of sensitiveness of biochemicalcomposition in long-term surveys might be supported byPeteiro et al. (2006) who detected long-term sublethal effectsthrough another physiological stress indicators at the end of thisexperimental culture (February, 2004). The latter parallel surveycarried out in the same experimental culture with the samepopulations reported slower growth for the population from thearea most affected by the spill (Pindo).

The lack of differences between Miranda and the referencepopulation (Redes) in their biochemical composition mayreflect that Miranda mussels were not affected or only hardlyaffected by the spill. Those agree with González et al. (2006)that located the less affected area at the north of the central zoneof the spill according to PAH concentrations in water, area thatincludes Miranda location. Labarta et al. (2005) obtainedsimilar results according to several physiological and biochem-ical stress indicators in mussels.

Excluding the onset of the culture, all populations followedsimilar trends in their biochemical composition, which mightbe determined by the reproductive and energetic reservecycles. Similar biochemical cycles in mussels were reportedin Ría de Arousa by Freites et al. (2002), and both studies are inagreement with seasonality of reproductive and energeticreserve cycles in M. galloprovincialis described by Villalba(1995) in Galician Rías. The latter author described the Ría deAres-Betanzos, area with only one annual spawn episode insummer although he also suggested the possibility of anautumn second spawning event when environmental conditionswere favourable. This pattern in reproductive cycle seems toagree with that observed in the present experimental culturebased on the maximums in triacylglycerol contents (May andSeptember, 2003) that coincided with condition index peaksdescribed by Peteiro et al. (2006) for the populations understudy.

The importance of the environmental conditions, specificallytemperature and food abundance versus the origin of thepopulations in the regulation of reproductive and energeticreserve cycles was extensively reported (Gabbott, 1983; Lowe,1988; Fernández-Reiriz et al., 1996). Moreover, Kopp et al.(2005) observed how M. edulis and M. galloprovincialis, spe-cies with no simultaneous reproductive cycles in their originaldistribution area, coupled their gametogenic and energeticreserve cycles when both were cultured in a common envi-ronment. Similar results were obtained by Beninger and Lucas(1984) in a similar experience with clams Tapes decussatus andTapes philippinarum.

Summarising, the biochemical pattern for the three popula-tions under experimental culture do not seem to be related to theprevious hydrocarbons exposure beyond the disturbancesdetected at the onset of the culture (March, 2003) in Pindopopulation. Lack of long-term alterations in Pindo populationcould be due to depuration of hydrocarbons stored or thedevelopment of certain tolerance to PAHs. Therefore, it is likelythat the fact of using the same zone for their culture seemed toregulate, in a higher magnitude, the energetic reserve cycle in along-term basis for the three populations than their origin ortheir previous biochemical status.

Acknowledgements

We would like to thank A. Ayala, B. González and L. Nietofor the biochemical analyses, H. Regueiro, M. García and O.Fernández-Rosende for technical assistance and PROINSAemployees for their help in sampling tasks. This study wassupported by the contract-project PROINSA, Code CSIC2004448, Galicia PGDIT03RMA13E.

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