M Laura Martín-Díaz

Julián Blasco

Diego Sales

T Ángel Delvalls

M Laura

Martín Díaz

THE USE OF A KINETIC BIOMARKER APPROACH FOR IN SITU MONITORING OF LITTORAL SEDIMENTS USING THE CRAB Carcinus maenas

Keywords: Metallothionein, Enzymatic activity, Oxidative stress, Carcinus maenas, Ecotoxicology, Dredged material, Invertebrates

Caged, transplanted, intermoult, female shore crabs (C. Maenas) were exposed to sediments from the Port of Cadiz (SW, Spain); the Port of Huelva (SW, Spain), the Port of Pasajes (NE, Spain) and the Port of Bilbao (NW, Spain) for a period of 28 days. Organisms were sampled on days 0, 7, 14, 21 and 28 allowing examination of different biomarkers to exposure to metals and organic compounds; Metallothioneins (MTs), Ethoxyresorufin Odeethylase (EROD), Glutathione-S-transferase (GST) and Glutathione peroxidase (GPX). Sediment samples were also analyzed to determine chemical concentration of metals (As, Cd, Cr, Cu, Hg, Mn, Ni, Pb and Zn), PAHs and PCBs. Metals such as As, Cu and Zn associated with mining activities (Port of Huelva), and contaminants such as Ni, Pb, Hg and PCBs, associated with oil spills and industrial activities (Ports of Cadiz, Pasajes and Bilbao), were found to interfere in detoxification and/or anti-stress oxidative defenses in C. maenas. Positive, significant (p < 0.05) induction of MTs concentration was associated with increasing sediment As, Cu, Zn and Hg concentrations. EROD activity was associated with PCBs, GST activity with Hg and GPX activity with As, Cu, Ni, Pb and Zn. The use of the parameter AR, which involves the kinetic patterns of the battery of biomarkers tested, was found to be a powerful and sensitive tool for evaluating the bioavailability and adverse effects of contaminants bound to sediments, enabling polluted and non polluted sites to be easily distinguished.

Introduction

Contaminated sediments are a risk to aquatic life, human health, and wildlife throughout the world.

Overwhelming evidence demonstrates that chemicals in sediments are responsible for toxicological and adverse ecological effects [START_REF] Andersen | Comparision of marine sediment toxicity test protocols for the amphipod Rhepoxonius abronius and the polychaete worm Nereis (Neanthes) arenaceodentata[END_REF]. Frequently, the chemicals causing these effects are present in the sediment as mixtures of organic, metal, and other types of compounds.

The adverse environmental effects associated with contaminated sediments in ports and estuaries have resulted in a number of international treaties and protocols for their environmental management. The OSPAR and Helsinki Conventions (North Sea, North-East Atlantic, Baltic Sea) propose strict guidelines to control the disposal of contaminated sediments. In recent years, remediation and management of contaminated sediments is becoming increasingly important, which has led to an increasing economical and technological burden on management agencies. Thus, there is a clear need for the development of science based tools for the identification of sediments that are impaired, allowing agencies to set priorities and make effective management decisions for dealing with contaminated sediments.

The most widely applied methods for the assessment of sediment toxicity are acute and chronic sediment bioassays. Although a wide range of acute toxicity tests are already recommended, standardized and included in sediment assessment regulatory frameworks, the development and application of chronic toxicity tests including the measurement of sub lethal responses (biomarkers), although widely supported, is still under investigation. The use of biomarkers in the evaluation of the toxicity of dredged material is under continuous development and improvements are still desirable.

An important advantage of biomarkers in assessing the impact of dredged materials is their inherent capability to detect the early occurrence of various stress conditions within the organism and monitor the temporal progression (or regression) of the disturbance at various levels of biological organization (Regoli et al., 2002). When assessing the exposure to a mixture of contaminants in dredged materials within the aquatic environment, biotransformation and antioxidant enzymes as well as metal-binding proteins such as metallothioneins (MTs) play an important role in identifying contamination by organic and metal pollutants respectively. However, ecotoxicological measurements obtained in laboratory studies can be difficult to translate into accurate predictions of toxicological effects in the natural environment. Since both overestimation and underestimation of effects may occur, laboratory observations must always be validated with field research. The transplanting and caging of bioindicator organisms provides a combination of the experimental control of laboratory bioassays and the environmental realism of field monitoring [START_REF] Salazar | Using caged bivalves to characterize exposure and effects associated with pulp and paper mill effluents[END_REF]. Thus, the combination of this methodology with biomarker measurements of suitable indicator species may provide a strong tool for the monitoring of contaminated sediments, allowing the determination of the total external load that is biologically available in the "real world" situation [START_REF] Van Der Oost | Fish bioaccumulation and biomarkers in environmental risk assessment[END_REF].

Still, studies that use biotransformation and antioxidant enzymes as tools to monitor polluted sites should be performed carefully. Various authors have pointed out the serious risk of misinterpretation associated with using a single "snap-shot" (i.e., a single measurement at the end of exposure) to rationalize oxidizing versus other effects of toxic contaminants in the environment [START_REF] Bouskill | Differentiating copper and arsenic toxicity using biochemical biomarkers in Asellus aquaticus and Dreissena polymorpha[END_REF]. Thus, a study of a continuous series of biomarker responses over time rather than a single measurement after concrete exposure time should be performed in order to overcome the problem mentioned above.

The aims of this study were to determine the measured biomarker responses within C. maenas over a 28-day exposure period to contaminated sediments. This protocol was chosen to: 1) fill the lack that exists from measuring a single time point,, 2) reveal temporal variation in oxidative injury related to increasing exposure time, and 3) allow the distinction and classification of sites with different magnitudes (and types) of sediment toxicity. To do this caged, female crabs were exposed in situ to sediments with different levels of metals and organic compounds from a number ports within Spain.

The responses of a suite of biomarkers, including metallothioneins (MTs), mixed function oxidase (EROD), glutathione-S-tranferase (GST), and glutathione peroxidase (GPX): antioxidant enzyme were determined during a 28-day exposure period and through a kinetic approach.

Materials And Methods

Study site

Eight sampling sites were chosen (Figure 1) for the assessment of sediment toxicity in Spanish ports.

Ca2 and Ca3 (negative toxicity control) were situated in the Port of Cádiz (SW, Spain), which has been studied in some depth [START_REF] Delvalls | Site-specific sediment quality values for the Gulf of Cádiz (Spain) and San Francisco Bay (USA), using the sediment quality triad and multivariate analysis[END_REF]Riba et al., 2004a,b) and is characterized by an absence of significant contamination. Hu2 and Hu3 were situated in the Port of Huelva (SW, Spain) and are characterized by extensive heavy metal contamination related to historic mining activity. Bi2 and Bi3 were situated in the Port of Bilbao (NNE, Spain), which is characterized by significant shipping activity with sediment contamination predominantly associated with organic contamination, especially hydrocarbons. Pa2 and Pa3 were situated in the Port of Pasajes (NNE, Spain), which is also characterized by significant shipping activity and by contamination associated with organic compounds similar to the Port of Bilbao.

General approach

Chemical characterization of the sediment

Analysis of sediment was performed before starting the experiment according to Spanish recommendations for dredged material (CEDEX, 1994). The dry weight fraction was measured using the weight loss at 105 ºC. In order to perform the rest of the analyses sediments were dried at 40ºC over 24h. Metal content in the different sediments was determined following the methods reported by Riba et al. (2004a,b);PCBs (congeners #28, 52. 101, 118, 138, 153 and 180) and PAHs content was analyzed according to U.S. EPA SW-846 method 8270/8082. All the analytical procedures were checked using reference material (MESS-1 NRC and CRM 277 BCR, for heavy metals and NCR-CNRC HS-1 for organic compounds).

In situ toxicity test

Intermoult, female, C. maenas of a standardized size (Martín-Diaz et al., 2004) Cages were exposed to sediments for 28 days. On days 0, 7, 14, 21 and 28, cages were collected and 10 individuals sampled. The temperature (15ºC±1ºC), pH (7.8-8.2), salinity (33.8±0.3) and dissolved oxygen (>5 mg•L -1 , 60% saturation) were measured.

Sublethal responses determination

Ten, intermoult, female C. maenas were collected from within each port on days 0, 7, 14, 21 and 28.

Crabs were dissected and their hepatopancreas were removed for biomarker determination.

Hepatopancreas tissues were homogenized 1:4 in cold homogenization buffer: Hepes-NaOH (10 mM) buffer containing 125 mM NaCl, 1 mM EDTA and 1 mM dithiothreitol, pH 7.4 at 4ºC at a 1:5 weight:volume ratio (Lafontaine et al. 2000). After homogenization, samples were centrifuged (4ºC, 10 000g, 30min) and the supernatant collected for biomarker determination (S9 fraction). Samples obtained to determine MTs content were centrifuged at 28 000 g for 40 min. The supernatant was utilized for total protein determination according the dye-binding principle (Bradford, 1976). 000 g for 40 min) was added to 0.9 mL of NaCl (0.9 %), heated to 95 ºC for 4 min, then centrifuged at 10 000 g for 15 min at 4ºC. The supernatant was stored at -80 ºC prior to MT concentration determinations using purified rabbit metallothionein (Sigma-Aldrich). MT concentrations were expressed as µg MT (mg total protein) -1 . Mixed function oxidize activity was measured using the adapted EROD assay initially adapted for fingerling rainbow trout [START_REF] Gagné | Hepatic metallothionein level and mixed function oxidase activity in fingerling rainbow trout (Oncorhynchus mykiss) after acute exposure to pulp and paper mill effluents[END_REF]. 50 µL of supernatant (homogenate 10 000 g for 30 min) were added to 10 µM 7-ethoxyresorufin and 10 µM reduced NADPH in 100 mM KH2PO4 buffer (pH 7.4). The reaction was started by the addition of NADPH, allowed to proceed for 60 min at 30ºC, and stopped by the addition of 100 µL of 0.1 M NaOH. The formation of 7-hydroxyresorufin was determined fluorometrically using 520 nm (excitation) and 590 nm (emission) filters. 7-hydroxyresorufin in the samples was achieved through a standard calibration curve developed with concentrations of 7-hydroxyresorufin. Results were expressed as pmol•min -1• (mg total protein) -1 . The procedure utilized for the determination of Glutathione S-transferase activity was adapted from the method of [START_REF] Mcfarland | Biomarkers of oxidative stress and genotoxicity in livers of field collected brown bullhead, Ameiurus nebulosus[END_REF]. The activity was analyzed using 42 mM 1-chloro-2,4-dinitrobenzene (CDNB) and 1 mM GSH as substrates and measured spectrophotometrically at 340 nm every 30 seconds for 3 min. Results were expressed as nmol min -1 (mg total protein) -1 . The methodology used for the determination of glutathione peroxidase activity was also adapted from [START_REF] Mcfarland | Biomarkers of oxidative stress and genotoxicity in livers of field collected brown bullhead, Ameiurus nebulosus[END_REF]. GPX activities were measured spectrophotometrically at 340 nm every 2 minutes for 10 minutes, using as substrate 1 mM cumene hydroperoxide. Results were expressed as nmol min -1 (mg total protein) -1 .

Data and statistical analysis

A continuous study of biomarker responses over time as opposed to a measurement of sublethal responses at a single time point was performed. Metallothioneins and enzymatic activity responses were fitted to different kinetic models using SIGMA PLOT 8.0 software package. Linear regression was used to determine the kinetic parameters of MTs at each site. GST ´s, EROD´s and GPX´s kinetic equations were obtained through hyperbolic regression analysis, where the different enzymatic activities were fitted to the best hyperbola equation.

In order to summarize the temporal variation of the different biomarkers, the area (AR) under the fitted kinetic curves was calculated using Mathematica 4.0 Differences between the AR of control and contaminated sediments were analyzed using SPSS/PC+. Significant differences between biomarker responses of crabs exposed to control and contaminated sediments were determined using one-way ANOVA. Post-hoc examination of significant differences between sites was carried out using Dunett´s test. The significance level was set at p < 0.01.

Significant correlations between chemical concentrations in sediments and biomarker responses (using AR parameters) were examined using a Pearson's correlation analysis using the SPSS/PC+ statistical package. The level of significance was set at p < 0.01.

Results

Chemical characterization

Summarized results of the chemical analysis are shown in Table 1. The chemical characterization of the sediment samples indicates that the sediments contain a mixture of contaminants (metals, PCBs and PAHs). Sediments from the port of Huelva were characterized by higher concentrations of metals

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Specimen survival

During the 28-day, in situ, bioassay exposure to sediments from four Spanish ports, no sedimentdependent mortalities were observed for intermoult female C. maenas. Survival in all treatments was more than 90%.

Biomarker responses

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Significant induction of EROD activity in crabs exposed to Pa3 (16.71.52% of control) (p<0.01), Pa2

(1.47%) (P<0.01), Bi2 (1.34%) (p<0.01), Bi3 (1.19%) (p<0.05) and Ca2 (1.12%) was also found. No induction was observed in Hu2 and Hu3. Finally, significant MTs increase was observed in crabs exposed to Hu3 (1.75% of control) (p<0.01), Hu2 (1.66%) (p<0.01), Ca2 (1.45%) p<0.01), Bi2 1998), which is the ratio between phase I and phase II enzyme activities was determined (Figure 4). EROD:GST in caged female crabs are maximally induced after 14 in Pa2, and after 21 days of exposure in Ca2 and Pa3 (Figure 4). As opposed to the EROD activity, the BTI values in crabs which belong to the mentioned sites decrease after 21 days of exposure due to the increased phase II activities (Figure 4).

Discussion

Comparisons between the temporal biomarker responses in female crabs (C. maenas) exposed to sediments from four Spanish ports and a new parameter, which involves biomarker responses over time, have been calculated. This new parameter, AR, allowed the comparison of temporal biomarker responses in organisms exposed to control and contaminated sites, as well as contaminants bound to sediment which have provoked adverse effects.

Metallothionein concentration (MT)

Significant induction (p<0.01) of MTs after 28-day exposure to sediments contaminated with As, Cd, Cu, Hg and Zn was found in Hu2, Hu3 and Ca2 (p<0.05). C. maenas has previously been shown to be a useful species in metallothionein biomarker testing studies, allowing the detection of the potential effects of metal contamination in situ [START_REF] Bebianno | Variation of metal and Metallothionein concentrations in a natural population of Ruditapes decussatus[END_REF]. High concentrations of metals were observed in the port of Huelva, presumably due to the historic mining activity in the area. Ca2 was also highly contaminated with Hg. MTs constitute a family of low-molecular-weight, cysteine-rich proteins functioning in the regulation of the essential metals Cu and Zn, and in the detoxication of these and other, non essential, metals such as Cd and Hg [START_REF] Roesijadi | Metal regulation in aquatic animals: mechanisms of uptake, accumulation and release[END_REF]. The cellular interactions involving MTs are expected to follow two general lines, the first being the interception and binding of metal ions that are initially taken up by the cell and the second being the removal of metals from nonthionein ligands that include cellular targets of toxicity. The latter may represent a detoxication function for structures, which have been reversibly impaired by inappropriate metal binding. The role of MTs in sequestering metals is well established, while their induction by exposure to a wide variety of metals (e.g. Cd, Cu, Zn, Hg, Co, Ni, Bi and Ag) is associated with their induction by exposure protection function [START_REF] Stegeman | Molecular responses to environmental contamination: enzyme and protein systems as indicators of chemical exposure and effect[END_REF]. Relatively few studies in marine environments have reported the induction of MTs associated with As.

Nevertheless, induction of MTs due to As has been described in the freshwater invertebrate D. polymorpha (Bauskill et al., 2006).

Ethoxyresorufin-O-deethlylase activity (EROD)

EROD activity is involved in the first phase of metabolism, unmasking or adding reactive functional groups, which involve oxidation and reduction or hydrolysis [START_REF] Goeptar | Oxygen reductase and substrate reductase activity of cytochrome P450[END_REF]. Induction is a clear sign of CYP1A1 enzyme activity, which is among the many known cytochrome isoforms. Increases in EROD activities have been reported in many species of invertebrates, including clam and crab species, following exposure to organic trace pollutants (Lafontaine et al., 2000). In the present study significant induction (p<0.01) of EROD activity was measured in hepatopancreas homogenates of C. maenas exposed to PCBs (p<0.01) bound to sediments from the port of Pasajes (Pa2, Pa3).

Gluthatione-S-transferase activity (GST)

Chemical compounds, such as heavy metals, are biotransformed to conjugates of reduced glutathione (GSH). Conjugation with GSH is a very important detoxification pathway that is catalyzed by the enzyme GST. A significant increase in GST activity was observed in crabs exposed to sediments from Ca2 (p<0.05) associated with the presence of Hg (p<0.01). This finding indicates that Hg (at the concentrations found at site Ca2), induces GST activity in the hepatoprancreas of C. maenas and may be used to elucidate the redox-activity generaterated by the oxidative stress produced by Hg (Elunalai et al. 2007).

Gluthatione peroxidase activity (GPX)

Many pollutants (or their metabolites) may elicit toxicity reactions related to oxidative stress. Oxygen toxicity may act as a strong oxidant, capable of reacting with critical cellular macromolecules, possibly leading to DNA damage and cell death. Defense systems that inhibit oxyradical formation include the antioxidant enzymes such as glutathione peroxidase (GPX) (van der Oost et al., 2000). GPX activity measured was significantly increased after exposure to sediments from sites Ca2, Ca3, Hu2, Hu3, Bi2, Bi3, Pa2 and Pa3. Interestingly, the induction of this biomarker ranged from 9.38% to 16.75% over control. The induction of this biomarker was correlated with high concentrations of sediment-bound metals, such as, As, Cu, Pb, Ni and Zn. Previous reports have also found that As [START_REF] Bouskill | Differentiating copper and arsenic toxicity using biochemical biomarkers in Asellus aquaticus and Dreissena polymorpha[END_REF]), Cu (Sánchez et al., 2005), Ni (Tsangaris et al., 2006), Pb (Alves de [START_REF] Alves De Almeida | Protective effect of phospholipid hydroperoxide glutathione peroxidase (PHGPx) against lipid peroxidation in mussels Perna perna exposed to different metals[END_REF] and Zn [START_REF] Elumalai | Enzymatic biomarkers in the crab Carcinus maenas from Minho River estuary (NW Portugal) exposed to zinc and mercury[END_REF] have the capacity to promote oxidative stress and modulates enzymatic and non-enzymatic antioxidant expression within biological tissues.

Integrated time-dependant biomarkers responses

By using a battery of biomarkers to evaluate sediment toxicity over a 28-day period, we were able to analyze the time dependent trends in the biochemical responses of C. maenas. According to [START_REF] Bouskill | Differentiating copper and arsenic toxicity using biochemical biomarkers in Asellus aquaticus and Dreissena polymorpha[END_REF], if the cellular effects of contaminants bound to sediment followed a simple linear etiology over time, initial EROD and MTs induction should protect cells from injury, followed by antioxidant enzymes induction, GST and GPX, to avoid oxidative stress and lipid peroxidation when MTs or EROD are overwhelmed. However, the biomarker responses observed in this study did not appear to follow this simple linear etiology. Determined biomarker responses vary over the 28-day exposure time due to differences in the contaminant levels for each site. Metallothioneins and EROD enzymatic activity were induced by As, Cd, Cu, Hg, Zn and PCBs, respectively. Since, for these biomarkers, the highest responses were found on day 28, their induction was slower than that of GPX and GST activities. This behavior suggests that after the beginning of the exposure, antioxidant enzymes such as GST and GPX were induced to avoid the oxidative stress caused by As, Hg, Cu, Ni, Pb and Zn, as found in previous studies. After the initial induction, a return of antioxidant enzymes to basal levels, suggests that MTs detoxification mechanisms were involved in the acclimation of C. maenas to contamination by As, Cd, Cu, Hg and Zn. In contrast, PCB exposure (at the concentration tested) did not produce significant oxidative stress.

The delay in the induction of MTs could be due to the sequestration of metals by MTs via the displacement of Zn, leading to a release of free Zn that, in turn, induces de novo MTs synthesis. Indeed, in previous studies of invertebrate species [START_REF] Correia | Studies on biomarkers of copper exposure and toxicity in marine amphipod Gammarus lacusta (Crustacea): 1. Induction of metallothionein and lipid peroxidation[END_REF]Rigwood et al., 1998) exposed to As, induction of MTs was not fast enough to prevent lipid peroxidation.

In the present study biomarker responses have been correlated with the contaminants measured in the sediments. Contaminants determined in the sediments tested are those recommended for dredged material characterization (CEDEX, 1994). Nevertheless, more studies should be performed in this direction since other contaminants not measured and synergetic and/or antagonic effects have not been determined and could have affected the results obtained.

The BTI values determined in crabs at the sites Pa2, Pa3 and Ca2, indicate that the actual hazards of environmental contaminants slightly decrease in time after an initial increase. These results support those described by [START_REF] Van Der Oost | Assessment of Environmental Quality and Inland Water Pollution Using Biomarker Responses in Caged Carp (Cyprinus carpio): Use of a Bioactivation:detoxication Ratio as a Biotransformation Index (BTI)[END_REF] which were found in caged carps exposed to polluted sites.

Conclusions

In summary, by examining the temporal evolution of a suite of biomarkers in the test organism C. maenas, it was revealed that As, Cu and Zn associated with mining activities and Ni, Pb, Hg and PCBs, associated with oil spills and industrial activities, interfere in detoxification and/or anti-stress oxidative defenses under field conditions, suggesting that these contaminants are likely to cause adverse effects in wild populations of C. maenas.

Different behaviors of the biomarker responses were observed related to the time-dependant induction of metallothioneins, phase I enzymes and phase II enzymes. It is of increasing demand that the study of biomarker responses over time and the search of a parameter that involves this temporal evolution be undertaken. By using the calculated parameter AR we were able to summarize the temporal progression of the ecotoxicological responses, allowing the comparison toxic contaminant bioavailability between polluted and non-polluted sites. Oxidative injury has a

  were enclosed in rectangular cages (50 x 25 x 10 cm) made of plastic net. During the experiment 24 crabs were placed in each cage and 2 cages were placed per site. Crabs were fed frozen mussels. No condition indexes were noted along the experiment. Low mortality was observed in individuals exposed to control sediments which indicated that crabs conditions were feasible for field caging experiments. The cages were anchored to the sediment at each site by a scuba-diver within each of the four Spanish ports: Port of Cadiz (SW, Spain) (Ca2, Ca3-control sediment), Port of Huelva (SW, Spain) (Hu2, Hu3), Port of Pasajes (NE, Spain) (Pa2, Pa3) and Port of Bilbao (NW, Spain) (Bi3, Bi2).

(

  As, Cu, Pb, Zn, Ni and Cd) compared with the reference site Ca3. Whereas, sediments from the ports of Pasajes and Bilbao were mostly characterized by high concentrations of PCBs and PAHs, although contamination by metals such as Cr, Mn, Ni, Pb and Hg was also found. Sediment from the port of Cadiz was not highly contaminated (Ca3), although high concentrations of Hg were determined in Ca2.

  Summarized results of biomarker responses determined in hepatopancreas tissues of C. maenas exposed to contaminated sediments (Ca2, Ca3, Hu2, Hu3, Bi2, Bi3, Pa2, Pa3) are shown in Figure 2. In this figure experimental biomarker responses over time and their approximation to kinetic models could be observed. Biomarker responses described different patterns over time for each biomarker response measured in C. maenas. GST and GPX activities increased until days 14 and 21, respectively, after which they declined reaching basal levels by the end of the experiment. EROD activity and MTs concentration increased during the 28-day exposure; with linear induction observed for MTs and exponential induction for EROD activity. No significant induction of detoxification and antioxidant parameters was observed in the control individuals (Ca3).The equations to the different biomarkers, the correlation coefficient and the representation of the areas (AR) calculated for each biomarker and sites are shown in Figure3. The results show a good correlation between the experimental data and those predicted by the results as described in Figure2. It confirms a good approximation of biomarker induction (MTs, GPX, GST and EROD) to Linear and Hyperbolic Kinetic models.Significant induction of GST activity (compared to controls) were observed in crabs exposed to Ca2(2.38% of control) (p<0.01), Pa3 (1.70%) (p<0.01), Bi2 (1.68%) (p<0.01), Hu3 (1.45%), Hu2 (1.40%) Bi3 (1.27%) and Pa2 (1.12%). Conversely, significant induction of GPX activity was also observed in crabs exposed to Bi3 (16.75% of control) (p<0.01), Pa3 (15.08%) (p<0.01), Hu2 (14.32%) (p<0.01), Hu3 (14.07%) (p<0.01), Bi2 (12.02%) (p<0.01), Pa2 (10.85%) (p<0.01) and Ca2 (9.38%) (P<0.01).

( 1 .

 1 28%), Pa3 (1.16%). No induction in Bi3 and Pa2 was detected. Significant positive correlations were observed between contaminants bound to sediments and biomarker responses (Table 2). A significant correlation between GST activity induction and Hg (p < 0.05) was observed. Induction of GPX activity was correlated with As (p<0.01), Cu (p<0.01), Pb (p<0.01), Zn (p<0.01) and Ni (p<0.01). Significant induction of EROD activity was only observed in individuals exposed to ∑PCBs. Whereas, MT induction was associated with the metals As (p<0.05), Cd (p<0.05), Cu (p<0.05), Zn (p<0.05) and Hg (p<0.01). The balance between bioactivation on the one hand and detoxication on the other can be crucial for the assessment of possible hazards due to the exposure to toxic substances (van der Oost et al. 1998). For this reason, in the present study the biotransformation index (BTI) (van der Oost et al.,

Figure 1 .

 1 Figure captions:

Figure 2 .

 2 Figure 2. Representation of metallothioneins (MTs) concentration, Ethoxyresorufin O-deethylase

Figure 3 .

 3 Figure 3. Representation of the area under the curve (AR) calculated for each biomarker and

Figure 4 .

 4 Figure 4. Biotransformation index: EROD : GST in C. maenas hepatopancres exposed over a 28-day

Table 1 .

 1 Summarized results of chemical concentrations of sediments from the Port of Cadiz (Ca2, Ca3), Port of Huelva (Hu2, Hu3), Port of Bilbao (Bi2, Bi3) and Port of Pasajes (Pa2, Pa3). Chemical CA2 30.77 1.32 14.94 202.80 1.98 201.60 20.14 86.90 378.25 0.11 0.11 CA3 16.61 1.23 8.43 46.76 0.28 294.40 16.90 17.61 135.50 0.01 0.01 HU2 532.27 2.50 24.10 149.70 1.99 303.60 7.10 384.70 1857.00 0.01 0.01 HU3 272.78 1.32 8.13 772.50 1.20 354.45 128.55 217.60 1176.00 0.01 0.01 BI2 104.49 2.00 23.11 204.10 1.43 396.60 32.00 147.50 777.50 0.23 66.77 BI3 21.71 0.04 3.48 23.03 0.18 191.35 15.72 285.90 122.35 0.01 13.90 PA2 28.76 0.70 23.42 167.10 1.29 180.00 28.48 293.70 763.00 0.74 1.06 PA3 23.78 0.04 18.61 162.50 1.36 152.60 19.61 246.00 576.00 0.24 0.26

	75	c
	76	o
	77	n
	78	c
	79	e
	80	nt
	81	ra
	82	ti
	83	o
	84	n

Table 2 .

 2 Significant positive Pearson´s correlation between biomarkers responses and contaminants of interest. Data were pooled across sites and areas (AR) of the fitted kinetic approaches. Asterisks indicate significant relationships (*p<0.05; **p<0.01).

	Biomarker/	GST	GPX	EROD	MT
	Contaminant
	As	-	0.48**	-	0.40*
	Cd	0.01	0.02	-	0.4*
	Cr	0.01	0.00	0.04	0.02
	Cu	-	0.46**	-	0.47*
	Hg	0.4*	0.27	-	0.51**
	Mn	-	0.15	-	0.25
	Ni	-	0.42**	-	0.15
	Pb	-	0.69**	0.25	0.23
	Zn		0.52**	-	0.4*
	ΣPCBs	-	0.00	0.73**	-
	ΣPAHs	0.13	0.04	0.30	0.09

temporal progression, which must be considered when using biotransformation and antioxidant enzymes as biomarkers of sediment toxicity. Thus, we recommend the use of biomarker assays that specifically consider the temporal progression of ecotoxiological responses, as these will provide a stronger basis for the identification of sediment toxicity, particularly when sediments contain a mixture of contaminants.

Acknowledgements.

This research was funded by a grant from the Spanish Ministry for Education and Science (REN2002-01699) and the Spanish Ministry for Development (BOE, 13-12-2002). L. M. was funded by the "Juan de la Cierva Programme" (Spanish Ministry for Education and Science). This manuscript has been English edited by Edward Morris PhD.

Bi3▪ Bi2▪

Iberian Peninsula


Damian Fernandez-Jover

Pablo Sanchez-Jerez

Just Bayle-Sempere

Pablo Arechavala-Lopez

Laura Martinez-Rubio

Jose Angel

Lopez Jimenez

Francisco Javier Martinez Lopez

Jose Angel Lopez Jimenez

Coastal Fish Farms Are Settlement Sites for Juvenile Fish

Keywords: Juvenile fish, fatty acid, biomarker, zooplankton, stomach content, diet, aquaculture impact

Two south-west Mediterranean fish farms were monitored over a period of 22 months to test if sea-cage fish farms act as settlement habitats for juvenile fish.

Twenty juvenile fish species were found to settle at farms throughout the year. Fish assemblage composition varied markedly over time and was dependent on the spawning period for each species. The most abundant species were Oblada melanura, Atherina spp., Diplodus sargus, Boops boops and Liza aurata. Up to 3783±1730 individuals/cage were found closely associated with the cages. Highest densities were observed during the warmer summer and autumn months. Zooplankton sampling and stomach content analyses of the most abundant species were done to assess prey availability, selectivity and diet overlap among species. Copepods were the main prey item for all juvenile fish species, irrespective of fish size. Ivlev's Index indicated that food was not a limiting factor for juvenile fish at farms. Furthermore, food pellets from the farm affected the food chain by modifying the fatty acid profiles of farm-associated zooplankton and juveniles of L. aurata and O. melanura. These results show that aquaculture can directly influence the body composition of juvenile fish that recruit to sea-cage fish farms.

INTRODUCTION

Settlement and recruitment of fish to nursery habitats may be determined by a combination of several factors, such as spawning strategies of breeding stocks [START_REF] Sherman | Spawning strategies of fishes in relation to circulation, phytoplankton production, and pulses in zooplankton off the northeastern United States[END_REF], chemical cues (Sweatman, 1988), or oceanographic features [START_REF] Govoni | Eulerian views of layered water currents, vertical distribution of some larval fishes, and inferred advective transport over the continental shelf of North Carolina, USA, in winter[END_REF], amongst others. Despite considerable research, factors leading to habitat choice by pre-settlement fish remain unclear. Fish recruit to a wide variety of natural environments, but also to artificial structures like docks, oil jetties [START_REF] Rilov | Fish assemblage on natural versus vertical artificial reefs: the rehabilitation perspective[END_REF], oil platforms [START_REF] Love | A pilot study of the distribution and abundances of rockfishes in relation to natural environmental-factors and an offshore oil and gas-production platform off the coast of southern California[END_REF], fish attraction devices (FADs; [START_REF] Gooding | Ecological significance of a drifting object to pelagic fishes[END_REF], artificial reefs [START_REF] Beets | Experimental evaluation of fish recruitment to combinations of fish aggregating devices and benthic artificial reefs[END_REF], and bivalve aquaculture rafts [START_REF] Powers | Macroalgal growth on bivalve aquaculture netting enhances nursery habitat for mobile invertebrates and juvenile fishes[END_REF]. In the case of FADs, the two most widely accepted explanations for this behaviour are the 'meeting-point theory' -where fish aggregate around a reference site in order to form schools and therefore gain protection; and the 'indicator-log theory' -where fish aggregate around drifting objects which accumulate in frontal zones where food may be more abundant [START_REF] Freon | Review of fish associative behaviour: toward a generalisation of the meeting point hypothesis[END_REF]. Factors which induce an individual to choose an artificial structure instead of a natural environment remain unclear, though this behaviour may have important consequences for population dynamics due to potentially differential predation rates or food availability between natural and artificial sites.

As sea-cage aquaculture production in the Mediterranean has continued to increase in recent years (e.g. >160,000 t y -1 of sea-bream and sea-bass; [START_REF] Fao | Cage aquaculture. Regional reviews and global overview[END_REF], so too have concerns regarding the potential impacts of aquaculture on the natural environment of the surrounding area. Impacts on wild fish populations have attracted considerable attention due to environmental and economical interests. Sea-cage aquaculture has a strong aggregative effect on wild fish [START_REF] Dempster | Attraction of wild fish to sea-cage fish farms in the south-western Mediterranean Sea: spatial and short-term temporal variability[END_REF]. Attracted fish actively feed on uneaten food pellets which fall through nets [START_REF] Fernandez-Jover | Seasonal patterns and diets of wild fish assemblages associated to mediterranean Coastal fish farms[END_REF], while carnivorous species are attracted by the increased abundance of prey [START_REF] Sanchez-Jerez | Interactions between bluefish Pomatomus saltatrix (L.) and coastal sea-cage farms in the Mediterranean Sea[END_REF]. Wild fish, which take advantage of the ready and constant availability of artificial food resources around fish farms, may also experience a change in their fatty acid (FA) composition with unknown consequences for their physiology.

To date, there is an almost total lack of knowledge regarding the role of fish farms as settlement habitats for juvenile fish. Therefore, to determine whether sea-cages are used by coastal fishes as settlement habitat, fish censuses and captures were carried out monthly over a 2-year period at two sea-bass and sea-bream farms in the south-western (SW) Mediterranean Sea. Stomach content analysis of captured individuals representing the dominant settling species were also carried out. Zooplankton tows immediately adjacent to the sea-cages were made in order to investigate changes in food utilization, food availability and prey selectivity by juvenile fish. Analysis of the FA composition of food pellets, zooplankton and juvenile fish were also made to test the hypothesis that lipids contained in food pellets influenced the food chain and led to changes in the physiology of settling juvenile fish around sea-cages.

MATERIALS AND METHODS

Study Sites

Two Mediterranean fish farms, separated by 45 km, were chosen off south-eastern Spain at Campello (38°25'12.58"N; 0°20'48.88"W) and Guardamar (38° 5'45.88"N; 0°36'15.84"W). The farm at Campello was located 3.2 km offshore in an average depth of 28.6 m. The farm consisted of 12 circular cages each having a diameter of 19 m and a height of 17 m, and producing 300 t yr -1 of fish. The farm at Guardamar was located 3.7 km offshore and sited in a depth of 24 m, and consisted of 24 circular cages, each having a diameter of 19 m and a height of 15 m, and producing 1100 t yr-1 of fish. Both farms reared sea-bass (Dicentrarchus labrax) and sea-bream (Sparus aurata).

Spatial and Temporal Variability in Aggregated Species

The composition and abundance of juvenile fishes (< 40 mm standard length-SL) around floating cages was estimated monthly from April 2006 to January 2008. A pilot study showed that juveniles aggregated around (<2-3 m) the floating cage tubes and in the first 3 m of the water column. Sampling was therefore restricted to this area.

Sampling was carried out on three randomly selected days each month. On each sampling occasion, one diver slowly swam around each of three randomly selected cages to census the composition, abundance, and estimates of the size classes of juvenile fishes. Spaces between cage structures were carefully searched for hiding individuals. A second diver then captured several schools of fish using a dipnet, separating each school sampled into separate bags based on species and size. Fish were sampled between 0900 and 1100 on each day of sampling.

Diet

Captured juveniles were stored in 70% ethanol. Once in the laboratory, they were identified (to species where possible) and standard length (SL) measured with a calliper to the nearest 0.1 mm. Stomachs were then removed, and all contents identified to the lowest taxonomic level possible and counted. Only entire organisms and detached heads were identified and counted. Some stomachs contained large numbers of copepod eggs, which were not used in calculation of the relative frequency of prey items. Overall, the stomachs of 82 Atherina spp., 54 Boops boops, 48 Diplodus sargus, 68 Liza aurata and 67 Oblada melanura were analyzed.

Prey Selectivity

To estimate food availability, zooplankton samples were collected using vertical tows (17 m -surface). Two tows were made on each sampling day using a 60 cm diameter plankton net having a 200 µm mesh size. Ivlev's index (E;[START_REF] Ivlev | Experimental Ecology of the Feeding of Fishes[END_REF] was used to estimate prey selectivity using the formula: E= (r i -p i )/(r i +p i ), where r i is the relative abundance of the food category i in the stomach (as a proportion of all stomach contents) and p i is the availability of this prey in the environment. Values range from -1 (total rejection or inaccessibility of the prey) to +1 (active selection of the prey), and zero indicates random feeding. Cage attached organisms (such as amphipods) were not used for calculations since it was virtually impossible to estimate their abundances from plankton tows. Prey that were absent from stomach contents were not included in calculations since their inclusion would have influenced the results for all other prey items [START_REF] Johnson | The comparison of usage and availability measurements for evaluating resource preference[END_REF].

Fatty Acid Composition

The FA profile of Liza aurata (12 individuals, 18-28 mm standard length -SL) and Oblada melanura (8 individuals, 15-20 mm SL), also collected with a dipnet, from around farms was compared to control populations captured at least 10 km away from the nearest fish farm. L. aurata were captured in September and October 2007, while O. melanura were captured in June and July 2007. Individuals captured at the two farms were pooled using the same number of fish from each location. Those captured at the two control locations were also pooled in this way. The FA composition of zooplankton was also analyzed. At each location, two zooplankton tows were made near farms and compared with tows made ≥3 km away from farms. Tows were done with the same methodology applied to the prey selectivity study. Once in the laboratory, samples were filtered and frozen at -18ºC.

After whole individual fish tissue was filter homogenised, the FA composition of the total lipid fraction was determined by fat extraction following the method of Folch et al. Peaks were identified by comparison of their retention times with appropriate FAME standards purchased from Sigma Chemical Company (St. Louis, MO, USA). Individual FA concentrations were expressed as percentages of the total content. [START_REF] Budge | Fatty acid composition of phytoplankton, settling particulate matter and sediments at a sheltered bivalve aquaculture site[END_REF] detected inputs of terrestrial organic matter in the marine environment using the ∑(18:2ω6, 18:3ω3) marker, whilst ∑(22:0, 24:0) has also been proposed to be useful as such a marker (e.g. [START_REF] Colombo | Lipid biogeochemistry in the Laurentian Trough -II. Changes in composition of fatty acids, sterols and aliphatic hydrocarbons during early diagenesis[END_REF][START_REF] Budge | Fatty acid composition of phytoplankton, settling particulate matter and sediments at a sheltered bivalve aquaculture site[END_REF].

These markers were therefore also analyzed in order to detect any influence of farm feeding routines on wild fish assemblages.

Permutational multivariate analysis of variance (PERMANOVA) was used to test for differences in the overall fatty acid composition among the potential sources of variation [START_REF] Anderson | PERMANOVA_2factor: a FORTRAN computer program for permutational multivariate analysis of variance (for any two-factor ANOVA design) using permutation tests[END_REF]). We tested whether the main FAs varied between farms and controls using univariate analysis of variance (ANOVA), the factor 'Farm' was considered a random factor with two levels -farm vs. control. Prior to ANOVA, heterogeneity of variance was tested with Cochran's C-test. As data were percentages, they were arcsine transformed (Underwood, 1997).

RESULTS

Spatial and Temporal Variability in Aggregated Species

Eighteen fish species, belonging to nine families, were found to settle at the Campello and Guardamar fish farms; Atherinidae, Carangidae, Labridae, Moronidae, Mugilidae, Pomatomidae, Serranidae, Sparidae and Syngnathidae (Fig. 1, Table 1). Juvenile fishes were consistently present around the cages in all months sampled except during April 1). Species that were present in lower abundances are included in Tables 1 and2.

Diet

The percentage of empty stomachs was 9.7% for Atherina spp.; 0% for Boops boops; 8.3% for Diplodus sargus; 16.1% for Liza aurata and 16.3% for Oblada melanura. The dominant prey item was adult copepods in all of the stomachs analyzed (Fig. 2). The predominance of copepods in the diet was especially obvious for B. boops, L. aurata and O. melanura of all sizes (>60% of the diet). After copepods, the diet of these three species also contained a considerable number of copepodites and unidentified crustaceans. Cladocerans were also an important component of the diet of the smallest B. boops. For the smallest sizes of O. melanura, low numbers of nauplius larvae and amphipods were also recorded. In contrast, Atherina spp. and D. sargus stomachs contained other prey items, such as nauplius larvae, copepodites and cladocerans, even though copepods were still the predominant prey item (29-52% for Atherina spp. and 35-57% for D. sargus). Amphipods were also a significant component of the diet for both species and, in the case of Atherina spp., this was restricted to the largest individuals. Fish eggs were also an important dietary item for D. sargus and made up 3-7% of the total items consumed.

Prey Selectivity

Ivlev's index showed that all five studied species actively rejected zoea larvae and cladocerans for consumption (Fig. 3). This pattern was specially marked for O. melanura, which selected other prey items such as copepodites (E = 0.18) and copepods (E = 0.20). Atherina spp. and B. boops both selected copepodites (E = 0.82 and 1 respectively), and Atherina spp. rejected nauplius larvae (E = -0.72). Juveniles of D. sargus preferred copepodites (E = 0.30) and fish eggs (E = 0.81). L. aurata rejected fish eggs and fish larvae, and preferred adult rather than juvenile copepods (E = 0.34 for copepods).

Fatty Acid Composition

The FA composition of the two types of food pellets used at Campello farm, and the single type used at Guardamar, are indicated in [START_REF] Fernandez-Jover | Changes in body condition and fatty acid composition of wild Mediterranean horse mackerel (Trachurus mediterraneus, Steindachner, 1868) associated with sea cage fish farms[END_REF]. As is common in the FA composition of commercial aquaculture food pellets, there were high levels of palmitic acid (16:0), oleic acid (18:1ω9), linoleic acid (18:2ω6) and eicosapentaenoic acid (EPA; 20:5ω3).

As given by the PERMANOVA test, there were significant differences between the FA profiles of control and farm-associated fish (F=6.57, p<0.01 for L. aurata, and F=6.68, p<0.01 for O. melanura). Significant differences were also detected for some of the individual fatty acids, and fatty acid ratios, for both L. aurata and O. melanura. The presence of palmitic acid (16:0) and linoleic acid (18:2ω6) were clearly higher in the tissue of farm-associated fish than in that of controls (Table 2). There was also significantly lower concentrations of arachidonic acid (20:4ω6) in farm-associated fishes than in controls. For O. melanura, there were significantly higher levels of EPA (20:5ω3) and total ω7 acids in farm-associated fish. Significant differences were also found for ∑(18:2ω6; 18:3ω3) in O. melanura due to increased levels in farm-associated fish (Table 3).

The FA profiles of zooplankton collected at the farms also showed significantly elevated levels of pellet-derived acids compared with zooplankton collected at control locations (Table 2). Indicator ∑(18:2ω6; 18:3ω3) was 6 times higher in zooplankton collected at farms than at control locations. The ratio w3/w6 was 15 times lower in zooplankton collected from farms than at control locations. This was due to high levels of docosahexaenoic acid (DHA) in control zooplankton communities.

DISCUSSION

This study has demonstrated that sea-cage fish farms are settlement sites for several fish The lack of information regarding both the settlement of these species, as well as stock information on adult populations across the Mediterranean mean that it is difficult to relate the influence of aggregations of this magnitude on the total settlement of these species in a particular area.

Our work shows that other species did settle at fish farms, however, they did so in much lower abundances (Table 1) (e.g. Diplodus spp., Seriola spp., Pomatomus saltatrix).

Similarly, the two species cultured at Guardamar and Campello farms, Sparus aurata and Dicentrarchus labrax did settle around the cages, however, are not detected as adults except when escape events occur [START_REF] Dempster | Attraction of wild fish to sea-cage fish farms in the south-western Mediterranean Sea: spatial and short-term temporal variability[END_REF][START_REF] Fernandez-Jover | Seasonal patterns and diets of wild fish assemblages associated to mediterranean Coastal fish farms[END_REF]. Factors that cause fishes to select certain settlement sites but not others, are difficult to assess, however, these fishes may use sound or chemical cues from resident fish (Sweatman, 1988;[START_REF] Dempster | Homing of pelagic fish to fish aggregation devices (FADs): the role of sensory cues[END_REF][START_REF] Mann | On the attraction of larval fishes to reef sounds[END_REF]. This does not seem to be the case for S. aurata and D. labrax as shown by the low numbers of these species found in the vicinity of the cages. However, many of the species detected as juveniles around cages are found in high abundances and biomasses around SW Mediterranean farms as adults. The presence of adult sparids (Boops boops, Diplodus spp., Oblada melanura), mugilids, and carangids (Trachinotus ovatus, Trachurus mediterraneus) around farms may indicate that some species-specific chemical cues are acting and inducing some species to settle around the cages but, at the moment, no data supports this theory. In addition, the floating cages may influence water currents in the vicinity of farms, leading to the retention of presettlement fish in the same way that coral reefs modify local currents favouring the retention and local settlement of coral reef fish [START_REF] Kingsford | Influence of tidally induced fronts and Langmuir circulations on distribution and movement of presettlement fishes around a coral reef[END_REF].

Movements between sea-cages and surrounding natural habitats are also important when juvenile fish reach a certain size. It has been previously reported that both Diplodus puntazzo and D. sargus usually settle on rocky reefs < 2 m deep, while D. vulgaris prefer areas > 2 m deep. The preference for a particular habitat type decreases as the juveniles grow and dispersal from this habitat occurs when individuals reach 45-55 mm in length [START_REF] Macpherson | Ontogenic shifts in habitat use and aggregation in juvenile sparid fishes[END_REF]. [START_REF] García-Rubies | Substrate use and temporal pattern of recruitment in juvenile fishes of the Mediterranean littoral[END_REF] showed that juvenile D. sargus, D. cervinus, D. puntazzo, D. vulgaris and Sarpa salpa prefer to settle in shallow habitats (< 2 m deep) consisting mainly of sand, gravel or small blocks. Furthermore, the size-atsettlement recorded by these authors coincided with that recorded for the same species in the present study (i.e. 5-15 mm). For all censused fish species, abundances were highest for the smallest size-classes of fish. Abundances, however, sharply declined in the following months. This may indicate a migration to other habitats when a greater swimming capacity has been attained [START_REF] Macpherson | Ontogenic shifts in habitat use and aggregation in juvenile sparid fishes[END_REF], or high mortality rates due to increased predatory pressure [START_REF] Finn | Two-phase recruitment of Apogonids (Pisces) on the Great Barrier Reef[END_REF] as a result of the presence of adult carnivorous fishes around the cages (e.g. Pomatomus saltatrix, [START_REF] Sanchez-Jerez | Interactions between bluefish Pomatomus saltatrix (L.) and coastal sea-cage farms in the Mediterranean Sea[END_REF]. If the mortality of juvenile fish is high around cages, there exists the potential for negative effects on local fish stocks, since many of the juveniles are species of commercial importance in the Mediterranean. In any case, the predatory behaviour of adult fishes seems to be highly prey size-dependent, and predation on small juveniles is likely to be moderate in the study region. Indeed, the predators Trachurus mediterraneus and Trachinotus ovatus found around cages mainly feed on uneaten food pellets [START_REF] Fernandez-Jover | Changes in body condition and fatty acid composition of wild Mediterranean horse mackerel (Trachurus mediterraneus, Steindachner, 1868) associated with sea cage fish farms[END_REF]2008). Only a few predator species prey upon wild fish around cages and the size of prey fishes has been shown to be always > 10 cm SL [START_REF] Sanchez-Jerez | Interactions between bluefish Pomatomus saltatrix (L.) and coastal sea-cage farms in the Mediterranean Sea[END_REF]Fernandez-Jover et al., unpublished data). If mortality of juveniles due to predation is moderate around sea-cages, then fish farms may provide shelter as well as appropriate settlement habitat for species that otherwise could have potentially drifted offshore to non-suitable settlement areas. This has been previously suggested to generate positive effects on the survivorship of juvenile fishes around oil platforms [START_REF] Emery | Do oil platforms off California reduce recruitment of bocaccio (Sebastes paucispinis) to natural habitat? An analysis based on trajectories derived from high-frequency radar[END_REF].

Very few empty stomachs were recorded for all fish species examined for dietary analysis and prey selectivity, indicating that food was not a limiting resource around fish farms. In addition, despite that all species had consumed high numbers of copepods and copepodites (except L. aurata), Ivlev's index did not show a clear preference for copepods, indicating that the demand for copepods is low relative to their availability.

The considerable overlap in diet among all studied species shows that food, and especially copepods, to be a non-limiting factor as all species were able to consume other food items at low rates. Food resources associated with cage nets (mainly amphipods such as Caprella spp. and Jassa spp.) are probably not exploited despite their high abundances on the nets (P. Sanchez-Jerez, pers. obs.). These amphipods, which are larger compared with pelagic food resources, may be oversized for the jaw width of most juvenile fishes. Consequently, our results do not support the suggestion that juvenile fishes settle around sea-cages because of the potential for high food availability associated with the structure of the farm itself (i.e. floating rings, nets, etc.).

The consumption of zooplankton by juvenile fishes aggregated around fish farms may affect the structure of local food webs. Zooplankton collected around fish farms showed elevated levels of linoleic acid (18:2ω6) and depressed levels of DHA (22:6ω3) relative to controls. This pattern is linked to the dramatic differences in ω3/ω6 ratio and total polyunsaturated acids between control and farm-associated zooplankton. However, this substantial difference may be due to high levels of DHA in zooplankton collected at control sites. Several studies have found similar DHA levels to those we found in zooplankton collected from around farms. [START_REF] Nanton | The effects of temperature and dietary fatty acids on the fatty acid composition of harpacticoid copepods, for use as a live food for marine fish larvae[END_REF] found DHA and linoleic acid values of 16% and 1.5%, respectively, for zooplankton from the Atlantic Ocean. [START_REF] Rossi | Lipid biomarkers and trophic linkages between phytoplankton and anchovy (Engraulius encrasicolus) larvae in the NW Mediterranean[END_REF] detected DHA and linoleic acid levels that ranged between 12-14% and 2-6%, respectively, for zooplankton from the Mediterranean. These reported values are similar to the DHA levels for farm-associated plankton we detected in this study, however a high proportion of our linoleic acid was of aquaculture food pellet origin. Increments in linoleic acid levels may therefore be a consequence of the consumption of particulate matter arising from uneaten pellets and the faeces of cultivated fish by zooplankton around fish farms. In addition, particulate and dissolved organic matter may influence zooplankton fatty acid composition through the microbial and protozoan loop [START_REF] Mazzocchi | Recurrent patterns in zooplankton structure and succession in a variable coastal environment[END_REF]Turner et al., 1988). This change in the FA composition of zooplankton was reflected in the FA profiles of juvenile fishes associated with the cages. We detected increased levels of linoleic and palmitic acids for both species analyzed. High levels of the terrestrial marker ∑(18:2ω6; 18:3ω3) were also detected for O. melanura and this was a result of the high proportion of copepods (with food pellets influence in their composition) in the diet of this species.

There was also a significant decrease in arachidonic acid (22:4ω6) for both species.

Marine fishes have specific requirement for dietary arachidonic acid [START_REF] Castell | Effects of purified diets containing different combinations of arachidonic and docosahexaenoic acid on survival, growth and fatty acid composition of juvenile turbot (Scophthalmus maximus)[END_REF][START_REF] Bell | Arachidonic acid in aquaculture feeds: current status and future opportunities[END_REF] because of its vital function as the main precursor to a wide variety of biologically active compounds, such as the eicosanoids (Tocher and Sargent, 1987). This acid is necessary for the optimal growth, development, and health of juvenile fishes [START_REF] Bell | Arachidonic acid in aquaculture feeds: current status and future opportunities[END_REF], and hence may contribute to the larval survivorship of marine teleosts. Further work using controlled experiments in the laboratory is necessary in order to better assess the incorporation rates of this acid in different species. Its residence time in fish tissues must also be quantified if arachidonic acid is to be considered as a potential biomarker for testing the influence of fish farms on the physiology of juvenile fish.

In summary, the results of this work clearly demonstrate that juvenile fishes use seacage fish farms as settlement areas in the SW Mediterranean Sea. The input of FAs of aquaculture origin may be associated with a change in the physiology of juvenile fish species which aggregate there. This also raises the question as to whether offshore aquaculture facilities in the Mediterranean are responsible for attracting juvenile fishes to settle in areas with high abundances of adult predators, or conversely, whether they provide a unique settlement habitat for drifting larvae that may otherwise have perished in the open sea before finding appropriate settlement habitat [START_REF] Emery | Do oil platforms off California reduce recruitment of bocaccio (Sebastes paucispinis) to natural habitat? An analysis based on trajectories derived from high-frequency radar[END_REF]. Table 1. Abundances (individuals/cage ± standard error) of fish species counted around the two studied fish farms, A) Guardamar and B) Campello, over a 22 month period. Species are (left to right): Dicentrarchus labrax, Diplodus cervinus, Diplodus puntazzo, Diplodus vulgaris, Unidentified sparids, Pomatomus saltatrix, Sarpa salpa, Seriola spp., Serranus scriba, Sparus aurata, unidentified syngnathids, Symphodus roisalli, Trachinotus ovatus and Trachurus mediterraneus. Densities of the most abundant species are shown in Figure 1.

Tables

A) GUARDAMAR

(

  1957), using a mixture of chloroform and methanol (1:1 for the first extraction and 2:1 for the second). Fatty acid methyl ester (FAME) samples were analyzed according to the method of Stoffel et al. (1959) by gas-liquid chromatography using a SP TM 2560 flexible fused silica capillary column (100 m length, 0.25 mm internal diameter and 0.20 µm film thickness; SUPELCO) in a Hewlett-Packard 5890 gas chromatograph.

  Figure 1) in November 2006. Finally, the sparid Oblada melanura reached the highest abundances for any juvenile fish recorded in the study in the summer months at both farms; Campello: 2355±1144 (7-20 mm SL) and 1778±331.7 (20-30 mm SL) individuals/cage, July 2006; Guardamar: 1287.1±892.2 (<7 mm SL) individuals/cage, June 2007, Figure1). Species that were present in lower abundances are included in

  species in the SW Mediterranean Sea. The almost complete absence of individuals in the open areas between cages suggests that juvenile fishes may associate closely with the floating ring structure of the cages in order to gain protection from predators. Our estimated densities indicate that the most abundant species (i.e. Boops boops, Atherina spp., and Oblada melanura), may reach abundances of > 10000, 20000 and 50000 individuals/farm respectively, depending on the number of cages at the farm studied.

  Dempster et al. (2002) found adult Boops boops, D. sargus, D. vulgaris, Pomatomus saltatrix, Trachinotus ovatus and Trachurus mediterraneus around the same facilities studied in this work. None of these species of between 60 and 150 mm SL were found around the cages in this study, which suggest that a connection between different habitats for different developmental stages exists for these species. Natural tagging experiments (e.g. using otolith chemistry; Gillanders and Kingsford, 2000) may be useful to test if individuals which settle at fish farms as juveniles return to the same offshore aquaculture facilities as adults.

Figure 1 .Figure 2 .Figure 3 .

 123 Figure 1. Species abundances (individuals/cage ± standard error) of juvenile fish of multiple sizes classes (mm SL) around Guardamar and Campello fish farms over the sampling period (April 2006-January 2008).

Table 2 .

 2 Fatty acid composition of juvenile Liza aurata, O. melanura and zooplankton collected from farm and control locations. Data is expressed as percentages ± standard deviations.

			D.	D.			P.		Seriola		S.
	YEAR	MONTH	cervinus	puntazzo D. vulgaris	U. sparid	saltatrix	S. salpa	spp.	S. scriba S. aurata	roisa
	2006	4						31.37±10.07	0.28±0.08
		5						4.11±0.96			17.36±2.41
		6
		7 0.13±0.02			12.91±1.67		27.05±4.79
		8						2.25±0.25	0.11±0.1
		9
		10				2.27±1.03					0.42±0.11
		11				132.1±48.71
		12
	2007	1
		2
		3
		4
		5
		6
		7		0.24±0.03
		8							0.31±0.04		0.1±0
		9		0.19±0.04
		10		1.48±0.27 41.18±11.44		1.04±0.26
		11			79.96±46.76
		12
	2008	1			1.46±0.38
	B) CAMPELLO
					D.				S.		U.
	YEAR MONTH D. labrax D. cervinus	puntazzo D. vulgaris U. sparid	P. saltatrix	salpa Seriola spp.	syngnathid T. ovatu
	2006	4
		5
		6			0.82±0.14
		7 0.14±0.02 0.57±0.11					0.14±0.02	4.13±0
		8 0.26±0.03	0.3±0.06							214.45±
		9
		10				10.58±3.65
		11				45.75±12.11
		12			0.74±0.19
	2007	1			0.23±0.06

Table 3 .

 3 ANOVA results for comparison of the main fatty acid composition of A) Oblada melanura and B) Liza aurata collected from fish farms and control locations. * p<0.05, ** p<0.01, *** p<0.001.

		Value of ANOVA F-statistic
		df Linoleic acid	DHA	∑(18:2ω6;18:3ω3) Palmitic acid ω3/ω6 Arachidonic acid EPA
	A) Oblada melanura
	Farm/Control	1 12.09**	0.49	9.14**	4.84*	3.18	108.8***	24.9***
	Residual	14
	Total	15
	B) Liza aurata
	Farm/Control	1 4.69*	0.76	1.55	8.38**	0.44	25.08***	0.52
	Residual	22
	Total	23

Acknowledgments

We thank the staff of the Cudomar (El Campello) and Martorres (Guardamar) fish farms for their help and to J. Hughes and F. Tuya for language corrections and C. Valle for his help during field sampling. Comments made by J. Dalsgaard helped to improve the manuscript. The study forms part of the ACUFISH project (Dpt. Marine Science and Applied Biology, University of Alicante) and was funded by Spanish Ministry of Science grant MYCT-REN2003-00794.

Liquid chromatography. Micromethod for preparation of methyl esters. Analytical Chemistry 31, 307-308. Sweatman, H., 1988. Field evidence that settling coral reef fish larvae detect resident fishes using dissolved chemical cues. Journal of Experimental Marine Biology and Ecology 124 (3), 163-