Abstract Estuaries are marine areas at greatcontamination risk due to their hydrodynamicfeatures. PAH are wide and ubiquitous contam-inants with a high presence in these marine en-vironments. Chemical analysis of sediments canprovide information, although it does not give adirect measure of the toxicological effect of suchcontaminants in the biota. Samples of Venerupispullastra, Cerastoderma edule, and Mytilus gallo-provincialis were collected from two locations inCorcubión estuary (Norhwest of Spain). The levelof PAH in sediment and biota, and its possible ori-gin were assessed. A moderate level of contamina-tion was observed with a predominance of PAH ofa pyrogenic origin. Genotoxic damage, measuredas single-strand DNA breaks with the comet as-say, was evaluated in gill tissue and in hemolymph.The values of DNA damage obtained showed ahigher sensitivity of clams and cockles to the pol-
J. Fernández-Tajes (B) · F. Flórez · S. Pereira ·T. Rábade · J. MéndezDepartment of Cell and Molecular Biology,Faculty of Sciences, University of A Coruña,A Zapateira s/n, 15071 A Coruña, Spaine-mail: [email protected]
B. LaffonToxicology Unit, Department of Phsycobiology,University of A Coruña, A Coruña, Spain
lution load level. These differences among speciesmake us suggest the use of some other speciescoupled with mussels as an optimal tool for bio-monitoring estuarine environments.
Estuaries are semi-enclosed sea areas with limitedself-renewal ability and therefore particularly atrisk from contaminant inputs (Vlahogianni et al.2007). Pollution in these marine environments isconsidered a critical issue because of the highvariation in several abiotic factors, such as salinity,pH, and temperature, that alter the bioavailabilityand, by consequence, the toxicity of pollutants(Monserrat et al. 2007). The Corcubión estuarycovers the municipalities of Cee and Corcubión,in the far west of the Galician seashore. A remark-able feature of this area is the presence of a smelt-ing industry, a shipyard and both commercial andfishing harbors. In addition to these facts, thereare two extractive areas for commercial clams andcockles located in the inner part of the estuary.Although it is possible that there are large inputsof pollutants in this area, data on the presence of
such contamination agents and on their effects onmarine biota are scarce.
PAH are ubiquitous widespread contaminantsand may be generated mainly by three processes:(1) combustion of organic matter at very high tem-peratures (pyrolitic origin); (2) release of petro-leum (petrogenic origin); or (3) diagenic processes(degradation of the organic matter; Neff 1979).Once formed, PAH may enter marine coastalareas through the spillage of petroleum, indus-trial discharges, atmospheric fallout, and urbanrunoff (Neff 1979). Because of their low watersolubility and their hydrophobicity, heavy PAHrapidly become associated with organic and inor-ganic suspended particles (Gschwend and Hites1981; Chiou et al. 1998) being incorporated intomarine organisms via filtering activities. Genotox-icity of PAH is mainly linked to the ability ofmarine species to biotransform them into reactivemetabolites that can directly affect DNA or in-duce free oxygen radicals that would produce ox-idative damage in macromolecules such as DNAor proteins (Farmer 2003; Rocher et al. 2006).Estimates of the relative rate of DNA damageindicate that single strand breaks are the mostprevalent type of genetic damage caused by PAH(Bernstein and Bernstein 1991; Cheng and White2004).
Marine organisms can take up contaminantsfrom bottom sediments, suspended particulatematerial, the water column, and food sources(Livingstone 1993; Laffon et al. 2006). Incorpora-tion rate will depend not only on the availabilityof the contaminants but also on several bioticand abiotic factors (filtration rate, metabolismtemperature, salinity, pH, etc.). In this context,biomonitoring constitutes a good alternative anda complement to chemical systems used for con-tamination evaluation. Bivalves may result quiteuseful for the genotoxic biomonitoring of the seaenvironment because of their suitable size, widedistribution in both hemispheres, their filtering ac-tivity that favors bioaccumulation of contaminants(Khadim 1990), and their contact with sediments(Rank et al. 2005). In fact, they are consideredas good sentinel organisms and are widely usedto monitor the presence of toxic compounds inmarine environments (Solé et al. 1994; Porte et al.2000; Serafim and Bebianno 2001). Exposure of
aquatic organisms such as bivalves to genotoxiccontaminants could be a risk to human healthvia their incorporation through the food chain(Lemiere et al. 2005) and could also constitutean ecological risk that may lead to heritable mu-tations and loss in the total genetic diversity (ei-ther intra- o inter-species) with significant impli-cations for the long-term survival of natural pop-ulations (Bikham et al. 2000). Therefore, there isan increasing need to develop an early, fast, easy,and effective methodology to detect the geno-toxic consequences of exposure and metabolismof chemicals. In this sense, DNA strand breakshave been recognized as suitable biomarkers ofcontaminant DNA damage (Speit and Hartmann1995; Mitchelmore and Chipman 1998; Frenzilliet al. 2009) and have been widely measured bydifferent methods such as alkaline elution (Bihariet al. 1992) or alkaline unwinding (Nacci et al.1992; Liepelt et al. 1995). Among them, the sin-gle cell gel electrophoresis (Singh et al. 1988),or comet assay, has been recognized in severalstudies as a convenient method for biomonitor-ing DNA damage in the marine environment(Shugart 1988; Accomando et al. 1991; Frenzilliet al. 2001; Taban et al. 2004; Rank 2009).
The aims of this study were (a) to make apreliminary analysis of the PAH pollution levelin the Corcubión estuary, (b) to evaluate the po-tential of the comet assay to be used as a methodfor detecting genetic damage in gill cells andhemocytes of the clam Venerupis pullastra andthe cockle Cerastoderma edule; (c) to evaluate thesensitivity of both cell types to DNA damage, andto compare their relative sensitivity with Mytilusgalloprovincialis as one of the most bivalve widelyused as a sentinel species in biomonitoring studies.
Materials and methods
Sample collection
Clams (V. pullastra), mussels (M. galloprovin-cialis), and cockles (C. edule) were collected fromtwo locations in the inner part of the Corcubiónestuary (Corcubión, 42◦56′42.89′′ N 9◦11′35.05′′ O;A Concha, 42◦57′08.52′′ N 9◦11′23.23′′ O) close tothe smelting industry and the fishing port (Fig. 1).
Fig. 1 Map showing the location of the sampling sites.A Corcubión sampling site, B A Concha sampling site; 1fishing harbor, 2 shipyard, 3 smelting industry
Together with organisms, sediment samples weretaken in opaque plastic bags. The collected bi-valves from both polluted sites and from hatcherywere characterized by a similar maximum lengthand size (4.21 ± 0.5 cm for clams, 6.57 ± 0.5 cmfor mussels, and 3.15 ± 0.5 cm for cockles). It was75–80% of the maximum size reached within eachpopulation. This approach guaranteed that com-pared individuals from same species had similarmetabolic conditions and the influence of physi-ological differences between the two populationswas less pronounced (Regoli 2000; Namiesniket al. 2008).
Samples were transported immediately to thelaboratory, and the bivalves were placed in aquar-iums with filtered seawater (total PAH content<5 ng L−1) inside of a photoperiod chamber at18◦C in cycles of 12 h light–darkness for 1 day,to clear gut contents prior to the experiments.Reference bivalves were obtained from a hatch-
ery belonging to the CIMA (Centre of MarineInvestigations, Ribadeo, Spain) and acclimated inthe same abovementioned conditions. For eachlocation, four to eight individuals (depending onthe species) were used for PAH content deter-mination, and nine individuals were randomly se-lected for DNA damage evaluation by means ofthe comet assay.
Analysis of PAH in sediment and biota
Determination of total PAH content (TPAH) insediment and biota was performed by means ofa gas chromatography coupled to tandem massspectrometry using a Trace GC 2000 gas chro-matograph (Thermo Finnigan, Walthman, MA,USA) equipped with a GC PAL Autosampler(CTC-Analytics, Zwingen, Switzerland), PTV andsplit/splitless injectors, and coupled to a ThermoFinnigan Polaris Q ion trap mass spectrometer.
Reagents and standards
PAH native stock solution (PAH-STK-A,4,000 ± 200 ng mL−1 in isooctane) contain-ing naphthalene, acenaphthene, fluorene,phenanthrene, anthracene, fluoranthene, pyrene,benz(a)anthracene, chrysene, benzo(b)fluorene,benzo(k)fluoreanthene, benzo(j)fluoranthene,benzo(a)pyrene, indeno (1,2,3-cd)pyrene, dibenzo(a,h)anthracene, was purchased from WellingtonLaboratories (Guelph, ON, Canada) and usedfor preparing calibrations solutions. Calibrationsolutions were prepared by dilution of PAH-STK-A standard with isooctane. Solid standardsfor other PAH were purchased by Supelco(Bellefonte, PA, USA).
PAH-labeled compound solution (PAH-LCS, 2,000 ± 100 ng mL−1 in isooctane)containing naphthalene–d8, acenaphthene-d10,fluorene-d10, phenanthrene-d10, anthracene-d10,fluoranthene-d10, pyrene-d10, benz(a)anthra-cene-d12, chrysene-d12, benzo (b)fluorene-d12,benzo(k)fluoreanthene-d12, benzo(a)pyrene-d12,indeno(1,2,3-cd)pyrene-d12, and dibenzo(a,h)-anthracene-d14 was purchased from WellingtonLaboratories (Guelph, ON, Canada) and used assurrogate standard.
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Reference material SRM 2977 ‘Mussel Tissue.Organic contaminants and trace elements’ wereprovided by NIST (Gaithersburg, MD; USA).
Operation conditions
PAH separation was achieved with a DB-XLBcolumn (60 m × 0.25 mm × 0.25 um filmthickness) and the GC oven temperature pro-gramme consisted of 50◦C (3 min) and increasedat 4◦C min−1 up to 325◦C (held for 20 min).The injection was carried out in PTV-splitless, theinjected volume was 9 μL, and the injector pro-gramme started at 55◦C and increased at 3◦Cs−1
up to 300◦C (held for 20 min).Mass spectrometer worked in MS/MS mode,
particular conditions (precursor ion, product ion,and collision energy and isolation time) were op-timised for each compound and the ionizationvoltage was 70 eV. Transfer line and ionisationsources were 300◦C and 250◦C, respectively.
Extraction procedure
Different sample preparation methods were ap-plied depending on the type of matrix analyzed:
1. Sediment samples: before extraction eachsample of frozen dried sediment (2 g) wasspiked with 5 μL of PAH-LCS standard.The extraction was carried out by MAE with15 mL of acetone–hexane (1:1, v/v). Sulfurwas removed treating the extracts with Cuactivated with HCl. The extracts were cleanedwith a 5 g silica (3% deactivated) columnand eluted with 15 mL of dichloromethane–hexane (20:80, v/v). Then they were concen-trated in a rotary evaporator, dried undernitrogen stream, and redissolved in 100 μL ofhexane.
2. Biota samples: before extraction each sampleof freeze-dried biota (5 g) was spiked with5 μL of PAH-LCS standard. The extractionwas carried out by MAE with 15 mL ofacetone–hexane (1:1, v/v). The extracts werecleaned with a 22 g aluminum oxide (10%deactivated) column and eluted with 90 mL ofhexane. Then they were concentrated in a ro-
tary evaporator, dried under nitrogen stream,and redissolved in 100 μL of hexane.
Comet assay
Cell viability assayed by the Trypan-blue exclu-sion method was observed in the range of 90–100% for all samples.
The gills of the three species were excised andsliced, using dissection scissors and tweezers, andplaced in a tube containing 3 ml of CMFS solution(20 mM HEPES, 500 mM NaCl, 12.5 mM KCl, and5 mM EDTA) and left for an hour shaking slowlyhorizontally. They were then placed in a verticalposition for 5 min in order to allow the piecesof tissue to settle. The supernatant containingthe dissociated cells was collected with a pipette,transferred to another tube, and centrifuged at1,500 rpm for 5 min at 4◦C. Having removed thesupernatant, the pellet was resuspended in 1.5 mLof Kenny’s Salt Solution (0.4 M NaCl, 9 mM KCl,0.7 mM K2HPO4, and 2 mM NaHCO3).
Hemolymph was withdrawn from the posterioracductor muscle into a hypodermic syringe con-taining Alsever (glucose 20.80 g/L, sodium citrate8.00 g/L, EDTA 3.36 g/L, NaCl 22.50 g/L, andpH 7.5) and kept in ice until centrifugation.
The alkaline comet assay was performed inisolated gill cells and hemocytes following themethod described by Pérez-Cadahía et al. (2004).
One hundred randomly selected cells from eachindividual were examined, using the image cap-ture and analysis QWIN Comet (Leica ImagingSystems, Cambridge, UK). Percentage of DNAin tail comet (DNAt) was used to evaluate DNAdamage.
Statistical analysis
Statistical analyses were performed using theSPSS for Windows statistical package, version 12.0(Illinois, USA). Kolmogorov–Smirnov goodness-of-fit was applied to compare the distributionof the variables obtained in the study with thenormal distribution. As the distribution of all vari-ables departed significantly from normality, non-parametric tests were deemed adequate for thestatistical analysis of the data. The existence of sig-nificant differences among groups was determined
Environ Monit Assess (2011) 177:289–300 293
by means of the Kruskal–Wallis test, followed bya Mann–Whitney U test, to evaluate the effect ofthe origin and tissue of the three species. Level ofsignificance was set at 0.05. Associations betweentwo variables were analyzed by Spearman’s corre-lation. Level of significance was set at 0.01.
Results and discussion
PAH and their origin in sediments
One of the aims of this study was to makea preliminary evaluation of the pollution lev-els in the estuary of Corcubión. The total PAH(16 USEPA priority PAH) content in sedimentswere 449.63 for Corcubión and 108.46 for AConcha. According to these values and to theclassification proposed by Bihari et al. (2006),Corcubión could be classified as fairly contam-inated (250 μg/kg < TPAH < 500 μg/kg), andA Concha as slightly contaminated (TPAH <
250 μg/kg). The 16 USEPA priority pollutants inthe sediments were present in the two samplingsites, excepting for the three rings acenaphthyleneand acenaphthene that were below the detectionlimit in both locations, and for the two rings PAH
Table 1 PAH concentrations (μg/kg d.w.) in sedimentfrom Corcubión and A Concha
naphthalene, which was only present in Corcubión(Table 1).
In this study, we also investigated the sourcesof PAH. The distribution of light and heavy PAHreflects the common practice in the literature toassess the source of PAH, since the presence oflighter PAH is usually linked to a petrogenic ori-gin (Soclo et al. 2000; Colombo et al. 2006), andthe sources of the heavier fraction are more likelyto be identified as the pyrolitic ones. Accordingto this classification, sediments for both locationswould have a pyrolitic origin since the proportionof the lighter PAH (two and three rings) wasbelow 13% of the total concentration (Fig. 2).Another method recently used to estimate theorigin of PAH compounds, is to apply molecularindices based on the thermodynamic stability ofvarious isomeric compounds. PAH of molecularmass 178 (anthracene and phenantrene) and 202(fluoranthene and pyrene) are commonly used todistinguish between combustion and petroleumsources (Sicre et al. 1987; Budzinski et al. 1997;Soclo et al. 2000; Yunker et al. 2002). Accordingto index for mass 178, anthracene to anthraceneplus phenanthrene (An/178) ratio <0.10 usually istaken as an indication of petroleum while >0.10indicates a dominance of combustion. Values of0.02 for Corcubión site and 0.08 for A Conchasite reflect a petroleum source. Although this in-dex indicates a petrogenic origin, emissions forlignite have reported values below 0.10 (Yunker
Fig. 2 PAH distribution pattern (accumulated percentage)in sediments from Corcubión and A Concha
et al. 2002). The presence of a smelting indus-try located in an area close to sampling site ofCorcubión, which indeed uses lignite as raw ma-terial, could indicate a pyrolitic origin instead ofa petrogenic one. For mass 202 fluoranthene tofluoranthene plus pyrene (Fl/Fl+Py) values ob-tained for Corcubión (0.49) and A Concha (0.46)are characteristic of combustion of liquid fossilfuel (vehicle and crude oil; Yunker et al. 2002).The benz(a)anhracene to benz(a)anthracene pluschrysene (BaA/228) ratio was above 0.50 in bothlocations (0.84 for Corcubión and 0.68 for A Con-cha) implying combustion source of sediments.For the indeno(1,2,3-cd)pyrene to indeno(1,2,3-cd)pyrene plus benzo(ghi)perylene (IP/IP+Bghi)ratio, values above 0.50 are indicative of grass,wood, and coal combustion (Yunker et al. 2002).According to the different indexes (An7178,BaA/228, Fl/Fl+Py, and IP/IP+Bghi), there is apredominance of combustion origin although withan additional contribution from petrogenic hy-drocarbons present in the marine environment.These petroleum inputs assessed by the An/178ratio could be a result of oil spills due to therelease of petroleum by ships. However, as it hasbeen mentioned above, combustion of lignite hasreported values below 0.10. The results suggestthat the sources of PAH are mainly related to ve-hicle and ship traffic, fuel and wood combustion.Atmospheric deposition may be one of the major
PAH transport pathways into the estuary for PAHderived from combustion processes. Combustion-derived PAH emitted to the atmosphere can en-ter the water column by gas exchange across theair–water interface, dry deposition of airborneparticulate matter, or wet deposition by rainfall(Gustafson and Dickut 1997; Karacık et al. 2009).The wood fires in 2005 together with the floodsin early 2006 close to the Corcubión estuary areprobably the source of PAH with combustion ori-gin found in the sediments.
PAH in mussels, clams, and cockles
Table 2 shows the individual and TPAH con-centrations in the mussels, clams, and cocklescollected from Corcubión and from A Concha.Total concentrations for the 16 priority PAH were1,280.12 and 583.40 in clams, 1,052.62 and 909.24in mussels, and 843.26 and 309.75 in cockles forCorcubión and A Concha, respectively. The mostimportant contributors to PAH burden were ace-naphtene and fluoranthene in mussels; acenaph-thene and phenantrene in clams; and acenaph-thene, fluoranthene, and phenanthrene in cockles.
Although the accumulation pattern of PAH insediments and in the three species is not corre-lated, a positive relationship exists between conta-mination level and content of PAH in the studiedbivalves. Thus, Corcubión exhibited the highest
Table 2 PAHconcentration (μg/kgd.w.) in tissues fromVenerupis pullastra (Vp),Mytillus galoprovincialis(Mg), and Cerastodermaedule (Ce) in Corcubión(C) and A Concha (AC)
levels of total PAH in sediment, and all bivalvescollected from this site showed the highest contentof PAH.
Figure 3 shows the PAH distribution patternin the three species for both locations. Surpris-ingly, light PAH were not dominant in the threebivalves and either location. The four, five, andsix rings PAH accounted for more than the 70%of TPAH in mussels from Corcubión, and above50% of TPAH in cockles from A Concha. Thesedifferences between species and locations couldbe due to different ways of PAH intake. Thelighter PAH possess a higher water solubility,allowing the direct absorption by water and inter-stitial water, while the heavy ones would be ab-sorbed on particulate matter and should be assimi-lated by the digestive system of marine organisms.The presence of higher levels of much less solublePAH, such as benzo(a)pyrene, in bivalves thanin sediment, reinforce the idea that heavy PAHshould be assimilated by ingestion of particles. Itshould be noted that some of those PAH, such asdibenzo(a,h)anthracene, are more accumulated inbivalves compared to sediments.
Regardless of the species, PAH compositionpattern was dominated by the presence of three-ring PAH, followed by those with four rings, ex-cept for the mussels from Corcubión, for whichthe most abundant were the four-ring PAH. Asimilar trend, with high predominance of three-and four-ring PAH, was also reported by Binelliand Provini (2003), and by Perugini et al. (2007)in some species of bivalves. The total PAH con-centrations in mussels, clams, and cockles werecomparable with those reported for moderately
contaminated areas (200–1,000 ng/g) of differentbays worldwide (Baumard et al. 1999; Chase et al.2001; Liu and Kueh 2005), and at least one orderof magnitude lower than in highly anthropogeni-cally impacted sites (Telli-Karakoç et al. 2002;Liu and Kueh 2005). Although these levels couldbe considered as moderate levels, mussels fromCorcubion exhibited higher values to those ob-tained by Nieto et al. (2006) in mussels collected3 days after the Prestige shipwreck.
The different values of molecular indices(An/178, Ba/228, Fl/FL+Pyr, IP/IP+Bghi) calcu-lated in mussels, clams, and cockles (Table 3)reflect a similar trend than that obtained for sed-iments. The combustion seems to be the mainsource for PAH, but with a certain petrogenicinput.
The three species of bivalves analyzed in theexposed location are commercially harvested.If we attend to the threshold level proposedby the Food Safety Administration (USA) forcommercial exploitation of these organisms(200 μg/kg d.w. of the sum of the six HAP:benzo(a)anthracene, benzo(b) and benzo(k)-fluorantene, benzo(a)pyrene, dibenzo(a,h)-anthracene, and indeno(1,2,3-c,d)pyrene),mussels exceed this limit. Thus, mussels harvestedfrom Corcubión estuary should be analyzed priorto human consumption to avoid possible adversehealth effects.
Comet assay
Table 4 shows the results obtained in the cometassay carried out with gills and hemocytes in the
Fig. 3 PAH distributionpattern (accumulatedpercentage) in tissuesfrom Venerupis pullastra(Vp), Mytilusgalloprovincialis (Mg),and Cerastoderma edule(Ce) from Corcubión andA Concha
A Concha V. pullastra 0.050 Petrogenic 0.458 Combustion/ 0.522 Combustion 1.000 CombustionPetrogenic
M. galloprovincialis 0.195 Combustion 0.956 Combustion/ 0.852 Combustion 0.997 Combustionpetrogenic
C. edule 0.018 Petrogenic 0.980 Combustion/ 0.001 Petrogenic 0.999 CombustionPetrogenic
An178 Anthracene to Anthracene plus Phenanthrene index, Fl/+Fl+Py Fluoranthene to Fluoranthene plus Pyrene index,BaA/228 Benzoanthracene to Benzoanthracene plus Crysene index, IP/IP+Bghi indeno(1,2,3-cd)pyrene to indeno(1,2,3-cd)pyrene plus benzo(ghi)perylene (IP/IP+Bghi) ratio
three species from both locations and those fromthe aquaculture farm (reference).
DNA damage in mussels from Corcubión andA Concha was significantly higher than in musselsfrom the reference site. The values of DNAt ob-tained were practically equal for both locations, AConcha being slightly higher. For clams and cock-les, DNA damage was also significantly greaterin the two polluted sites than in the reference(Fig. 4). Although the three species showed in-creased genetic damage for both exposed sites,the levels of this damage differed, M. galloprovin-cialis being the most affected species, followedby C. edule and V. pullastra. The backgroundlevel of DNA damage, determined as the DNAtvalue obtained in the reference site, was higherin M. galloprovincialis (Table 4). The differenceamong species probably reflects differences in ex-posure (clams and cockles are buried), but mayalso reflect differences in physiology (metabolism,filtration rate, retention capabilities of PAH, etc).
Downs et al. (2002) reported species-specific re-sponses in mussel and clams collected from oil-impacted sites. They found differences betweenoiled and unoiled sites and between the re-sponse of the two bivalves in metabolic activa-tion and clearance of benzene, benzo(a)pyrene,and benzo(a)pyrene diol epoxide adducted pro-tein. They suggested that the differences in thehabitat might have contributed, the clams beingexposed to a higher concentration of pollutantsbecause of their location in the inner sediments.Other authors, such as Thomas et al. (2007) havealso found species-specific differences betweenmussels and clams, attributing the obtained re-sults to the habitat and the different physiologi-cal capacities of each species. In 2003, Woottonet al. undertook a study to evaluate the PAH-induced immunomodulation in three species, andthey concluded that the immunomodulation in thesentinel specie Mytilus edulis was no typical ofthe other two species studied, Ensis siliqua and
Table 4 Results of the comet assay carried out with gills and hemocytes of three species
Gills Hemolymph
Control Corcubión A Concha Control Corcubión A Concha
aSignificant differences between the sampling site and the control group (p value <0.05)
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Fig. 4 DNAt values in Venerupis pullastra (V. pullastra), Cerastoderma edule (C. edule), and Mytilus galloprovincialis (M.galloprovincialis) in gill cells and hemolymph. *p < 0.05, significant differences between the exposed and control groups
C. edule, suggesting that these would be moresensitive, and therefore, could reflect the gen-eral immune response to PAH more accurately.Cheung et al. (2006) also observed a high level ofDNA damage in hemocytes of C. edule comparedto those of M. edulis. These authors explain theresults as a consequence of differences in the an-tioxidant defense mechanism among species.
Hemolymph cells always exhibited lower levelsof DNA damage than gill cells except for C. edule,in which the highest levels of damage were indeedfound in this tissue. The observed difference be-tween tissues has been reported in other studies,in which the circulating cells were described asless sensitive than gill cells (Steinert et al. 1998;Coughlan et al. 2002; Rank et al. 2005). This fea-ture is likely to be due to either: (a) gills are in di-rect contact with the dissolved and particle-boundcontaminants, representing the most importanttissue in the uptake of contaminants (Frenzilliet al. 2009), or (b) the difference is related tothe different capacities in the detoxification ofpollutants, DNA-repair capacity/ability, and cellturnover (Akcha et al. 2004). Nevertheless, Rank
and Jensen (2003) found similar levels of DNAdamage in both cell types. They proposed the useof hemocytes for biomonitoring since they aremore easily manipulated and do not require celldissociation prior to the comet procedure. Thisdissociation procedure could have the potential ofintroducing damage through mechanical manipu-lation to obtain isolated cells (Frenzilli et al. 2009).In our case the differences obtained betweenboth tissues were not statistically significant.Thus, we recommend the use of hemocytes be-cause they are easier to obtain through relativelynon-invasive techniques that do not introducesupplemental DNA damage and, due to theirphysiological role in the transport of endoge-nous and exogenous substances and in immunedefense, are directly exposed to contaminants(Mersch et al. 1996) making them very convenientfor biomonitoring studies.
Only clams and cockles showed higher DNAtvalues in the location with a higher TPAH contentin sediment. Likewise, a high PAH body burdendid not directly imply a higher genetic damagefor any of the three species analyzed. This fact
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was indeed surprising, since accumulated PAHshould be, at least in part, responsible for thegenotoxic effects detected. Some authors havereported similar results (Large et al. 2002; Akchaet al. 2004). Laffon et al. (2006) did not find cor-relation between tissue levels of PAH and DNAdamage and suggested that DNA damage inducedby PAH could not be produced immediately, andthat a certain extension of it could already havebeen repaired in the moment of undertaking thecomet assay. Thomas et al. (2007) reported thatanalyses of PAH loads in tissues do not measureany form of PAH metabolites, especially from achronic exposure; hence, values of PAH body bur-den would be probably underestimates of the to-tal active damaging compounds. Likewise, Wesselet al. (2010) stated that the determination of thePAH tissue concentration is not suitable for theevaluation of individual exposure to PAH sincetheir genotoxicity is produced for the reactivemetabolites and not for the parental compound.It should also be taken into consideration thatmany chemicals that interact with DNA in manydifferent ways and that were not analyzed mightbe present in both water and sediments, constitut-ing a very complex scenario.
Conclusion
In summary, the levels of the 16 priority USEPAPAH observed in the estuarine environment ofCorcubión reflected a moderate level and a lowlevel of pollution for sampling sites from Cor-cubión and A Concha, respectively. Despite thefact that levels of carcinogenic PAH were lowin sediments, high levels of those same PAHwere detected in body burden of mussels, clams,and cockles, and mussels exceeded the thresholdlevel recommended for the human consumptionproposed by the US Food Safety Administrationfor commercial exploitation of these organisms.The DNA damage, evaluated by the comet assay,showed a good relationship with the pollutionload level at both sampling sites, except for M.galloprovincialis. The two cell types evaluated,hemocytes and gill cells, showed similar results.Consequently, we recommend the use of hemo-cytes, since they require less handling. The higher
sensitivity of clams and cockles, as compared tomussels, makes us propose the use of other speciescoupled with M. galloprovincialis for the optimalbiomonitoring polluted marine environments.
Acknowledgements This work was funded by a07MMA013103PR grant from the “Conselleria deInnovacion e Industria (Xunta de Galicia)”. We aregrateful to “Cofradía de Pescadores y mariscadores deCorcubión” for providing samples in this study and to Mrs.Ceres Fernandez for her kindly revision of the Englishgrammar style.
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