Ecotoxicology and Environmental Safety 73 (2010) 1279–1285

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Ecotoxicology and Environmental Safety

0147-65

doi:10.1

n Corr

E-m

journal homepage: www.elsevier.com/locate/ecoenv

In vivo genotoxicity and stress defences in three flatfish speciesexposed to CuSO4

H. Chairi a, C. Fernandez-Diaz b, J.I Navas c, M. Manchado b, L. Rebordinos a,n, J. Blasco d

a Laboratorio de Genetica, Facultad de Ciencias del Mar y Ambientales, Universidad de Cadiz, Polıgono Rıo San Pedro, s/n, 11510, Puerto Real, Cadiz, Spainb IFAPA Centro ‘El Toruno’, Junta de Andalucıa, Ctra Tiro Pichon s/n, 11500 El Puerto de Santa Marıa, Cadiz, Spainc IFAPA, Centro Agua del Pino, Junta de Andalucıa, Ctra, Punta Umbrıa-Cartaya s/n, 21459, Cartaya, Huelva, Spaind Instituto de Ciencias Marinas de Andalucia (CSIC), Polıgono Rıo San Pedro, s/n11510, Puerto Real, Cadiz, Spain

a r t i c l e i n f o

Article history:

Received 14 April 2010

Received in revised form

14 July 2010

Accepted 17 July 2010Available online 3 August 2010

Keywords:

Genotoxicity

Flatfish

CuSO4

Antioxidant enzymes

Stress protein

Comet assay

13/$ - see front matter & 2010 Elsevier Inc. A

016/j.ecoenv.2010.07.028

esponding author. Fax: +34956016448.

ail address: laureana.rebordinos@uca.es (L. Re

a b s t r a c t

We have used the comet assay to analyse, after 3 h, 24 h and 6 days, the genotoxic effect in vivo of

applying a single intraperitoneal injection of CuSO4, at a concentration of 2 mg/kg, to adult specimens of

Solea senegalensis, Dicologlossa cuneata and Scophthalmus rhombus. Metals content (Cu, Zn and Cd) in

liver was also measured. The activity of key stress defences was evaluated by analysing antioxidant

enzyme activity (catalase (CAT), superoxide dismutase (SOD), total glutathione peroxidase (t-GPX),

glutathione reductase (GR), glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate

dehydrogenase (6PGDH)), metallothionein (MT) and heat shock proteins (HSP70 and HSP60).

The results show that CuSO4 intake generates high and cumulative levels of genotoxicity throughout

the 6 days in all 3 species. After 6 days, metals content detected in specimens showed significant

differences from controls. Inter-species differences were detected in enzyme activity (Po0.05). A clear

response to CuSO4 was detected only in S. rhombus, with an increase of MT and a decrease of HSPs.

Variations in antioxidant defence levels and their comparative responses to the stress-inducing agent

are discussed.

& 2010 Elsevier Inc. All rights reserved.

1. Introduction

Pleuronectiformes (flatfish) constitute a broad taxonomic groupcomprising 11 families and about 500 species worldwide (Froeseand Pauly, 2007; Helfman et al., 1997). All these species share incommon an asymmetrical body development and a bottom-dwelling mode of life. Some of these species, such as Atlantichalibut (Pleuronectidae: Hippoglossus hippoglossus), turbot(Scophthalmidae: Psetta maxima), Dover sole (Soleidae: Solea solea)and Japanese flounder (Paralichthyidae: Paralichthys olivaceus) areof significant commercial interest in fisheries and aquaculture.Moreover, additional flatfish species are being studied for aqua-culture diversification in southern Europe including wedge sole(Soleidae: Dicologlossa cuneata), brill (Scophthalmidae: Scophthal-

mus rhombus) and Senegalese sole (Soleidae: Solea senegalensis).Copper sulphate (CuSO4) is a compound commonly used as an

algaecide, antifungal and antiparasite agent in aquaculture,especially in extensive production systems such as ponds.By other hand, copper is a ubiquitous metal which is present inestuary and coastal ecosystems as consequence of inputs from

ll rights reserved.

bordinos).

agricultural, mining, industrial activities and urban sewage.This metal is bioaccumulated by marine organisms and it canexert toxic effects. To produce these effects the reactive cationCu2 + seems to bind non-specifically to proteins and nucleic acidsin the cell, as well as increasing the rate of free radical formation,to promote cytotoxicity. However, copper sulphate could havecollateral effects in fish by inducing DNA strand breakages, andgenotoxicity in a tissue- and species-specific way (Arkhipchukand Garanko, 2005; Cavas et al., 2005; Gabbianelli et al., 2003;Gravato et al., 2006; Oliveira et al., 2008). The comet assay is atoxicogenetic technique considered to be a good indicator ofgenotoxicity. It was originally developed and applied in medicalresearch (Neri et al., 2006; Kopjar and Garaj-Vrhovac, 2005), but itis now widely used for environmental biomonitoring (AbouChak-ra et al., 2007; Pruski and Dixon, 2007). This assay has proved tobe useful for measuring DNA strand breaks in marine andfreshwater species in the presence of genotoxic compounds(Nogueira et al., 2006; Cabrita et al., 2005).

Reactive oxygen species (ROS) and Cu interact in the redoxcycling Haber–Weiss reaction to form highly reactive radicals,which in turn cause membrane lipid peroxidation and membranedisruption (Luza and Speisky, 1996). In order to maintain integrityand homoeostasis, cells possess diverse protective and antiox-idant defences. The antioxidant enzymes catalase and superoxide

H. Chairi et al. / Ecotoxicology and Environmental Safety 73 (2010) 1279–12851280

dismutase represent an important defensive barrier that isactivated in stressful situations (Livingstone, 2001). Metallothio-nein (MT) is a protein involved in metal homoeostasis that alsoplays an essential role in the detoxification of non-essentialmetals within the cell. MT levels can be enhanced by exposure tometals and by oxidants (Viarengo et al., 1999); this makes it apotential biomarker for evaluating the effects of Cu in fish. Finally,heat shock proteins (HSPs) are chaperones that play a key role inthe transport, folding and assembly of proteins. They can beinduced under abnormal conditions by various causal agentsincluding metals and oxidative stress (Basu et al., 2002). TheHSP70 and HSP60 families in particular are involved in proteinhomoeostasis as they stabilise and refold damaged proteins, andtarget mis-folded proteins in specific degradative pathways(Daugaard et al., 2007).

The aim of this work is to evaluate the effect of coppersulphate in three flatfish species. Genotoxicity was evaluatedusing the comet assay and for assessing the inter-speciesdifferences in defence mechanisms, additional biomarkers in-volved in the defensive cellular responses against oxidative stress(antioxidant enzymes, MT and HSPs) were also studied. Theinformation obtained will be useful for understanding the actionmechanisms of copper in fish and to determine the effect on thehealth and welfare status of these species in aquaculture and inwild fish.

2. Materials and methods

2.1. Animals

Juvenile individuals of Senegalese sole, wedge sole and brill (Table 1) were

obtained from the IFAPA Centro Agua del Pino facilities (Cartaya, Huelva, Spain).

Fish rearing conditions are reported in Jimenez-Cantizano et al. (2008). Animals

were dosed via a single intraperitoneal injection with 2 mg kg�1 of copper

sulphate (CuSO4 Sigma-Aldrich) dissolved in phosphate buffered saline (PBS). Fish

controls were injected with PBS. The copper dose was chosen as it has been seen to

induce oxidative stress into the sea bass (Dicentrachus labrax) at the times used in

this experiment (Romeo et al., 2000). Prior to manipulation, fish were

anaesthetized with phenoxyethanol (100 mL/m3). After injection, animals

recovered normally without any signs of stress. Fish specimens (n¼30) from the

treated and control groups were sampled at 3 h, 24 h and 6 days post injection

(p.i.). For the comet assay, livers were extracted (Table 1) and kept on ice until

analysis. For the other analyses, livers were frozen in liquid nitrogen and stored at

�80 1C until use. All animals were treated humanely with regard for alleviation of

suffering, and all laboratory procedures involving animals were carried out in

compliance with the Guidelines of the European Union Council (86/609/EU) and

Spanish laws at the time of the experiment.

2.2. Biochemical determinations

Livers (pooled wet mass of three individuals per treatment) from each

sampling point were separated into six groups for the assessment of the different

variables. Liver aliquots were homogenised in 50 mM Tris–Cl pH 7.8 buffer

containing 5 mM EDTA, 1 mM DTT and Cocktail inhibitor proteases (Sigma P8340).

Samples were cooled in an ice bath and centrifuged (100,0000g; 1 h; 4 1C). Upper

layer of fat was eliminated and aliquots were finally frozen at �80 1C.

Catalase (CAT) and superoxide dismutase (SOD) enzyme activities were

measured using a Lambda 25 spectrophotometer (Perkin–Elmer) at 25 1C. Assays

were run at least in duplicate. CAT activity was determined by measuring the

decrease in hydrogen peroxide concentration at 240 nm (Aebi, 1974). SOD was

measured by the xanthine–xanthine oxidase system as described by McCord and

Table 1Some details of the sampling (wet weight, with mean7SE, liver weight, n¼30).

Species Age Total weight (g) Liver weight (g)

Senegalese sole 2 years 63.372.8 1.10

Wedge sole 2 years 43.073.1 0.60

Brill 1 year 39.374.1 0.25

Fridovich (1969). One unit of SOD activity is defined as the amount of sample

causing a 50% inhibition of cytochrome c under these conditions.

Total glutathione peroxidase (t-GPX), glutathione reductase (GR), glucose-6-

phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase

(6PGDH) measurements were carried out using a microplate reader (Genios,

Tecan). Assays for these four enzymes were run at least in triplicate in 96-well

format (McFarland et al., 1999). t-GPX activity was measured according to Flohe

and Gunzler (1984) using cumune hydroperoxide as substrate, and GR activity

according to Cohen and Duvel (1988), measuring the rate of NADPH oxidation.

G6PDH activity was determined according to Lorh and Waller (1974) and 6PGDH

according to Bonamusa et al. (1992), measuring in both cases the appearance of

NADPH.

2.3. Comet assay

The comet assay was performed essentially according to the procedure of Tice

(1996). To prepare the cell suspension, a small piece of liver was placed in 1 mL of

cold Hank’s buffered solution (HBSS) containing 20 mM EDTA and 10% DMSO.

Then, it was minced into fine pieces, and after it had settled, the cell suspension

was removed (Tice, 1996). To evaluate the rate of DNA damage, slides were stained

by adding 40 mL of ethidium bromide (2 mg/mL). For each individual, images of 300

randomly selected cells (100 cells �50 per replicate—from each of three

individuals) from the inoculated and control groups were analysed using an

Axioskope 2 plus microscope equipped with a Photometrics CoolSNAP camera.

Imaging was performed using specialized analysis Comet Assay Software Project

(CASP) (University of Wroclaw, Poland) (Konca et al., 2003; Garcia et al., 2007). To

quantify the DNA damage, the tail length and the fraction of DNA in the comet tail

were estimated. Tail moment was calculated according to the following formula:

Tail moment¼(% DNA in tailntail length)/100.

2.4. Chemical analysis

Metals were analysed in digested samples according the procedure described by

Martın-Dıaz et al. (2005). The results were checked using reference material (DOLT1

of NRC Canada). The recovery ranged between 97.2578.95 for Cu and 103.2714.4

for Cd. Certified values and our values are very close for all analysed metals. The

detection limits were: 0.9 mg/L for Cu; 0.6 mg/L for Cd and 0.2 mg/L for Zn. Metal

concentration was analysed by ICP-OES (Perkin–Elmer 2000DV). The results are

expressed as mg g�1 dry weight.

2.5. Protein determination

Total protein content (TPC) for normalisation of biochemical determinations

was determined in a multiwell plate according to the Bradford (1976) method

using bovine serum albumin (BSA) as standard. Metallothionein (MT) levels were

determined according to the procedure described by Olafson and Olsson (1987) on

the cytosol fraction (100,000g�60 min) after denaturation at 95 1C for 4 min. The

results are expressed as mg MT mg�1 protein. Stress proteins HSP70 and HSP60

were measured according to Sole et al. (2004). Bands were visualised using the

alkaline phosphatase revealing mixture using NBT (p-nitroblue tetrazolium) and

the same amount of BCIP (5-bromo-4chloro-3-indolyl phosphate) and semi-

quantified by scanning with a Laser Densitometer (Epson GT-8000). Absorbance of

the samples was expressed as arbitrary units/mg protein.

Fig. 1. Genotoxic effect of CuSO4 in the three flatfish species, Senegalese sole

(S. senegalensis), wedge sole (D. cuneata) and brill (S. rhombus). Tail moments of

treated and control groups at 3 h, 24 h and 6 days p.i. are represented. Values with

the same superscript are not significantly different (Po0.05).

H. Chairi et al. / Ecotoxicology and Environmental Safety 73 (2010) 1279–1285 1281

2.6. Statistical analysis

Data were expressed as the mean7standard deviation (S.D.). For the comet

analysis, a Kruskal–Wallis test followed by a post hoc analysis was performed. For

biochemical and metal analyses, a two-way ANOVA to test difference due to

species and treatment was carried out. The Tukey test post hoc was applied to

identify significant differences among groups compared with the corresponding

controls. In all cases Po0.05 was considered statistically significant.

3. Results

3.1. Genotoxic effects of copper sulphate

The genotoxic effects of CuSO4 were evaluated in sole, wedgesole and brill using the comet assay. After CuSO4 injection allthree species suffered severe DNA damage, as determined by thetail moment in liver cells. Tail moments increased significantly

Fig. 2. Metal content (Cu, Zn and Cd) in liver samples of, S. senegalensis, D. cuneata and S

solid bars experimental conditions (CuSO4 injection). Different letters denote statistica

(nPo0.1; nnPo0.05).

early, at 3 h, in wedge sole and brill. These values increasedprogressively at longer exposure times in the three species, andshowed the highest values at 6 days post injection (p.i.) (Fig. 1).Tail moments were higher in Senegalese sole than in the othertwo flatfish species. No differences were observed in the controls(PBS injection), at any time p.i., with values ranging between0.6 and 0.8.

3.2. Metal content of flatfish species

Metal concentrations (Cu, Zn and Cd) in the liver of fish from thecontrol and copper-treated groups at 6 days p.i are shown in Fig. 2.D. cuneata presented a significantly higher Cu content than theother two flatfish species. No differences between S. senegalensis

and S. rhombus were found (P40.05). CuSO4 injection increasedCu levels significantly in D. cuneata and S. rhombus but not inS. senegalensis (Po0.01 and Po0.05, respectively).

. rhombus at six days p.i. Open bars represent control conditions (PBS injection) and

l significance at Po0.05. ‘‘ n ’’ indicates significant differences between treatments

H. Chairi et al. / Ecotoxicology and Environmental Safety 73 (2010) 1279–12851282

For zinc, the highest basal contents were found in S. rhombus,followed by S. senegalensis and D. cuneata; no differences were foundbetween the two latter species. CuSO4 treatment increased Zn levelsin S. rhombus and D. cuneata (Po0.05 and Po0.01, respectively). Forcadmium, S. senegalensis showed the highest basal levels. Only S.

rhombus and D. cuneata presented differences in cadmium levelsafter CuSO4 injection (Po0.01 and Po0.05, respectively).

3.3. Activity of antioxidant enzymes

Analysis of the activity of antioxidant enzymes revealed cleardifferences between species (Fig. 3). The basal activity of theantioxidant enzymes t-GPX, G6PDG and 6PGDH was significantlyhigher in S. rhombus (Po0.05), that of GR in S. senegalensis (Po0.05)and that of SOD in D. cuneata (Po0.05). Only CAT activity levelswere similar in the three species analysed. With respect to theCuSO4 treatment, no significant differences in the activity of theseenzymes was observed, except for 6PGDH in S. senegalensis.

3.4. Stress protein response

MT concentrations in the control and copper-treated group forthe three flatfish species are presented in Fig. 4a. Steady-statelevels were similar in the three species. Copper treatmentsignificantly increased MT levels only in S. rhombus.

Levels of stress protein HSP70 and HSP60 measured byimmunoblotting are shown in Figs. 4b and c, respectively; levelsof HSP70 values were similar in the three species whereas HSP60was higher in D. cuneata. CuSO4 treatment reduced significantlyHSP70 and HSP60 levels in S. rhombus (Po0.05).

Fig. 3. Antioxidant enzyme activity in S. rhombus, S. senegalensis and D. cuneata of catala

(G6PDH), 6-phosphogluconate dehydrogenase (6PGDH), glutathione reductase (GR) and

(PBS injection) and solid bars experimental conditions (CuSO4 injection). Results repre

‘‘ n ’’ indicates significant differences between treatments (Po0.05).

4. Discussion

Copper is a bio-essential metal necessary for the normalfunctioning of cells. However, it can also catalyse the formation ofhydroxyl free radicals (dOH) in the Fenton and metal-catalysedHaber–Weiss reactions, resulting in oxidative DNA damage andDNA strand excision (Galaris and Evangelou, 2002; Lesser, 2006).The liver is a target organ for metal detoxification and storage infish (Arellano et al., 1999). Previous studies in sole using coppersulphate have demonstrated significant hepatic cellular damage(Oliva et al., 2007, 2009). In this work we show that copper canalso provoke severe DNA damage in this organ. Althoughsignificant effects can be detected at 3 h in Dicologoglosa cuneata

and S. rhombus, the magnitude of damage increased at 24 h andlater at 6 days after treatment. These two species that sufferedDNA damage sooner also presented higher copper levels thancontrols in liver after treatment (Fig. 2); this suggests that theaccumulation of Cu is a species-specific factor directly correlatedwith the appearance of DNA damage. These differences in Custorage also could explain the temporal differences in the cometassay between the Senegalese sole and the other two flatfishspecies, although the magnitude of damage was similar acrossspecies.

Although the specimens were not injected with zinc andcadmium in the experimental design used, zinc is an essentialmetal which plays an important role in enzymes involved in thedefence mechanisms and in metallothionein induction; cadmiumwas found in the control fishes and cadmium content can beaffected by the variations of MT induction and displacementresulting from copper contamination. In line with these con-siderations, zinc and cadmium levels have also been measured.

se (CAT), total glutathione peroxidase (t-GPX), glucose-6-phosphate dehydrogenase

superoxide dismutase (SOD) at six days p.i. Open bars represent control conditions

sent mean7S.E; bars with different letters are statistically significant (Po0.05).

Fig. 4. Stress protein levels in liver of S. rhombus, S. senegalensis and D. cuneata of (a) metallothionein (MT) concentrations; (b) and (c) represent the immunodetection of

HSP70 and HSP60, respectively, at 6 days p.i. Open bars represent control conditions (PBS injection) and solid bars experimental conditions (CuSO4 injection). Results

represent mean7S.E.; bars with different letters are statistically significant (Po0.05). n indicates significant difference between treatments (Po0.05).

H. Chairi et al. / Ecotoxicology and Environmental Safety 73 (2010) 1279–1285 1283

It was observed that in S. rhombus, the higher Zn level and theincrease in enzyme activity that depended on glutathion meta-bolism (t-GPX, G6PDH and 6PGDH) suggest that this specie has abetter defence system than the others. The high MT, together withlow HSP70 levels exhibited at six days after CuSO4 treatment, givethis species an acquired tolerance to stress and protection againstcopper, as demonstrated by low cytotoxicity values. In contrast,the down regulation of HSP70 expression suggest the existence ofa protective compensatory mechanism, and that HSP70 is lessinvolved in protection following a large single copper injection, inS. rhombus. However, in the rainbow trout, the hepatotoxicity ofcopper has been related to the impairment of HSP70 response tosubsequent stressors which is crucial for protecting cells fromproteotoxicity (Feng et al., 2003).

Fader et al. (1994) reported that the expression of HSP70 inresponse to seasonal temperature fluctuations is species-specific,indicating that each species has constitutive levels in accordancewith its ecological and physiological background. For S. rhombus,the increases found in zinc and cadmium levels could be relatedto metallothionein induction, since significant increases in levelsof these were reported in the copper-treated samples, comparedwith the controls. Among other species (rainbow trout, commoncarp and gibel carp) clear difference in metal handling have beenpointed out, with high liver residues and liver accumulation ratesfor the most sensitive species (De Boeck et al., 2004). It seems thatthis behaviour does not apply in the flatfish studied, at leastconsidering the DNA damage at six days p.i. as an indicatorof sensitivity. The lack of induction of metallothionein inS. senegalensis may be related to the toxicity of the administereddose; George et al. (1996) found that cadmium injected i.p. inacute doses (4200 mg/kg) in Scophthalmus maximus provoked adecrease in the MT gene transcription and protein translation,

thus reducing the MT synthesis due to cytoxicity at high dosage.High dosage of copper (500 mg/kg) in D. labrax provoked a toxiceffect, and lower MT levels were reported in copper-treatedsamples than in controls (Romeo et al., 1997) evident for thisgenotoxico absence.

The liver has been described as a target organ of contaminationbecause of the role it plays in xenobiotic metabolism, and itsuffers an accumulation of contaminants in general (Cogun et al.,2003; Monteiro et al., 2009).

Published data show that the accumulation of metal depends onthe concentration and the period of exposure as well as severalother factors such as salinity, temperature, and the metabolicactivity of tissue (Ay et al., 1999; Zhou et al., 2001). CuSO4 alsoinduced an increase in the comet parameters of the erythrocytes ofSparus aurata (Gabbianelli et al., 2003) and in erythrocytes of trout,as detected by an increase of the micronucleus (Bagdonasand Vosyliene, 2006). Our results show that the species with thelowest amounts of copper in the liver suffers the highest genotoxiceffect.

The results obtained vary according to the species. This reflectsthe different immune responses, DNA repair rate and metabolicactivities of each type of fish, and explain the divergencesbetween and within species.

Although inter-site differences in DNA strand break levels mayalso be related to eco-physiological differences, the formation ofDNA strand breaks in flatfish liver cells could result from theabsorption and the biotransformation of genotoxic stressful agents.Various mechanisms of DNA strand break formation havebeen proposed for higher vertebrates. More information isnecessary to determine its possible effects by applying the copperin baths so that the optimal amounts do not induce damage tothe DNA.

H. Chairi et al. / Ecotoxicology and Environmental Safety 73 (2010) 1279–12851284

The three fish species analysed showed a high variability inrespect of background levels. The concentrations of accumulatedmetals depend upon the metal accumulation strategy of thespecies concerned, and they fall along a gradient (Rainbow, 1990).D. cuneata showed the highest values for copper in the liver. Theratio between copper concentration in the liver of D. cuneata andthe other two fish ranged between 4:1 and 35:1. Concentrationsin the same range for liver in D. cuneata were found by Blascoet al. (2003) in specimens collected in the Gulf of Cadiz. Thisstrong accumulation capacity should be accompanied by physio-logical pathways for storage and detoxification, and reduction ofthe toxic effect. However, this does not seem to be associated withmetallothionein induction because the protein levels were similarin the control specimens for the three species. The lack ofvariation between analysed metals in control and copper-treatedsamples in S. senegalensis may be related to efficient mechanismsof homoeostasis for metals in this species. The opposite may bethe case in S. rhombus and D cuneata, which showed significantincreases in the liver at 6 days after copper administration. Thiscapacity may be related to age and especially to fish size, sinceindividuals of S. senegalensis are heavier than individuals of otherspecies, or it may be a simple species-specific response. The latterseems to be the more likely explanation of these variations ofmetal levels in tissues, genotoxic damage and enzymaticresponses presented each species; Widianarko et al. (2000)investigated the relationship between the concentration of threemetals (Pb, Zn, Cu) and fish size (in Poecilia reticulata) and foundthat there was a significant decline in lead concentrations withthe increase in size, whereas concentrations of copper and zincdid not depend on body weight. They concluded that bodyconcentrations of copper and zinc are regulated and maintained ata certain concentration.

The specific characteristics of the response to CuSO4, such asthe speed of response and the level of induction, appear to bespecific for each species and are probably a consequence of themechanism of toxicity of the compound. Questions concerningthe specificity of stress proteins to copper are, however, notanswered and could be subject of future research.

5. Conclusion

Copper treatments are used as a chemotherapeutic agent inaquaculture; however the information on the effect of copper onfish health is scarce. The present study has demonstrated thatthree species of flatfish (S. senegalensis, D. cuneata and S. rhombus)injected with copper sulphate showed statistically significantdifferences with respect to controls at 6 days after treatment,when subjected to the comet assay, analysis of heavy metalsaccumulation, and tests of antioxidant defences.

This research provides information that should be useful foraquaculture in designing disease treatments, the genotoxic effectsof which can be the source of other diseases, such as skeletalmalformations that seriously affect flatfish.

Acknowledgments

This work has been supported financially by the RegionalGovernment (Junta de Andalucia, Spain) in a research project(AGR-516). The authors thank Esmeralda Ramos and Ma del MarBarrios for their collaboration in taking and analysing samples,and Dr. Ismael Cross for assisting in the statistical treatment of thedata. We thank Royston Snart for reviewing and correcting themanuscript.

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