Montserrat Solé

Meritxell Antó

Miguel Baena

Maite Carrasson

Francesc Joan E Cartes

Francesc Maynou

Hepatic biomarkers of xenobiotic metabolism in eighteen marine fish from NW Mediterranean shelf and slope waters in relation to some of their biological and ecological variables

Keywords: antioxidant enzymes, xenobiotic metabolism, ecological parameters, marine fish, NW Mediterranean

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Hepatic biomarkers of xenobiotic metabolism in eighteen

marine fish from NW Mediterranean shelf and slope waters in relation to some of their biological and ecological variables

Introduction

In the marine environment, pollution monitoring studies are currently carried out mainly in coastal areas due to their proximity to the point sources of anthropogenic chemicals. However, transport of chemicals from coastal sites to greater depths by sea currents as well as the global atmospheric transport, contribute to the presence of pollutants offshore and in remote areas (UNEP/ [START_REF] Unep/Map ; A | Molecular biomarkers of oxidative stress in aquatic organisms in relation to toxic environmental pollutants[END_REF]. The NW Mediterranean shelf and slope are subject to intense fisheries, and the occurrence of anthropogenic chemicals in their waters/sediment/biota has been well studied within the Mediterranean context [START_REF] Albaigés | Persistent Organic Pollutants in the Mediterranean Sea[END_REF]Gómez-Gutiérrez, 2007). Nevertheless, studies reporting the effects of chemicals on commercial fish are scarce as most of them focus on the sentinel red mullet in coastal sites [START_REF] Porte | Assessment of coastal pollution by combined determination of chemical and biochemical markers in Mullus barbatus[END_REF][START_REF] Martin-Skilton | Evidence of endocrine alteration in red mullet, Mullus barbatus from the NW Mediterranean[END_REF][START_REF] Zorita | Assessment of biological effects of environmental pollution along the NW Mediterranean Sea using red mullets as sentinel organisms[END_REF], with less attention being paid to species from fishing grounds (García de la Parra et al., 2000;[START_REF] Solé | Esterase activities and lipid peroxidation levels in offshore commercial species of the NW Mediterranean Sea[END_REF]2009a;2009b). Thus, we have investigated the presence of organic pollutants (PAHs, PCBs and DDTs) in sediment from two areas: shelf and slope. We have also determined the activities of hepatic markers involved in the metabolism of foreign compounds (xenobiotics), as well as antioxidant defences, in several fish species from these two habitats. The biomarkers selected are among those currently adopted in pollution monitoring studies [START_REF] Cajaraville | The use of biomarkers to assess the impact of pollution in coastal environments of the Iberian Peninsula: a practical approach[END_REF][START_REF] Van Der Oost | Fish bioaccumulation and biomarkers in environmental risk assessment: a review[END_REF]. The main role of the antioxidant defences is to prevent the action of an excess of oxyradicals resulting from exposure to xenobiotics (Valavanidis et al., 2006).

Among the antioxidant defences, catalase (CAT) transforms H 2 O 2 in water and O 2 .

Glutathione reductase (GR) is an antioxidant enzyme that maintains the balance GSH/GSSG favouring the reduced form (GSH). GSH is considered a key soluble antioxidant by itself but also as it is a cofactor in several enzymes. As opposed to CAT, GR requires NADPH, and it is therefore a more energy demanding enzyme. Although

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4 not considered in the present study, aquatic organisms possess many other defences either enzymatic (e.g. superoxide dismutase, glutathione peroxidase) or non-enzymatic oxyradicals scavengers (e.g. glutathione, carotenoids, vitamins C and E) that can also play an antioxidant role.

Another hepatic marker selected in our study is part of the cytochrome P450 superfamily. In particular the CYP1A form is a terminal protein in the mixed function oxygenase system, indicative of phase I metabolism (mostly hydroxylation reactions).

In fish, its main catalytic role is frequently measured as ethoxyresorufin O-deethylase (EROD) activity and it is a well accepted biomarker of dioxin-like compounds and PAHs exposure. However, diet and some environmental and physiological factors can modulate the response of the total cytochrome P450 content. This is specially evident in the response of the CYP1A dependent EROD activity [START_REF] Whyte | Ethoxyresorufin O-deethylase (EROD) activity in fish as a biomarker of chemical exposure[END_REF].

Carboxylesterases are also classified as phase I enzymes involved in the hydrolysis of many chemicals and have a relevant role in detoxification and cell protection from chemicals action [START_REF] Satoh | Structure, function and regulation of carboxylesterases[END_REF][START_REF] Al-Ghais | Differential inhibition of xenobioticmetabolizing carboxylesterases by organotins in marine fish[END_REF]. Foreign chemicals, but also endogenous compounds (e.g. sex hormones), that undergo phase I metabolism (e.g. hydroxylation, oxygenation reactions) can be further conjugated with endogenous molecules, such as GSH, via glutathione S-transferases (GSTs) and consequently, be more readily excreted (phase II metabolism). Moreover, GST activity has also an antioxidant role as peroxidase [START_REF] George | Enzymology and molecular biology of phase II xenobioticconjugating enzymes in fish[END_REF]. Both, phase I and II systems are seen to vary seasonally, although variations in GST are much less marked than EROD activity. Protein yield (PY) was adopted as an unspecific marker indicative of hepatic protein synthesis. A ratio between bioactivation (phase I) and detoxification (phase II) named biotransformation index (BTI) has been proposed in fish as indicative of the balance of both paths [START_REF] Van Der Oost | Assessment of environmental quality and inland water pollution using biomarker responses in caged carp (Cyprinus carpio): use of bioactivation:detoxification ratio as a biotransformation index (BTI)[END_REF].

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The selection of the fish species was based on their availability and frequency in the catch by the trawling fleet in the selected sites, their economic and ecological interest were also considered. Moreover, the choice of a broad spectrum of species contributes to fill in the gap concerning xenobiotic biotransformation enzymes in marine fish [START_REF] Fitzsimmons | A compilation of in vitro rate and affinity values for xenobiotic biotransformation in fish, measured under physiological conditions[END_REF], as most data, particularly in the Mediterranean, refers to the red mullet Mullus barbatus and few attempts have been made to use other sentinels. To name a few studies considering species also adopted in this present survey: e.g Helicolenus dactylopterus [START_REF] Amato | An integrated ecotoxicological approach to assess the effects of pollutants released by unexploded Chemicals ordnance dumped in the southern Adriatic (Mediterranean Sea)[END_REF], Phycis blennoides (Garcia de la Parra et al., 2000) and Merluccius merluccius [START_REF] Pietrapiana | Evaluating the genotoxic damage and hepatic tissue alteration in demersal fish species: a case study in the Ligurian Sea (NW-Mediterranean)[END_REF]. The reason for enlarging the number of species, as potential sentinels, and extending the area of study offshore is justified for the interest, in the case of pollution events (e.g oil spills, dredging activities, dumping operations), of having previously characterised the natural variations on these hepatic parameters currently applied in pollution monitoring studies.

On a previous study, the relationship between xenobiotic metabolism enzymes and fish diet, habitat, phylogeny was pointed out (Solé et al., 2009a). The present study aims to further explore and strengthen, or rebate, these former observations by considering more species (from 9 to 18) as well as by adding temporal (seasonality) and spatial (two sites comparison) variations as novel factors.

Materials and methods

Sampling area and fish collection

The sampling took place seasonally: winter (February), spring (April), summer m (41º 12'N, 2º 28'E) and 1000 m (41º 10'N, 2º 30'E). During the summer cruise, fish from another area (Vilanova) were also sampled on the shelf (41º 14'N; 1º 76'E) and slope (41º 07'N; 2 º21'E). Once on board, fish were immediately sexed, weighed, measured and dissected and their livers frozen in liquid nitrogen and transported to the laboratory where they were kept at -80ºC until biochemical analysis. A list of the species selected is given in Table 1. More information, including their phylogeny, common name and depth of occurrence, as well as a map of the selected sites is detailed elsewhere [START_REF] Solé | Muscular cholinesterase activities and lipid peroxidation levels as biomarkers in several Mediterranean marine fish species and their relationship with ecological variables[END_REF].

Physiological markers

Hepatosomatic index (HSI) was calculated as: (liver weight/body weight)x100, condition factor (CF) as: (body weight/length 3 )x100 and gonadosomatic index (GSI) as:

(gonad weight/body weight)x100. Protein yield (PY) of the liver was calculated as: mg protein/g wet weight.

Sample preparation and biochemical determinations

A portion of the liver (0.2-0.5 g) was homogenised in phosphate buffer using a polytron® blender and the supernatant (S10) obtained after centrifugation at 10,000g for 30 minutes was used for biochemical analysis. For comparative purposes, assay conditions were kept similar and only the sample volume varied in order to achieve linearity in the enzymatic measurements. All assays were carried out in duplicate or triplicate at 25ºC with the S10 supernatant, with the exception of CAT, CbE and GST activities which required a further dilution. A description of the methodologies for sample preparation and enzymatic determinations: catalase (CAT; EC 1.11.1.6),

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7 glutathione reductase (GR; EC 1.6.4.2), 7-ethoxyresorufin O-deethylase activity (EROD; EC 1.14.14.1), carboxylesterases (CbE; EC 3.1.1.1), glutathione S-transferase (GST; EC 2.5.1.18) as well as total protein content in the S10 fraction was as described elsewhere [START_REF] Solé | Esterase activities and lipid peroxidation levels in offshore commercial species of the NW Mediterranean Sea[END_REF]2009a). Briefly, CAT, GR, CbE and GST were measured spectrophotometrically at the wavelength of 240, 340 (as decreases), 405 and 340 (as linear increases in optical density), respectively, over a one to 5 minutes period using a microplate reader model TECAN Infinite200. EROD was measured fluorimetrically after 30 minutes incubation at 30ºC and using resorufin as standard. Activity was expressed in relation to the total protein measured [START_REF] Bradford | A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein dye binding[END_REF].

Statistical analysis

Physiogical parameters (Table 1) are presented as mean ± SD and hepatic biomarkers (Table 2) as mean ± SEM. Species differences were tested using parametric one-way (Fig 1 ) or two-way ANOVA (Table 2) followed by post-hoc test (Newman-Keuls) using the software Statistica v. 7.0. Pairwise comparisons using t-test were applied to site comparisons (Besós and Vilanova). Pearson correlation coefficients were computed to analyse the relationship between pairs of biomarkers.

Canonical Correspondence Analysis (CCA; Legendre and Legendre 1998) was performed with the R library vegan to associate biochemical markers with season and quantitative ecological variables in the multidimensional space. CCA is a multivariate technique that allows examining in a reduced dimensional space the relationship between variables (in our case, biomarkers) and linear constraints (in our case, season and ecological variables). The results of the CCA analysis can be visualised through a graph called biplot that allows depicting simultaneously the linear constraints and the variables. Significance of the ordination plot was tested through the pseudo-F test

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proposed by Legendre and Legendre (1998) with 1000 permutations. The significance of each linear constraint was likewise assessed with the permutational envfit routine of the vegan package (J. Oksanen, http://cc.oulu.fi/~jarioksa/softhelp/vegan.html).

Results

Ecological and physiological parameters

Biological and ecological features of the fish species analyzed such as size, habitat depth, swimming capacity, trophic level, and the contribution of different faunistic compartments (benthos, suprabenthos and zooplankton) in the fish diet were presented as tables 1 and 2 elsewhere [START_REF] Solé | Muscular cholinesterase activities and lipid peroxidation levels as biomarkers in several Mediterranean marine fish species and their relationship with ecological variables[END_REF]. Detailed definition of stomach fullness (F) as an indicator to estimate the amount of food consumed by fish (indicating the trophic condition of fishes) are in [START_REF] Cartes | Changes in the diet and feeding of the hake Merluccius merluccius in the shelf-break of Balearic islands (Western Mediterranean): influence of the mesopelagic-boundary community[END_REF].

Herein we report on physiological parameters such as: condition factor (CF), hepatosomatic (HSI) and, in females the gonadosomatic (GSI) indexes (Table 1). The HSI was seen as species dependent and varied seasonally. The HSI in perciforms (0.5-3.3) was lower than in gadiforms (0.9-6.5) and in the elasmobranchs was the highest (4.8-11.5). On the contrary, the CF (indicator of the nutritional status) was higher in perciforms (0.4-1.3) than in gadiforms (0.2-0.8) and it also displayed seasonality. The GSI was measured in females and tentatively used as indicative of the reproductive period for the species. For shallow species with a full seasonal data set, summer was seen the reproductive period for M. barbatus and T. draco, whereas in S. maena it was winter. In slope species M. poutassou and P. blennoides a higher GSI corresponded to the autumn period, whereas in T. scabrus, N. aequalis and G. melastomus it was winter.

Species differences in hepatic markers

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Due to unbalanced sampling data sets, to test for species differences on the activities of the hepatic markers: PY, CAT, GR, EROD, CbE and GST, only fish from the winter sampling were selected. This was based on the fact that the winter sampling included to a more complete set of species and did not consider the temporal variations (as a potential confounding factor) due to the observed seasonality in some of the Enzymatic activities considering all the seasons are indicated (Table 2) for each biomarker and species in order to define trends as it enlarged, considerable for some species, the number of individuals considered and thus allowing a more robust statistical analysis. A classification in two main phylogenic groups (teleosts and elasmobranchs) was also made. Clear differences were seen in both groups in all enzymatic activities with the exception of GST activity (Table 2).

Seasonality was also attempted in eleven species (seven from the shelf and five from the slope) as they occurred in, at least, three samplings. All biomarkers varied seasonally although a clear pattern for species and enzymatic activity could not be evidenced. Overall, in the winter sampling, EROD and CbE activities were significantly higher while, in the summer it was CAT (Table 2). No site related differences were seen for most biomarkers, with the exception of P. blennoides from the Vilanova site, where CAT was significantly inhibited (F=5.26, p=0.034) while GST and CbE were enhanced (F=5.99, p=0.025 and F=13.82, p=0.0016, respectively). However, consideration has to be given to the fish size (34.5 ± 7.8 and 26.5 ± 2.2 cm TL for Besós and Vilanova specimens, respectively). Fish of comparable sizes from both sites, such as M. barbatus, exhibited elevated CbE while T. draco had reduced GR activity, both species sampled at the Vilanova station.

Statistical differences between habitats, groups and seasons are indicated in

Relationship with ecological parameters

Canonical correspondence analysis (CCA) was first attempted with all the species with a complete set of data (14 out of 18) and considering the temporal variation. However, this attempt did not indicate any clear pattern. Thus, we separated fish according to their habitat (shelf and slope) and, in this case, the relationships between biomarkers against season and ecological variables, as explanatory factors, were all significant, explaining 57.4% of the variance in shelf biomarkers and 47.2% in slope biomarkers (fig. 2). Pearson product-moment correlations (r) between individual biomarkers and ecological variables in the shelf and slope are shown in Table 3, 12 relationship with ecological variables and in particular with the main components of the diet (benthos, suprabenthos, zooplankton) was not as clear as in the shelf group, and also the strength of the ordination was lower, with less than 50% of the data variance explained by the first 2 axes. However, GR and GST were positively related to swimming, as occurred in the shelf, and to feeding on zooplankton, while they were inversely related to feeding on benthos. PY was also positively related to F. Slope species feeding on suprabenthos exhibited higher TL and lower F.

Discussion

Transport of pollutants from coastal point sources to greater depths is well documented in the Mediterranean (review of [START_REF] Albaigés | Persistent Organic Pollutants in the Mediterranean Sea[END_REF]. Some chemicals can reach (in some species and for some chemical classes) concentrations closer to those recorded in coastal fish [START_REF] Escartín | Hydroxylated PAHs in bile of deep-sea fish. Relationship with xenobiotic metabolising enzymes[END_REF][START_REF] Borghi | Organotin pollution in deep-sea fish from the northwestern Mediterranean[END_REF][START_REF] Storelli | Polychlorinated biphenyl residues in deep-sea fish from Mediterranean Sea[END_REF]2008;2009). Thus the modulation of xenobiotic metabolising enzymes and antioxidant defences in fish inhabiting these offshore areas is likely to occur. In fact this study evidences that, in general terms, the antioxidant enzymes (CAT and GR), GST and CbE, although not EROD, were more elevated in fish inhabiting greater depths.

Depth-related changes in metabolism have largely been studied in pelagic fish but less is known on benthic and benthopelagic species, coincident, the latter group, with the habitat of the fish in this study. [START_REF] Drazen | Depth-related trends in metabolism of benthic and benthopelagic deep-sea fishes[END_REF] described a decrease in some muscular enzymatic activities in fish in relation to depth, specially for the first 600 m in a study carried out in Pacific waters. This trend being more obvious for pelagic rather than for benthic and benthopelagic fish. Among the reason(s) for the reported lower activities in deeper adapted fish, one is based in the visual-interactions hypothesis and, consequently, would affect those enzymes involved in motility. In addition, most

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13 studies carried out relating fish metabolism and depth are conducted in regions of the world, other than the Mediterranean. In those other regions, changes on environmental parameters (temperature, oxygen concentration) are large and the depths considered elsewhere went up to of 4000 m (Drazen and Seibel, 2007 and references within). In this current study (60-1000 m), a clear decrease in hepatic protein content, indicated as PY (Fig 1A) as well as in muscle protein content [START_REF] Solé | Muscular cholinesterase activities and lipid peroxidation levels as biomarkers in several Mediterranean marine fish species and their relationship with ecological variables[END_REF] coincide with observations for other regions and fish species [START_REF] Childress | Depth-related enzymatic activities in muscle, brain, and heart of deep-living pelagic teleosts[END_REF]Janssens et al., 2000, Drazen and[START_REF] Drazen | Depth-related trends in metabolism of benthic and benthopelagic deep-sea fishes[END_REF]. Despite this coincidence, in the present Mediterranean study, the physical parameters in water experienced little fluctuations.

That is, mean annual temperatures measured at 5 metres above the sea bottom (mab) at both contrasted depths (shelf and slope) within each season was only 1-2ºC apart, except in autumn which was almost 4ºC different. Likewise, oxygen at 5 mab only varied from 0.1 to 0.2 mg/L, as it is characteristic in the Mediterranean. But, what is more relevant for contrasting purposes is the fact that the enzymes here considered refer to those involved in hepatic metabolism rather than to muscular markers, more involved in motility. In short, as a clear decrease in protein over depth did not coincide with a decrease in hepatic activities, xenobiotic metabolism is not likely to be compromised in fish from greater depths.

This study also evidenced great species differences in hepatic markers, regardless of habitat and group. As far as antioxidant parameters concerns, former studies had indicated a decrease in some antioxidant defences: SOD and GPX, although not CAT, in deep sea fish sampled up to 1300 m in Atlantic waters [START_REF] Janssens | Reduced enzymatic antioxidative defences in deep-sea fish[END_REF]. This trend was not seen in this Mediterranean study, as CAT and GR activities were, on the contrary, significantly enhanced in some slope fish including N. aequalis and L. lepidion. In fact, these results agree with the hypothesis of a metabolically

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14 cheaper antioxidant defence (CAT) in fish inhabiting poorer environments [START_REF] Janssens | Reduced enzymatic antioxidative defences in deep-sea fish[END_REF]. Great species differences are coincident with another Mediterranean study that contrasted antioxidant defences and xenobiotic metabolism in strictly deep-sea fish (1500-1800 m), in which species particularities were attributed to their biology/diet rather than to site trends, as chemical exposure was also similar [START_REF] Porte | Xenobiotic metabolising enzymes and antioxidant defences in deep-sea fish: relationship with contaminant body burden[END_REF].

Species differences were also evidenced in P. blennoides and the flatfish Lepidorhombus boscii sampled in the same NW Mediterranean region and at 350-450 m depth range and, also in this case, fish were exposed to a similar pollution load (García de la Parra et al., 2000). The a priory expected site differences in chemical exposure between the two selected sites (Besós and Vilanova) was not confirmed by the hepatic responses, neither by the bile PAH levels [START_REF] Insausti | Biliary fluorescent aromatic compounds (FACs) measured by fix wavelength fluorescence (FF) in several marine fish species from the NW Mediterranean[END_REF], nor by the muscular markers or the chemical analysis of the sediment [START_REF] Solé | Muscular cholinesterase activities and lipid peroxidation levels as biomarkers in several Mediterranean marine fish species and their relationship with ecological variables[END_REF]. Besós site is under the influence of the river with the same name and well-known for its high pollution load [START_REF] Castells | Occurrence of polychlorinated naphthalenes, polychlorinated biphenyls and short-chain chlorinated paraffins in marine sediments from Barcelona (Spain)[END_REF]. Vilanova, on the contrary, is a fishing port with a lower industrial influence from the nearby coast. However, it is likely that this port, situated south of the Llobregat river (with an important industrial catchment area), receives the Llobregat river influence as a result of the north-south water currents in the NW Mediterranean.

Turbulence events that would enhance pollutants biodisponibility were higher in spring and winter and in the Vilanova site. Neither chemical analysis nor turbulence events support significant Besós-Vilanova site differences or consistent temporal trends, as far as the persistent chemicals analysed concerns [START_REF] Solé | Muscular cholinesterase activities and lipid peroxidation levels as biomarkers in several Mediterranean marine fish species and their relationship with ecological variables[END_REF]. This also implies that greater pollution gradients are necessary to be reflected in the adopted biomarkers. In fact, field studies to successfully relate biomarker responses to pollution in the Mediterranean, refer to sites closer to land discharges and therefore subject to greater pollution loads [START_REF] Porte | Xenobiotic metabolising enzymes and antioxidant defences in deep-sea fish: relationship with contaminant body burden[END_REF], or linked to particular pollution events: e.g dumping

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15 operations [START_REF] Regoli | Application of biomarkers for assessing the biological impact of dredged materials in the Mediterranean: the relationship between antioxidant responses and susceptibility to oxidative stress in the red Mullet (Mullus barbatus)[END_REF][START_REF] Amato | An integrated ecotoxicological approach to assess the effects of pollutants released by unexploded Chemicals ordnance dumped in the southern Adriatic (Mediterranean Sea)[END_REF]. The levels of PCBs (0.5-5.8 ng/g d.w.), DDTs (1.4-14.4 ng/g d.w.) and PAHs (176-466 ng/g d.w.) encountered in the sediment of the selected sampling sites are in the low range of those reported in sediments from other NW Mediterranean areas [START_REF] Cardellicchio | Organic pollutants (PAHs, PCBs) in sediments from the Mar Piccolo in Taranto (Ionian Sea, Southern Italy)[END_REF][START_REF] Gómez-Gutierrez | Assessment of the Mediterranean sediments contamination by persistent organic pollutants[END_REF][START_REF] Castells | Occurrence of polychlorinated naphthalenes, polychlorinated biphenyls and short-chain chlorinated paraffins in marine sediments from Barcelona (Spain)[END_REF].

As it was observed for the antioxidant defences, phase I and II enzymes also evidenced greater species differences rather than site trends. While EROD was particularly enhanced in fish from the shelf, CbE and GST were better represented in slope species. Other studies in the Mediterranean, contrasting deep-sea fish (L. lepidion included) with coastal fish also revealed comparable enzymatic activities [START_REF] Escartín | Hydroxylated PAHs in bile of deep-sea fish. Relationship with xenobiotic metabolising enzymes[END_REF]. The same trend occurred in an Atlantic study with fish sampled from 300 to 3500 m [START_REF] Stegeman | Cytochrome P450 1A expression in midwater fishes: potential effects of chemical contaminants in remote oceanic zones[END_REF]. Among gadiforms, T. scabrus displayed high EROD activity (considering is a slope species), as well as the highest CbE and one of the highest GST activities. T. scabrus is one of the species distributed deeper among our targeted ones, and it has a high feeding activity over Calocaris macandreae, a burrowing shrimp living into the mud [START_REF] Cartes | Changes in the diet and feeding of the hake Merluccius merluccius in the shelf-break of Balearic islands (Western Mediterranean): influence of the mesopelagic-boundary community[END_REF]. As CbE is a general hepatic metabolism marker, high levels of CbE in T. sabrus could be related to higher exposure to sediment bound pollutants in bathyal-muddy bottoms and/or to a particularly high metabolism in this species. On the contrary, the lowest enzymatic defences were clearly displayed by elasmobranchs, confirming earlier observations (Filho et al., 1993a;1993b[START_REF] Gorbi | Antioxidant efficiency and detoxification enzymes in spotted dogfish Scyliorhinus canicula[END_REF], Solé et al., 2009a). Nevertheless, due to their large liver (about 10 g) in the species included in this study, the total hepatic functions in these elasmobranchs might not differ so much from the shelf fish with smaller livers (0.5-2 g). Nevertheless, it has to be taken into account that the liver in elasmobranchs has also an important function in swimming and floatability, thus our observation is merely

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Another important factor to consider when comparing shelf and slope is that all our measurements were carried out under the same assay conditions of substrate concentrations, pH, temperature and pressure while in the natural environment fish will experience particular conditions depending on their habitat that will affect enzymes kinetics and protein interactions, specially in those from greater depths [START_REF] Gibbs | Biochemistry at depth[END_REF].

In this study, the ratio EROD/GST termed biotransformation index (BTI) and used as an estimation of phase I/phase II metabolism, was also adopted [START_REF] Van Der Oost | Assessment of environmental quality and inland water pollution using biomarker responses in caged carp (Cyprinus carpio): use of bioactivation:detoxification ratio as a biotransformation index (BTI)[END_REF]. While in the biotransformation process EROD can generate chemicals more reactive than the parent compound, GST is always a detoxification step. Due to the larger variations in EROD than in GST activities between species, this index mostly followed EROD trends (Table 2). This BTI ratio could predict that perciforms are more prone to form toxic metabolites, whereas T. minutus showed the greatest detoxification capacity. Coincident with T. scabrus, T. minutus also feeds on prey distributed on or in the mud (e.g. the burrowing shrimp Alpheus glaber: authors' unp. data). BTI as an index has been successfully used contrasting sites using a single sentinel species (van der Oost et al 1998), while in the present study several species are contrasted and, the nature of the particular P450s involved, could greatly differ. Nevertheless, this index could be useful as an estimation of an overall biotransformation trend.

Seasonal variation in the hepatic biomarkers was also attempted in this study.

Although a common and clear pattern was not observed, enzymatic activities in winter were, in general, higher than in summer (Table 2). Other studies have revealed that the parameter most affected by seasonality is EROD activity, as it is modulated by sex hormones, and this would reflect the reproductive season, mostly in females, by expressing a significantly lower EROD activity [START_REF] Whyte | Ethoxyresorufin O-deethylase (EROD) activity in fish as a biomarker of chemical exposure[END_REF]. An attempt to correlate EROD activity with the GSI in females in two species, one from the shelf, M.

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barbatus, and one from the slope, T. scabrus was made. Nevertheless, EROD activity was not related to GSI as in a mixed group of males, females and immature specimens.

When grouping them by sex the sample size was too small to predict any relationships.

In future studies more frequent samplings (monthly) and larger sample sizes should be considered in order to test for such correlations.

Due to the large number of species (18), biomarkers (7) and ecological variables (6) considered, a multivariate CCA was carried out in order to relate biomarker responses with ecological parameters in shelf and slope fish. Significant relations were observed (Fig. 2), although some differ from those in a former study (Solé et al., 2009a), despite enzymatic activities in the common species and sampling period were fully coincident. To account for this difference, it could be (1) the fact that fish from shelf and slope are now considered independently, (2) the ecological parameter TL is now given a numeric value and (3) samples from several seasons are included. In this current approach, relationships between biomarkers and ecological variables were all significant (p<0.001) although the ecological variables chosen only explained around 50% of the variance observed in the biomarkers. Thus other parameters not considered here are likely to play an important role in the biomarker responses. Overall, our observations confirm the role of seasonality in modulating biomarker responses and the need for adequate references when applying these biomarkers in pollution monitoring.

Another important observation extracted from the CCA is that the relationships between hepatic biomarkers and trophic variables were clearer in shelf than in slope fish. This is probably because of the lower prey availability on deeper environments than drives to an increase in the trophic diversification of slope species [START_REF] Cartes | Feeding strategies and partition of food resources in deep-water decapod crustaceans (400-2300 m)[END_REF]. That is, slopedwelling fish exploit, with a similar intensity, prey from different ecological compartments (benthos, zooplankton). In this way, TL was better related to hepatic

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18 biomarkers in shelf than in slope fish. In turn, this coincides with a more simple organization (trophic chain) over the shelf while more complex one (food webs) is present at greater depths. In the slope, in addition to scarcer food there are also more unpredictable pulses of food availability. Moreover, liver in slope fish can also be regarded to act as a storage organ (i.e. liver and HSI in slope fish is bigger than in those from the shelf) and this may differentially affect hepatic enzymes. Even if physical parameters (temperature, oxygen, salinity) at the slope in the Mediterranean remain fairly constant, seasonality is still an important force influencing the fish diet, feeding activity and reproduction [START_REF] Cartes | Changes in the diet and feeding of the hake Merluccius merluccius in the shelf-break of Balearic islands (Western Mediterranean): influence of the mesopelagic-boundary community[END_REF]. On the contrary, in the Mediterranean shelf, significant differences in physical parameters between periods of homogeneity (winter and spring) and stratification (mostly summer) can be encountered due to existence of a thermocline at about 150 m in the Mediterranean [START_REF] Cartes | Changes in the diet and feeding of the hake Merluccius merluccius in the shelf-break of Balearic islands (Western Mediterranean): influence of the mesopelagic-boundary community[END_REF].

Swimming activity also played, as expected, a more marked role in shelf fish.

Nevertheless, this role could be enhanced due to the presence in the shelf of B. boops, a species which feeds exclusively on pelagic prey while, at the slope, due to above mentioned dietary diversification, an equivalent species was not present [START_REF] Cartes | Feeding strategies and partition of food resources in deep-water decapod crustaceans (400-2300 m)[END_REF].

Conclusion

This study evidences great species differences in hepatic biomarkers that contrast with little variations in physical environmental parameters and pollution gradients. Most enzymatic activities, except EROD, were enhanced in slope species (mostly gadiforms) in contrast to shelf species (mostly perciforms), despite pollutant loads in the sediment at both depths being similar. This study further explores relationships between hepatic biomarkers and ecological parameters formerly outlined (Solé et al., 2009a). As the CCA revealed, in the shelf seasonality plays a more swimming, fullness-F and type of diet and cruise code). Ecological variables as in Table 1 in [START_REF] Solé | Muscular cholinesterase activities and lipid peroxidation levels as biomarkers in several Mediterranean marine fish species and their relationship with ecological variables[END_REF]. A) shelf fish and B) slope fish. All relations are significant and the ecological variables explain 57.4 % and 47.2 % of the variance for hepatic biomarkers from shelf and slope fish, respectively.

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Table 1. Mean and standard deviations of morphological markers: Hepatosomatic Index (HSI), Condition Factor (CF) and Gonadosomatic Index (GSI) in fish species collected at the fishing grounds of Besós. The number of individuals analysed is the same of Table 2 from [START_REF] Solé | Muscular cholinesterase activities and lipid peroxidation levels as biomarkers in several Mediterranean marine fish species and their relationship with ecological variables[END_REF], except for the GSI in females which is indicated in brackets (n). n.a. not available. .31 ± 1.73 (3) 7.20 ± 3.12 0.31 ± 0.05 0.32 ± 0.27 (5) 5.86 ± 2.66 0.30 ± 0.03 0.09 ± 0.08 (2) 4.80 ± 1.42 0.29 0.04 0.49 ± 0.53 (6)

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(

  July) and autumn (October) in 2007. It was carried out on research (Garcia del Cid-CSIC) and trawling commercial (Stella Maris III) vessels in front of the Barcelona coast (NE Spain) offshore the Besós river on the continental shelf at depths of 50-65 m (41º 2º 16'E) and 200 m (41º 20'N, 2º 15'E) and on the continental slope at 600-850

  biomarkers (Fig 1; see also below Section 3.3). Nevertheless, N. aequalis (summer) and B. boops (spring and summer) are also included here for species comparison as no winter samples were available. In addition, in the winter sampling, M. merluccius was present at two depth ranges: 50-60 (shelf) and 600-850 m (slope). A clear decrease in PY content in relation to habitat depth was evidenced (Fig 1A). This relationship was significant and negative between PY and HSI (r=-0.726; p<0.05; n=16), all teleosts groups considered. Antioxidant defences such as CAT (Fig 1B) were, in general, lower in shallow fish (except in C. linguatula, T. minutus and M. merluccius), whereas GR activity (Fig 1C) was species-dependent regardless of habitat. Extreme GST activities (Fig, 1D) were recorded in T. minutus, which was 7.5-fold higher than in C. linguatula (the species with the lowest GST activity). Phase I EROD (Fig 1E) activity was significantly elevated in the perciforms: P. acarne, P. bogavareo and M. barbatus. Among slope fish, only T. scabrus expressed EROD activity comparable to shallow fish. As for CbE (Fig 1F), T. scabrus and H. dactylopterus from the slope displayed, by far, the highest activity. Homogeneous groups are indicated by the same letters in the respective figures.

  Biomarkers in relation to habitat(shelf and slope), group (teleosts and elasmobranchs) and season(winter, spring, summer and autumn) Two main groups were made: those from the shelf 50-60 m (including P. bogavareo from 200 m) and those from the slope (600-850 m) which also considered the most strictly deep-sea L. lepidion and N. aequalis (1000 m). Shelf fish are mostly represented by perciforms while slope fish are mainly gadiforms and the two elasmobranchs included are each one representative of a habitat: S. canicula (shelf) and G. melastomus (slope). In Table2results for the biomarker responses in relation to habitat are also indicated. Depth influenced all enzymatic activities: CAT, GR and CbE were significantly lower in shelf fish while EROD activity was elevated.

  3.4. Site differencesSix species were fished at both sites (Besós and Vilanova) in a sample size big enough (n=10) to allow comparison: M. merluccius, M. barbatus, S. maena from the M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 11 shelf and P. blennoides, T. scabrus and G. melastomus from the slope.

  which can help assess the strength and direction of correlation between biomarkers and ecological variables shown in the CCA ordination. CCA analysis from the shelf species (Fig 2A) revealed that swimming was positively related to GR and GST (first axis) and negatively to CAT, while EROD and CbE were positively related to feeding on zooplankton and benthos, respectively, which essentially determined ordination axis 2. Trophic level (TL) was inversely related, in turn, to fullness (F) and suprabenthos feeding. For the slope species (Fig 2B), the

Figure 1 .

 1 Figure legends

Figure 2 .

 2 Figure 2. Results of the Canonical Correspondence analysis (CCA) ordination of

  Fig 2A. Shelf

Table 2

 2 by using different superscripts.

Table 2 .

 2 Biomarker activities considering fish from all the samplings (n). Different letters indicate statistically significant differences (p<0.05).

	Species (n)	PY 1	CAT 2	GR 3	GST 3	EROD 4	CbE 3	BTI 5
	P. acarne (19)	77.0 ± 3.3 f	1048 ± 53 abc	12.5 ± 0.7 bhi	416 ± 37 abcd

Table 3 .

 3 Pair-wise correlations between biomarkers and environmental variables used in the CCA analysis (Fig.2). Bold type indicates significant correlation at the p<0.05 level.

	SHELF	Benthos	F	Suprabenthos swimming TL.isotopy Zooplankton
	BTI	0.202	-0.075	-0.227	-0.346	0.200	0.184
	catalase	-0.261	0.387	0.277	-0.407	-0.088	-0.072
	CbE	-0.140	-0.108	0.097	-0.162	-0.159	0.188
	EROD	0.241	-0.068	-0.241	-0.238	0.225	0.128
	GR	-0.211	-0.221	0.157	0.274	-0.419	0.225
	GST	-0.071	0.128	0.142	0.469	-0.055	-0.318
	PY	0.449	-0.289	-0.301	-0.181	0.383	0.210
	SLOPE
	BTI	0.332	-0.185	-0.069	-0.129	-0.067	-0.264
	catalase	0.490	-0.481	0.407	-0.054	0.231	-0.531
	CbE	0.541	-0.284	0.017	-0.142	-0.069	-0.466
	EROD	0.345	-0.310	-0.118	-0.034	-0.161	-0.263
	GR	0.064	-0.269	0.008	0.339	0.027	-0.041
	GST	0.105	-0.424	-0.052	0.270	-0.034	-0.069
	PY	0.319	0.189	0.027	-0.372	0.016	-0.280

Acknowledgements

This work was financed by the Spanish Science and Technology Ministry project BIOMARE (CTM2006-13508-CO2-02/MAR) and Complementary Action (CTM2006-28145-E/MAR). ACA (Agencia Catalana de l'Aigua, Generalitat de Catalunya) is acknowledged for providing data on organic pollutants in sediment. P. acarne P. bogavareo P. erytrinus B. boops M. barbatus S. maena T. draco C. linguatula H. dactylopterus T. minutus M. merluccius shelf M. merluccius slope M. poutassou P. blennoides T. scabrus N. aequalis L. lepidion S. canicula G. melastomus


Eunate Puy-Azurmendi

Asunción Navarro

Alba Olivares

Denise Fernandes

Elena Martínez

Miren López De Alda

Cinta Porte

Miren P Cajaraville

Damià Barceló

Benjamin Piña

ORIGIN AND DISTRIBUTION OF POLYCYCLIC AROMATIC HYDROCARBON POLLUTION IN SEDIMENT AND FISH IN THE BIOSPHERE´S RESERVE OF URDAIBAI (BAY OF BISCAY, BASQUE COUNTRY, SPAIN)

Keywords: Dioxin-like activity, Recombinant yeast assay, GC/MS, PAH metabolites

The Urdaibai estuary is an UNESCO's biosphere reserve impacted by recreational, agricultural and industrial activities. Polycyclic aromatic hydrocarbons (PAHs) are major pollutants in Urdaibai, and their control and the identification of their sources is central on the preservation of the area. Chemical analysis by GC-MS showed a significant pollution by PAHs in Urdaibai sediment samples, mainly from pyrolytic sources, with minor contributions from oil spills. Measurement of the dioxin-like activity using a yeast-based bioassay showed an excess of biological activity in sediment samples from the inner part of the estuary. Analysis of PAH metabolites in bile of thicklip grey mullet (Chelon labrosus) showed a large excess of 2-naphthol over 1-naphthol or 1-pyrenol, suggesting a specific contamination in the inner Urdaibai estuary by some industrial process. Therefore, the combination of these three techniques defined different PAH pollution sources in Urdaibai: A major pyrolytic origin, occasional oil spills, and specific industrial activities.

Introduction

The estuary of Urdaibai (43º23'N -2º41'W) is located in the Bay of Biscay, in the Northern Iberian Peninsula (Figure 1). It is 13 km long and an average width of 500m, and presents a limited maritime traffic. The main human activity in the zone is agriculture but a variety of industries can be found such as metallurgic industries, shipyards, treatment of surfaces, dyes and manufacture of cutlery, concentrated in the surroundings of the main town in the area, Gernika-Lumo. Due to its unique landscape and to its good balance between human activities and the natural environment, it was declared a Biosphere's Reserve by UNESCO in 1984. There are several environmental menaces looming over the Urdaibai ecosystem.

Contamination by polycyclic aromatic hydrocarbons (PAHs) is of special concern, as they can been derived from both human activities (industrial byproducts, incomplete combustion of carburants) or from oil spills, either internal (shipping, carburant handling) or external. In this regard, massive oil spills, like the Prestige spill in 2002, are known to affect the estuary in the recent past [START_REF] Cortazar | Distribution and bioaccumulation of PAHs in the UNESCO protected natural reserve of Urdaibai, Bay of Biscay[END_REF][START_REF] Orbea | Effects of the 'Prestige' oil spill on cellular biomarkers in intertidal mussels: results of the first year of studies[END_REF]. Therefore, the continuous surveillance of PAHs pollution and the identification of its possible sources is central on the maintaining of the environmental health of the Urdaibai estuary. The present work addresses these issues using a combination of chemical analyses and bioassays directed to determine PAHs levels and composition in sediments, evaluate their biological activity, and analyze metabolites diagnostic for their origin and possible sources.

Due to their hidrophobicity, PAHs accumulate both in sediments and in the fat of exposed animals. They are considered hazardous for human health and a number of them are known carcinogenic compounds [START_REF] Freire | Urinary 1-hydroxypyrene and PAH exposure in 4-year-old Spanish children[END_REF]IARC, 1983). PAHs constitute a very extended family of compounds, from which regulatory bodies have defined more or less comprehensive lists of priority compounds. The sixteen compounds listed by the US Environmental Protection Agency (EPA) or the eight ones included by the Water Framework Directive of the European Union (EU WFD) are used to define annual average and maximum allowable concentrations in surface waters [START_REF] Ec | Directive 2008/105/EC of the European Parliament and of the Council of 16 December 2008 on environmental quality standards[END_REF]. In contrast, no comparable quality standards for sediments have been defined yet, despite of the tendency of these compounds to accumulate in hydrophobic compartments. In this work, we analyze the sixteen EPA PAHs along the Urdaibai estuary to identify areas of accumulation and to infer possible sources.

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The carcinogenic effect of different PAHs is a consequence of the ectopic up regulation of the oxidative metabolism in liver and other tissues from vertebrates. This effect is mediated by the binding of the pollutant to the aryl hydrocarbon receptor (AhR) [START_REF] Gonzalez | The aryl hydrocarbon receptor: studies using the AHR-null mice[END_REF]Shimizu et al., 2000;van der Oost et al., 2003). The capacity of substances or environmental samples to activate the AhR system (the so-called dioxin-like activity, as dioxins are known to bind to AhR with extremely high affinity) can be quantified by a variety of aryl hydrocarbon-responsive reporter assays based on either in vivo animal tests (Willey et al., 1998) or different vertebrate cell lines (Villeneuve et al., 2002). Yeast-based bioassays represent a fast and cheaper alternative, although reports of their use for detection of AhR ligands in environmental samples are still scarce [START_REF] Boronat | Biological activity of aryl hydrocarbon receptor ligands in sediments from remote European lakes[END_REF][START_REF] Lee | Characterization of polycyclic aromatic hydrocarbons (PAHs) in different size fractions in deposited road particles (DPRs) from lake Biwa area, Japan[END_REF][START_REF] Murahashi | Human aryl hydrocarbon receptor ligand activity of 31 non-substituted polycyclic aromatic hydrocarbons as soil contaminants[END_REF].

Yeasts lack AhR or any equivalent receptor, but their genetic make-up can be easily modified to reconstruct at least partially the mammalian pathway [START_REF] Miller | A human aryl hydrocarbon receptor signaling pathway constructed in yeast displays additive responses to ligand mixtures[END_REF][START_REF] Miller | Expression of the human aryl hydrocarbon receptor complex in yeast. Activation of transcription by indole compounds[END_REF]. In the present study we used a yeast strain expressing the human AhR and the cofactor ARNT to activate a reporter gene (ß-galactosidase) upon the presence of AhR ligands in the cell culture [START_REF] Miller | Expression of the human aryl hydrocarbon receptor complex in yeast. Activation of transcription by indole compounds[END_REF]. In this genetic setup, the production of the reporter gene is diagnostic for the presence of AhR ligands in the medium, and can be quantified as an enzymatic activity with high precision (Noguerol et al., 2006a). With these tools, we determined AhR ligand activity in sediment samples from Urdaibai and compared these values with the observed chemical burden, to evaluate the importance of PAH pollution in the total toxicity of the sediments.

Fish living in PAH-contaminated environments absorb PAHs through the body surface and gills or by ingestion of contaminated sediment or food [START_REF] Orbea | Effects of the 'Prestige' oil spill on cellular biomarkers in intertidal mussels: results of the first year of studies[END_REF][START_REF] Raingeard | Cloning and expression pattern of peroxisome proliferator-activated receptor alpha in the thicklip grey mullet Chelon labrosus[END_REF]Varanasi et al., 1989). In this work, we assessed the exposure to PAHs in the thicklip grey mullet (Chelon labrosus, Mugilidae), a coastal fish present in eastern Atlantic Ocean and abundant in Urdaibai and other estuaries. This species is a good sentinel of human impact, as it feeds mainly on benthic diatoms, algae, small invertebrates and detritus. Direct measure of PAHs in tissues is impaired by the high capacity to metabolize and excrete PAHs of fish liver [START_REF] Meador | Bioaccumulation of polycyclic aromatic hydrocarbons by marine organisms[END_REF].

Quantitation of PAHs excreted through the bile as conjugated metabolites is a suitable alternative to assess PAHs exposure in fish, both in the field and in laboratory experiments, since bile metabolites can easily be detected with high precision and sensitivity [START_REF] Escartin | Assessment of PAH pollution in coastal areas from the NW Mediterranean through the analysis of fish bile[END_REF]Fernandes et al., 2008a;[START_REF] Fernandes | The combined use of chemical and biochemical markers to assess water quality in two low-stream rivers (NE Spain)[END_REF]Fernandes et al., 2008b;Yu et al., 1995). This methodology not only allows an

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estimation of the total PAH burden, but it also reveals information of the composition of this pollution, a central issue in the evaluation of the possible pollution sources.

Materials and Methods

Study area and sample collection

Surface sediment samples (one per site) were collected in April 2007, October 2007, and April 2008, in order to explore temporal trends, at six localities within the Urdaibai estuary, from its upstream limit to the mouth (Figure 1): Gernika, Murueta, Arteaga, Txatxarramendi, Mundaka and Laida. Murueta is a small town in the middle part of the estuary, where a medium size shipyard is placed. Arteaga is a small rural village, whereas Txatxarramendi is a small island in the outer part of the estuary. Mundaka is a small town in the mouth of the Oka River whose main activities are fisheries, agriculture and tourism. Laida is a beach at the mouth of the estuary, in front of Mundaka. For comparison purposes, sediments were also sampled in the leisure harbour of Arriluze, in the Abra estuary in the Basque Coast (43º23'N -3ºW, Figure 1), a site known by its high levels of pollution by PAHs [START_REF] Cearreta | Human activities along the Basque coast during the last two centuries: geological perspective of recent anthropogenic impact on the coast and its environmental consequences[END_REF]. Surface sediment samples, collected in precleaned amber-glass bottles, were transported to the laboratory in refrigerated containers, and then frozen at -20 °C. Fish (thicklip grey mullet Chelon labrosus) were sampled at approximately 1 Km distance from Gernika-Lumo urban center, near a sewage treatment plant, and in the Arriluze harbor, to compare PAH burden inside and outside Urdaibai. Six animals were captured in each site and campaign (36 animals in total). Fish were anaesthetized by immersion in a saturated solution of aminobenzocaine. Length (29.5±5.5 cm) and weight (395.9±184.6 g) of each fish were recorded, and the bile was taken, frozen in liquid nitrogen and stored at -80ºC until analysis.

Analysis of PAHs in sediments

Sediments samples were lyophilized during 48 h (10 -2 mbar vacuum) (Lioalfa, Telstar, Spain). The freeze-dried material was ground and homogenized using a mortar. Samples were sieved through 500 and 120 µm mesh to obtain an homogeneous sediment material and stored in precleaned glass bottles at -20 °C until extraction. Analysis of PAHs in the

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sediments was performed as described (Martinez et al., 2004). Sediment samples (1 g) were extracted by ultrasonication with 320 mL of a mixture of hexane:dichloromethane (1:1), and the resulting extracts were cleaned-up by solid phase extraction (SPE) using neutral alumina, reconstituted in 250 µL hexane, and analyzed of the resulting purified extracts by gas chromatography/mass spectrometry (GC/MS) with electron impact ionisation (70 eV) acquiring 3 characteristic ions per compound.

Quantitation, based on peak areas, was performed by the internal standard method using surrogate deuterated standards. This method allows the determination of the 16 EPA PAHs at ng/g levels (limits of detection between 0.1 and 2 ng/g) with satisfactory precision (relative standard deviation calculated from 3 replicates below 20%) and accuracy (recovery percentage above 50% for all compounds except Naphthalene (20%)

and Benzo[g,h,i]Perylene (47%). Analysis (n=3) of a certified freshwater harbour sediment material (BCR535) within the sample sequence yielded concentrations matching the certified values with a percentage of error below 12%.

Ratios of the PAH isomers Phenanthrene to Anthracene (Ph/An), Fluoranthene to Pyrene (Fl/Py) and Benzo[a]Anthracene to Chrysene (BaA/Chrys) were calculated as indicators of PAH origin. Values of Ph/An>50, Fl/Pyr<1 and BaA/Chrys<1 are typical of samples of petrogenic (oil spill) origin, whereas the corresponding ratios of samples originated in combustion processes (pyrolytic) are typically Ph/An<10, Fl/Pyr>1 and BaA/Chrys>1 [START_REF] Karacik | Polycyclic aromatic hydrocarbons and effects on marine organisms in the Istanbul Strait[END_REF][START_REF] Poster | Determination of PAHs in combustion-related samples and in SRM 1597, complex mixture of PAHs from coal tar[END_REF]Webster et al., 2001).

Recombinant yeast assay

Aryl hydrocarbon receptor (AhR)-based recombinant yeast assays (AhR-RYA) were performed as previously described (Noguerol et al., 2006a;Noguerol et al., 2006b). The YCM1 yeast strain [START_REF] Miller | Expression of the human aryl hydrocarbon receptor complex in yeast. Activation of transcription by indole compounds[END_REF] was grown overnight in minimal medium (6.7 g/L) yeast nitrogen base without amino acids plus ammonium sulfate (DIFCO, Basel, Switzerland) supplemented with 0.1 g/L of prototrophic markers as required and with galactose as a carbon source to express AhR and the cofactor ARNT [START_REF] Miller | A human aryl hydrocarbon receptor signaling pathway constructed in yeast displays additive responses to ligand mixtures[END_REF]. The final culture was adjusted to an optical density (OD) of 0.1 and split into 100 μL aliquots in 96-well polypropylene microtiter plates (NUNC™, Roskilde, Denmark; [START_REF] Garcia-Reyero | Monitoring of endocrine disruptors in surface waters by the yeast recombinant assay[END_REF] previously silylated by overnight exposure to an atmosphere of dimethylsilane. We performed a serial dilution scheme based on 1:3 dilution steps and using glass microcapillary tubes rather than pipette tips for all liquid

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transfers, to eliminate contact with plastic surfaces [START_REF] Garcia-Reyero | Monitoring of endocrine disruptors in surface waters by the yeast recombinant assay[END_REF]. Plates were incubated for 4 hours at 30°C under mild shaking. After incubation, 100 μL YPER™ (PIERCE™, Rockford, IL, USA) were added to each well and further incubated at 30°C for 30 min. Afterwards, 100 μL of assay buffer supplemented with 0.1% 2-mercaptoethanol and 0.5% of the 4-methylumbelliferone ß-Dgalactopyranoside (MuGal) solution (both from the FluorAce™ beta-galactosidase Reporter Assay Kit, Bio-Rad Laboratories, Hercules, CA, USA) were added to the lysed cells. After brief centrifugation, plates were read in a Victor3 Wallac spectrofluorometer (Perkin Elmer Inc., Wellesley, MA, USA), at 355nm excitation and 460nm emission wavelengths. Fluorescence was recorded for 15 to 20 min (one measurement per min); ß galactosidase activity values were calculated as rates of the increment of arbitrary fluorescence units with time, using standard linear regression methods. To test possible inhibitory activity (false negatives), yeast cultures were incubated for 4h with 2 μM of ß-naphthoflavone, added to a 1:30 dilution of each sample, and processed as before. No sample exhibited inhibitory activity high enough to significantly distort the results (not shown). In this particular system, the ratio of false positives is negligible [START_REF] Miller | Expression of the human aryl hydrocarbon receptor complex in yeast. Activation of transcription by indole compounds[END_REF]Noguerol et al., 2006a); several negative controls (wells with only vehicle plus yeast culture) were included in each plate. Samples were tested in triplicate. AhR-ligand activity values were calculated as naphthoflavone equivalents, NFEQ, defined as the concentration of ß-naphthoflavone required to elicit the same response as the sample in the assay. These values were calculated from dose-response curves by adjusting ß galactosidase values to a first-order Hill equation, as described (Noguerol et al., 2006a), using at least 9 determinations for each value.

Analysis of PAH metabolites in fish bile

Hydroxylated PAH metabolites were analyzed by a modification of a previously described method [START_REF] Escartin | Assessment of PAH pollution in coastal areas from the NW Mediterranean through the analysis of fish bile[END_REF]. Briefly, 100 mg of bile (6 samples per area and sampling campaign) were incubated for 1 h at 40ºC in the presence of 1 ml 0.4 M acetic acid/sodium acetate buffer pH 5.0, containing 2000 U of ß-glucuronidase and 50 U of sulfatase. Hydrolyzed metabolites were extracted with 1 ml of ethyl acetate (x3), the extracts recombined and concentrated under a nitrogen stream. Dry residues of bile metabolites were then derivatized by the addition of 100 ml of bis-(trimethylsilyl) trifluoroacetamide (BSTFA), heated for 1 h at 70ºC, and further reduced under a

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nitrogen stream. Analyses were carried by gas chromatography-mass spectrometry electron impact mode (GC-MS-EI) in a Fisons GC 8000 Series chromatograph interfaced to a Fisons MD800 mass spectrometer using a 30 m x 0.25 mm internal diameter HP-5MS column crosslinked with 5% PH ME siloxane (Hewlett-Packard, USA). The instrument was programmed from 90ºC to 140ºC at 10ºC min -1 and from 140ºC to 300ºC at 4ºC min -1 , using helium at 80 kPa as carrier gas. The injector temperature was 250ºC and the ion source and the analyzer were maintained at 200ºC and 250ºC, respectively. Just prior to sample analysis, calibration curves were performed with reference compounds and operating in selected ion monitoring mode (SIM). Hydroxylated-PAHs were identified by comparison of retention times and spectra of reference compounds. The silylation derivative ions used for monitoring and quantification of hydroxylated-PAHs were: m/z 216 and 201 for 1-and 2-naphthol; m/z 254 and 165 for 9-fluorenol; m/z 266 and 251 for 9-phenanthrol; and m/z 290 for 1pyrenol. Recovery of extraction procedure was higher than 90% for all the compounds tested (1-naphthol, 9-fluorenol and 9-phenanthrol), except for 1-pyrenol which was 85% [START_REF] Escartin | Assessment of PAH pollution in coastal areas from the NW Mediterranean through the analysis of fish bile[END_REF]. Concentrations are expressed as nanograms per gram of bile.

Statistics

All statistical calculations, including linear and non-linear regression methods, were performed using the SPSS v. 17 package (SPSS Inc., Chicago, Ill.) Unless otherwise noted, significance levels were set at p<0.05. Normality of distributions was tested with the Kolmogorov-Smirnov Z test. Most variables analyzed showed a non-normal distribution by the Z-test; therefore, non-parametric test were preferred. In cases in which parametric tests were required (e.g., ANOVA plus Tukey's test), they were performed using logarithmic transformants of the data, which followed a normal distribution with uniform variances for all categories tested (Z-and Levene's tests).

Results and Discussion

3.1.-PAH levels in sediments

Total PAH levels in Urdaibai sediments varied some 50-fold among sampling sites (Table 1, profiles in Figure 2A, bars). Except for Mundaka, which showed an extremely

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high variability, results from the three campaigns were similar for all sites, as revealed by K-means cluster analysis (Fig. 2B). The three sites at the inner part of the Urdaibai stuary showed similar, relatively low PAHs burdens, compared to Txatxarramendi and Arriluze sites (Figure 2). The two outer sites, Laia and Mundaka showed low PAH burdens comparable to those from the Urdaibai inner sites, except for the October 2007 sample from Mundaka, which we consider directly contaminated with oil (see next paragraph). A similar survey performed in years 2002-2004 recorded PAHs levels between 1,000 and 10,000 ng/g, with some occasional samples largely exceeding these values [START_REF] Cortazar | Distribution and bioaccumulation of PAHs in the UNESCO protected natural reserve of Urdaibai, Bay of Biscay[END_REF]. These results suggest that PAH levels remained essentially identical since at least 2002, suggesting a continuous, chronic contamination by PAHs, not necessarily linked to episodic oil spills.

Analysis of ratios between PAH isomers indicates a mainly pyrogenic origin of PAHs found in Urdaibai sediments (Table 1). 20 out of 21 samples showed Ph/An ratios well below 10, indicative of a combustion process. Similarly, 14 out of 18 samples showed BaA/Chr ratios over 1, also diagnostic of pyrogenesis [START_REF] Karacik | Polycyclic aromatic hydrocarbons and effects on marine organisms in the Istanbul Strait[END_REF][START_REF] Poster | Determination of PAHs in combustion-related samples and in SRM 1597, complex mixture of PAHs from coal tar[END_REF]Webster et al., 2001). In contrast, Fl/Pyr ratios below 1 shown by 11 out of the 21 samples suggest some contribution from oil spills (petrogenic) in at least some of them (Table 1). Petrogenic pollution, albeit never predominant, seemed to be more important in Gernika, Murueta and in the leisure harbor of Arriluze (Table 1), likely from seafaring and occasional oil spills from different sources. The October 2007 sample from Mundaka, which showed the highest PAH contents in all the series, is the only one with a Ph/An ratio close to 50, indicative of petrogenic origin (Table 1, Figures 2A and2B). We concluded that this particular sample may have been contaminated by an oil spill, probably a local one. With the exception of this particular sample, our results indicate that most PAHs found in Urdaibai and Arriluze have a pyrolytic origin, with a possible additional contribution of petrogenic hydrocarbons. Similar conclusion was reached in previous reports of PAH contents in Urdaibai sediments [START_REF] Cortazar | Distribution and bioaccumulation of PAHs in the UNESCO protected natural reserve of Urdaibai, Bay of Biscay[END_REF] and in the Istanbul Strait [START_REF] Karacik | Polycyclic aromatic hydrocarbons and effects on marine organisms in the Istanbul Strait[END_REF], both showing PAHs levels and isomeric ratios similar to the ones observed in our study.

AhR ligand activity in sediments

All samples from Urdaibai sediments were positive for AhR ligand activity, indicating a general contamination by dioxin-like compounds (Figure 2). Maximal activities

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corresponded to Txatxarramendi and Arriluze sites, whereas minimal values were found in Mundaka and Laida sites (Figure 2, continuous line). Differences between samples collected at a given site during the three campaigns were relatively small (less than a factor of two), except for some few exceptions (Figure 2). K-means clustering of data clearly distinguished the two outer sites (Mundaka and Laia) as the ones with the lowest AhR-ligand activity, followed by the three Urdaibai inner sites (Gernika, Arteaga and Murueta). In the same analysis, Txatxarramendi appeared as the most contaminated site, surpassing even the Arriluze site (Figure 2B). The Abra estuary is much more heavily industrialised than the Urdaibai estuary, receiving considerable amounts of industrial, urban and agricultural untreated wastewater during about 150 years. Since 1980 a sewage treatment plant was set up and the environmental quality of the estuary started a recovery process [START_REF] Gorostiaga | Recovery of benthic communities in polluted systems[END_REF], although levels of pollution by PAHs and other chemicals are still considered very high [START_REF] Cearreta | Human activities along the Basque coast during the last two centuries: geological perspective of recent anthropogenic impact on the coast and its environmental consequences[END_REF]. Therefore, the presence in Urdaibai of a site with pollution levels similar to (if not even higher than) those from the Arriluze site, both in terms of chemical content and biological activity, argues for the requirement of a more strict control of pollution in the generally regarded as preserved Urdaibai estuary.

Comparison between AhR ligand activity and PAH burden profiles showed some significant similarities as well as differences. For example, both profiles show an increasing tendency from the innermost Urdaibai site, Gernika, to Txatxarramendi, close to the mouth of the estuary (Figures 2A and2B, map in Figure 1). This last site showed values for both parameters comparable to the ones from the polluted Arriluze site in both profiles. In contrast, Mundaka and Laida showed a rather poor correlation between chemical and bioassay data, especially for the Mundaka site. In both cases, the observed low AhR ligand activity was at variance with their relatively high PAH burden. These discrepancies were diminished when the chemical data was converted to predicted dioxin-like activity, measured as TCDD equivalents (TEQ, Figure 2, discontinuous line), using relative effector potency (REP) values previously defined for AhR-RYA [START_REF] Murahashi | Human aryl hydrocarbon receptor ligand activity of 31 non-substituted polycyclic aromatic hydrocarbons as soil contaminants[END_REF]. By comparing the three profiles in Figure 2, we concluded that sediment samples from Mundaka and Laida were particularly poor in biologically active PAHs. This was particularly significant for the very highly polluted October 2007 Mundaka sample, which shows a very low biological activity.

Conversely, most samples from the estuary, from Gernika to Txatxarramendi, may

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contain some unidentified AhR ligand to explain their higher-than-predicted dioxin-like activity in the AhR-RYA assay (Figure 2). 2). Most of these compounds are known to bind the human AhR and to activate AhR-regulated genes, both in vitro and in vivo [START_REF] Murahashi | Human aryl hydrocarbon receptor ligand activity of 31 non-substituted polycyclic aromatic hydrocarbons as soil contaminants[END_REF]Villeneuve et al., 2002;Willey et al., 1998). Consistently, we found a good correlation between AhR-RYA results and the predicted TEQ values (Table 2). These results therefore indicate that a substantial fraction of the total AhR ligand activity present in Urdaibai sediments can be explained by their contents in the 16 EPA PAHs.

PAH metabolites in fish bile

Levels of PAH hydroxylated metabolites in bile of fish captured in Urdaibai and in Arriluze reflected the relative concentration of PAHs found in the sediments from both sites (Figure 3A). Fish collected in Arriluze showed values of PAHs metabolites in bile (734 -4079 ng/g of bile) about three times higher than those from Gernika (283 -1616 ng/g), although the variation between the three campaigns makes difficult to draw definite conclusions (Figure 3A). The general tendency for both sites was a decrease in OH-PAHs along the sampling seasons, however this decrease was more pronounced in Gernika, where the lowest levels of OH-PAHs were observed in April 2008 (Figure 3A). Metabolite profiles differed in both sites, suggesting different sources of PAHs.

For example, 2-naphthol was the dominant PAH metabolite detected (38 -71%) in fish from Gernika, whereas in fish from Arriluze the major metabolite detected was of 1pyrenol (52 -92%) (Figure 3B). 1-Pyrenol is considered a key PAH metabolite in fish bile to monitor PAH exposure [START_REF] Ruddock | Bile metabolites of polycyclic[END_REF]; consistently, its concentration in bile was significantly higher in fish from Arriluze than from Urdaibai (Mann-Whitney U test plus Bonferroni correction, p=0.001). Conversely, levels of 2-naphthol were higher in Gernika than in Arriluze (Mann-Whitney U test plus Bonferroni correction, p<0.001). Both 1-naphthol and 2-naphthol originate from naphthalene at very similar ratios through a 1,2 epoxy intermediate (Varanasi et al., 1989;Yang et al., 1999), and significant deviations on the concentrations of any of them are attributed to

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contamination from other sources unrelated to PAH metabolism, at least for human urine [START_REF] Meeker | Utility of urinary 1-naphthol and 2-naphthol levels to assess environmental carbaryl and naphthalene exposure in an epidemiology study[END_REF]Serdar et al., 2003). Although it is possible that the ratios of synthesis and/or excretion of the two isoforms may differ between the two examined mullet populations, the almost 40-fold excess of 2-naphthol over 1-naphthol in the Gernika population (but not in the Arriluze population) argues for a specific contamination by some industrial process liberating 2-naphthol or a precursor substance. The fact that 2-naphthol made up to 70% of total biliar OH-PAHs in Gernika fish (Figure 3B) also argues for a specific source of this compound.

Biliary levels OH-PAHs in grey mullet were 3 to 45-fold higher than those detected in bile of Trisopterus luscus, a benthoplagic fish, collected along the inner shelf of the Basque Country coast (Fernandes et al., 2008a), suggesting a significantly higher anthropogenic pressure in the reserve of Urdaibai compared to the coastline. OH-PAHs levels found in grey mullet from Urdaibai were also similar or even higher than those detected in Mullus barbatus (91 -1589 ng/g of bile) from the NW Mediterranean coast [START_REF] Escartin | Assessment of PAH pollution in coastal areas from the NW Mediterranean through the analysis of fish bile[END_REF]. Direct comparison between different fish species is always difficult due to differences in habitat, metabolism and diet. This notwithstanding, the data suggest a significant contamination by PAHs in the Urdaibai endogenous fish population compared to the one observed in fish population from the coast, in line with our data on PAHs content and dioxin-like activity.

General discussion and conclusions

Sediments from Urdaibai showed a significant contamination by PAHs, particularly at the outer locality of Txatxarramendi. This site showed PAH levels comparable to those found in Arriluze, located in the neighbouring highly polluted Abra estuary. Blue mussels (Mytilus galloprovincialis) from Txatxarramendi show a high prevalence of oocyte atresia in developing or ripe bivalves (Puy-Azurmendi et al.), a condition that has experimentally been linked to the exposure to petroleum hydrocarbons [START_REF] Ortiz-Zarragoitia | Biomarkers of exposure and reproduction-related effects in mussels exposed to endocrine disrupters[END_REF]. These results were a first indication that levels of PAHs and other pollutants in Urdaibai may be exceeding tolerance levels for at least some animal species.

The origin of PAHs in Urdaibai and Arriluze seems to be mainly pyrolytic, with some contribution of petrogenic sources in Gernika, Murueta and Arriluze. Comparison between chemical data with predicted and observed dioxin-like activity values defined Mundaka and Laida, showed a low proportion of dioxin-like PAHs, although their total PAH burden was similar to (or even higher than) that of other samples with much stronger dioxin-like activity. Conversely, many samples from inside the estuary presented dioxine-like activity values higher than predicted by their content in PAHs, suggesting a contribution of dioxin-like compounds from other sources unrelated to PAH pollution. Finally, relative concentrations of 1-pyrenol, 1-naphthol and 2-naphthol in fish bile could indicate a specific industrial impact in Gernika relative to Arriluze, which may be related to the excess of dioxin-like activity observed in the inner Urdaibai samples. We conclude that PAHs are responsible for at least a substantial part of the AhR ligand activity we observed in the AhR-RYA assay, consistently with their property of being powerful AhR ligands. The substantial amounts of PAH metabolites found in bile of fish from Gernika and Arriluze confirms recent exposure to PAHs, although their actual physiological impact remains to be elucidated.

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  AhR ligand activity values showed strong positive correlation with the concentration of two groups of individual PAHs: The three-ringed Phenanthrene and Anthracene as one group and the four-ringed Benzo[a]anthracene, Benzo[b]fluoranthene, Benzo[k]fluoranthene and Benzo[a] pyrene as the second one (Table

  samples. Sampling points with strong marine influence, like

Figure 1 .

 1 Figure1. Map of sampled sites. Sediment samples were collected at four localities inside the Urdaibai estuary (Gernika, Murueta, Arteaga and Txatxarramendi, detailed map on the right), Mundaka, at the river mouth, and Laida, outside the sand barrier limiting the estuary (grey areas in the detailed map). The leisure harbour of Arriluze, in the Abra estuary, was sampled as polluted external site. Fish were sampled only in Gernika and Arriluze. The approximate outline of the main city in Urdaibai, Gernika, is indicated with a discontinuous line at the bottom of the detailed map.

Figure 2 .

 2 Figure 2. A) Profiles of EPA PAHs contents (bars, left axis), AhR-ligand activity in NFEQ (ß-naphthoflavone equivalents, calculated by YCM-RYA; continuous line, right axis) and predicted TEQ values (discontinuous line, arbitrary units) in sediment samples from 6 localities in Urdaibai and from Arriluze, taken during three consecutive campaigns. B) Value classification by K-means clustering (4 groups). Letters from "a" to "d" indicate samples belonging to statistically differentiated clusters ordered by increasing average (centroid) values; shadows of gray indicate the degree of pollution, from the cleanest cluster (white, labeled "a") to the most contaminated one (black, labeled "d").

Figure 3 .

 3 Figure 3. A) Biliary levels of hydroxylated-PAHs detected in thicklip grey mullets collected in Gernika and Arriluze. Bars indicate total OH-PAHs content, SEM values are represented by lines. Letters correspond to homogeneous subsets of data (ANOVA plus Tukey's test, logarithmic transformants).

  Figure 1 Click here to download high resolution image

Table 1 .

 1 Concentration (ng/g) of EPA PAHs and isomer ratios in sediments from the Urdaibai estuary

	Ph/An Fl/Pyr BaA/Chr

Table 2 .

 2 Spearman's rank correlation between AhR-ligand activity and concentration of PAHs in Urdaibai samples

	Table 1

a) Two-tailed significance level. *, p<0.05; **, p<=.01; ***,p<0.005

Table 2

 2

Acknowledgements

This work was supported by Catedra UNESCO of the University of the Basque Country (UPV/EHU) through the project DERBiUr (UNESCO06/19), by the Basque Government through the project ETORTEK-IMPRES and a grant to consolidated research groups (GIC07/26-IT-393-07), and by the Spanish Ministry for Science and Innovation (CGL2008-01898/BOS, CGL2007-64551/HID and Consolider-Ingenio 2010 CSD2009-00065). Denise Fernandes acknowledges a postdoc fellowship (SFRH/BPD/34289/2006) from the Portuguese Fundação para a Ciência e Tecnologia (FCT) of the Ministry of Science and Technology of Portugal.We greatly acknowledge the help of Dr. Maren Ortiz-Zarragoitia in coordinating the samplings and of all the students in the Laboratory of Cell Biology and Histology of the University of the Basque Country that participated in samplings.