E Kerambrun
email: elodie.kerambrun@univ-littoral.fr

Are biochemical biomarker responses related to physiological performance of juvenile sea bass (Dicentrarchus labrax) and turbot (Scophthalmus maximus) caged in a harbour polluted area?

Keywords: biochemical biomarker, fish growth, condition indices, metals, PAHs, caging

Biomarker responses to toxic exposure have been used for decades to indicate stress in aquatic organisms, or the magnitude of environmental pollution. However, little has been done to compare the simultaneous responses of both biochemical and physiological biomarkers. The purpose of this study was twofold. Firstly to analyse the responses of several biochemical biomarkers measured on juvenile sea bass and turbot caged in a northern France harbour at a reference and contaminated stations. Several biotransformation parameters (Ethoxyresorufin-O-deethylase -ERODand Glutathione S-transferase -GST) and an antioxidant enzyme (Catalase -CAT) were analysed. Secondly, to compare their responses to several growth and condition indices, measured on the same fish. In the contaminated station, EROD and GST activities were found to be significantly higher, and a decrease of CAT activity was observed for both species. For individual sea bass, biochemical biomarkers showed numerous significant correlations with growth and condition indices, such as the Fulton's K condition index, the RNA:DNA ratio and the lipid storage index. On the contrary, there were only a few significant correlations for turbot, suggesting a species-specific response. Our study indicates that the analysis of the simultaneous responses of both biochemical and physiological biomarkers can be useful for monitoring complex exposure and to assess habitat quality.

Introduction

A multitude of xenobiotics contaminate the marine environment, and although chemical analyses are able to measure many of these compounds qualitatively and quantitatively, complex mixtures of these chemical pollutants cannot be fully assessed. Furthermore, chemical analyses alone do not reveal the impact of chemical pollution on the aquatic environment because of potential synergistic/antagonistic effects of complex mixtures of chemical pollutants. In this context, utilisation of biomarkers as an early warning of pollution or degradation in ecosystems has increased over the past 20 years [START_REF] Adams | Biological indicators of aquatic ecosystem stress[END_REF].

Identifying a suitable biomarker of toxicity represents a major challenge in current ecotoxicological research [START_REF] Cheung | The use of scale increment as a means of indicating fish growth and growth impairment[END_REF]. Many parameters have been investigated to assess disturbances of various physiological functions linked to chemical exposure [START_REF] Van Der Oost | Fish bioaccumulation and biomarkers in environmental risk assessment: a review[END_REF]. [START_REF] Peakall | Biomarkers: The way forward in environmental assessment[END_REF] defined biomarkers as "biological responses than can be related to an exposure to, or toxic effect of, an environmental chemical or chemicals". The subdivision of biomarkers in literature is rather diffuse since the impact of toxic xenobiotics on fish has been analysed with various types of exposure and effect biomarkers, ranging from molecular, through cellular and physiological responses, to behavioural changes. In order to avoid confusion, in this paper, biomarkers acting at the subcellular level as biotransformation process or oxidative stress protection will be referred to as "biochemical biomarkers" and biomarkers reflecting fish growth or physiological condition will be referred to as "physiological biomarkers".

Because many toxic effects initially occur at the subcellular level, there has been an increasing use of biochemical biomarkers that help to determine causative agents responsible for altered cellular function [START_REF] Schlenk | Correlation of hepatic biomarkers with whole animal and population.community metrics[END_REF]. Among the biochemical biomarkers described in relating literature, phase I and phase II biotransformation parameters such as EROD (Ethoxyresorufin-O-deethylase) and GST (Glutathion-S-transferase) activities are currently used in environmental risk assessment [START_REF] Sanchez | Assessment of seasonal variability of biomarkers in three-spined stickleback (Gasterosteus aculeatus L.) from a low contaminated stream: Implication for environmental biomonitoring[END_REF]. Biotransformation of chemicals is a requisite for detoxification and excretion [START_REF] Gravato | Dicentrarchus labrax biotransformation and genotoxicity responses after exposure to a secondary treated industrial/urban effluent[END_REF]. The first step is usually catalysed by cytochrome P450-dependent monooxygenases (phase I) and their products, or several others organic pollutants, are subsequently coupled to endogenous metabolites (phase II) [START_REF] Buhler | The role of biotransformation in the toxicity of chemicals[END_REF][START_REF] Landis | Introduction to Environmental Toxicology: Impacts of Chemicals upon Ecological Systems[END_REF]. Antioxidant enzymes are also commonly used to understand the associated toxic-mechanisms of xenobiotics [START_REF] Sanchez | Copper-induced oxidative stress in three-spined stickleback: relationship with hepatic metal levels[END_REF][START_REF] Oliveira | Organ specific antioxidant responses in golden grey mullet (Liza aurata) following a short-term exposure to phenanthrene[END_REF]. Many pollutants exert their effects through redoxcycling, resulting in the production of reactive oxygen species (ROS). The role of antioxidant systems is to protect the cells from this oxidative stress. Thus, measurement of components of the antioxidant defence system may be helpful to determine organism exposure to pollutant [START_REF] Bilbao | Effects of exposure to Prestige-like heavy fuel oil and to perfluorooctane sulfonate on conventional biomarkers and target gene transcription in the thicklip grey mullet Chelon labrosus[END_REF].

Attempting to relate biomarker responses of individual organisms to increasing pollutant exposure and stress, both in the laboratory and in situ, offers considerable potential for improving the ecological relevance of ecotoxicological test procedures [START_REF] Depledge | Assessment of Trace Metal Toxicity Using Molecular, Physiological and Behavioural Biomarkers[END_REF]. However, although the role of biochemical biomarkers as early warning tools is recognised, it is difficult to understand their significance at higher levels of biological organisation. Indeed, in spite of their rapid responsiveness and sensitivity to contaminant exposure, biochemical biomarkers have questionable ecological relevance, as a result of being endpoints at a low level of biological organisation [START_REF] Castro | An In Situ Bioassay Integrating Individual and Biochemical Responses Using Small Fish Species[END_REF]. On the contrary, changes in physiology and fitness seem to be a common response in marine organism exposed to stressful pollutants [START_REF] Alquezar | Effects of metals on condition and reproductive output of the smooth toadfish in Sydney estuaries, south-eastern Australia[END_REF][START_REF] Faucher | Impact of chronic cadmium exposure at environmental dose on escape behaviour in sea bass (Dicentrarchus labrax L.; Teleostei, Moronidae)[END_REF]. Pollutants can induce various biological responses in fish, affecting the organisms from the biochemical to the populationcommunity levels [START_REF] Adams | Biological indicators of aquatic ecosystem stress[END_REF]. For example, many investigations on biological responses of fish populations to contamination indicated a general decrease of the relative fecundity, the growth rate and condition factor in contaminated estuaries [START_REF] Laroche | Genetic and physiologic responses of flounder (Platichthys flesus) populations to chemical contamination in estuaries[END_REF][START_REF] Marchand | Responses of European flounder Platichthys flesus populations to contamination in different estuaries along the Atlantic coast of France[END_REF][START_REF] Amara | Growth and condition indices in juvenile sole Solea solea measured to assess the quality of essential fish habitat[END_REF]. It is likely that changes in individual health manifest themselves at higher levels of ecological organisation, leading to reduced fish recruitment, abundance and production.

It has long been suggested that biochemical biomarkers should be used in conjunction with measurements of fitness [START_REF] Anderson | Genetic and molecular ecotoxicology: a research framework[END_REF][START_REF] Depledge | Assessment of Trace Metal Toxicity Using Molecular, Physiological and Behavioural Biomarkers[END_REF]. However, few studies have demonstrated correlative relationships between biochemical biomarkers responses and reduced fitness of aquatic organisms exposed to toxicants [START_REF] Depledge | Assessment of Trace Metal Toxicity Using Molecular, Physiological and Behavioural Biomarkers[END_REF][START_REF] Lesser | Oxidative stress DNA damage and p53 expression in the larvae of Atlantic cod (Gadus morhua) exposed to ultraviolet (290-400 nm) radiation[END_REF][START_REF] Fonseca | Effect of copper exposure on growth, condition indices and biomarker response in juvenile sole Solea senegalensis[END_REF]. Biological impairment (e.g. embryonic malformations, growth and condition depletion, fecundity and pollution tolerance) may directly affect the survivorship of organisms. Therefore, this type of multifaceted approach is important because it will improve our ability to use molecular biomarker responses of organisms to predict higher-level consequences of toxicant exposure [START_REF] Rose | Assessment of seasonal variability of biomarkers in three-spined stickleback (Gasterosteus aculeatus L.) from a low contaminated stream: Implication for environmental biomonitoring[END_REF].

Although biochemical or physiological biomarkers are intended to be useful tools for environmental assessment in the field, most of them have been developed under laboratory controlled toxic conditions. These studies often fail to recreate the mixed exposure situations occurring in nature. However, in field situations, the migration of many fish species for feeding and breeding creates uncertainty about how individuals sampled in some habitat truly reflects the water quality around the site of capture. In comparison, a caging strategy may, in specific situations, give more realistic results in studies of bioavailability, bioaccumulation and biological effects of contaminants in fish. The technique of caging offers advantages over organism chemical exposure [START_REF] Oikari | Caging techniques for field exposures of fish to chemical contaminants[END_REF] with a control of the precise location and duration of exposure while environmental field conditions are preserved. The use of cage-held animals from a common source (e.g. hatchery) also removes the potential for genotype adaptation, which is a distinct possibility in feral fish populations exposed to contaminants [START_REF] Winter | Tissue levels and biomarkers of organic contaminants in feral and caged chub (Leuciscus cephalus) from rivers in the West Midlands[END_REF]. The development of caging field experiments would provide an indication of the impact of contamination on marine fish and information on the applicability of this experimental strategy for assessing habitat quality.

The present study was designed to analyse the responses of two biotransformation parameters (EROD and GST activities) and an antioxidant enzyme (catalase) on the basis of a caging field experiment of two marine fish species in a polluted area (Boulogne sur Mer). In a second part, the responses of these enzymatic biomarkers were compared to different growth rates (in length and weight) and condition indices, measured on the same individual fish and analysed in a previous study (Kerambrun et al., submitted for publication). We used three condition indices: the Fulton's K condition factor; the RNA:DNA ratio which is used in numerous studies as indices for nutritional condition and growth assessment in larvae and juvenile fish [START_REF] Buckley | RNA-DNA ratio: an index of larval fish growth in the sea[END_REF][START_REF] Gwack | Developmental changes in RNA:DNA ratios of fed and starved laboratory-reared Japanese flounder larvae and juvenile, and its application to assessment of nutritional condition for wild fish[END_REF][START_REF] Amara | Growth and condition of 0-group European flounder, Platichthys flesus as indicator of estuarine habitat quality[END_REF]; and a lipid storage index based on the ratio of the quantity of triacylglycerols (TAG; reserve lipids) to the quantity of sterols (ST; structural lipids) in fish [START_REF] Amara | Growth and condition indices in juvenile sole Solea solea measured to assess the quality of essential fish habitat[END_REF].

Material and Methods

This experiment was conducted in accordance with the Commission recommendation 2007/526/EC on revised guidelines for the accommodation and care of animals used for experimental and other scientific purposes. The University of Littoral Côte d'Opale is authorised to conduct experimentation on animals in its capacity as a certified establishment; according to the administrative order N° B62-160-2.

Experimental caging technique

Cage placement was carried out in June 2009, in an important northern France harbour (Boulogne sur Mer) (Fig. 1). This harbour is impacted by municipal and industrial discharges, fishing and shipping activities, and marinas. Two locations were chosen: station A, in front of the harbour (depth 5 -10 m according to tide) and station B, in the inner part of the harbour (depth 4 m). Previous data have shown that sediments from station A contain relatively low levels of metals and PAHs whereas those from station B are significantly more contaminated with metals and PAHs. The distance between the two stations was about 2.5 km. 48 juvenile sea bass (Dicentrarchus labrax), (Weight: 2.84 0.79 g; Age: 4 months) and 48 juvenile turbot (Scophthalmus maximus), (Weight: 4.62 0.72 g; Age: 3 months) were obtained from hatcheries (Aquanord and France Turbot, in France). Before the caging procedure, the fish were acclimatised for 2 weeks in a clean tank (2.7 m 3 ), supplied with an open seawater circuit.

The water was aerated with air pumps, and the photoperiod was set at a 10 h light and 14 h dark cycle. During the acclimation, water temperature was about 13 ± 1 °C and the fish were progressively fed with artemia and pieces of shrimps and mussels to acclimate them to natural prey consumption. One day before the caging experiment, they were anaesthetised (2phenoxyethanol), weighed (near to 0.01 g), measured for total length (near to 0.1 mm) and individually marked (Visual Implant Tag, 1.2 mm x 2.7 mm, Northwest Marine Technology).

For each species, fifteen fish were sampled before the beginning of the experiment to establish t 0 values, used as reference. Cylindrical cages (0.7 m height, 0.65 m diameter, 230 L volume), made from polypropylene mesh (10 mm) were used for the sea bass. Rectangular cages (0.3 m height, 0.6 m width, 0.9 length, 160 L volume) were chosen for the turbot, benthic fish, in order to increase the surface area in contact with the sediment. At each station, the two types of cage were immersed with fish randomly placed and then fixed to the bottom with a screw anchor by scuba-divers. Precisely, 15 sea bass and 17 turbot were placed in station A, and 18 sea bass and 16 turbot, in station B (initially, 18 fish of each species were provided in each cage, but some fish escaped when they were put in the cages due to bad weather conditions). Sea water physicochemical parameters (temperature, salinity, oxygen and pH) were measured, and bottom sediment sampled for chemical contaminant analysis. Following the 38-days exposure, all the fish were rapidly transferred to the laboratory (within 2 h), anaesthetised (2-phenoxyethanol), identified (tag), weighed and measured for total length. Livers were sampled, frozen in liquid nitrogen and stored at -80 °C. Muscles were sampled and preserved at -20 °C and otoliths (sagittae) extracted and preserved in ethanol (95%).

Sediment analysis

Sediment samples were collected near to each cage location using a Van Veen grab (250 cm² sampling area) in order to determine metals, PAHs and PCBs contents.

In order to determine selected metals (Cd, Cr, Cu, Ni, Pb, and Zn) in the total fractions, sediments were dried at 40 °C to constant weight and were ground into a powder. About 0.250 g ground sediments were digested with HF (Suprapur, Merck) at 110 °C for 48 h followed by a mixture of concentrated acids HCl:HNO 3 (3:1, v:v, Suprapur Merck) at 120 °C for 24 h, operation renewed once. Each heavy metal was measured by inductively coupled plasma-atomic emission spectrometry (ICP-AES, VARIAN Vista Pro, axial view).

Total Hg was measured in dry and ground sediment samples (without any pre-treatment) by means of atomic absorption spectroscopy (AAS) using an AMA 254 solid phase Hg-Analyser (Altec Ltd., Prague, Czech Republic) [START_REF] Ouddane | A comparative study of mercury distribution and methylation in mudflats from two macrotidal estuaries: The Seine (France) and the Medway (United Kingdom)[END_REF].

The persistent organic pollutants, including PAHs (EPA's 16 priority PAHs) and PCBs (7 congeners) were analysed. In brief, organic compounds were extracted from 2 g of dried sediment by microwave (120 °C for 15 min, 1200 W) assisted extraction with 40 mL of a mixture of acetone and hexane (1:1, v:v). Simultaneous determination of PAHs and PCBs was performed on a gas chromatography-mass spectrometry (GC-MS, VARIAN, CP 3800 -1200 MS TQ). A ZB-MultiResidue column (30 m, 0.25 mm, 0.25 µm) were used (Phenomenex). Identification of PAH compounds and PCB congeners was based on the comparison of their GC-retention times and their mass spectrum, with appropriate individual standards.

Biochemical biomarkers analysis

Livers were homogenised in an ice-cold phosphate buffer (0.1 M, pH 7.8) with 20% glycerol and 0.2 mM phenylmethylsulfonyl fluoride as a serine protease inhibitor. The homogenates were centrifuged at 10,000 g at 4 °C, for 15 min and the post-mitochondrial fractions were used for biochemical assays. Total protein concentrations were determined using the method of [START_REF] Bradford | A rapid and sensitive method for quantification of microgram quantities of protein utilising the principle of protein-dye binding[END_REF] with bovine serum albumin (Sigma-Aldrich Chemicals, France) as a standard.

Ethoxyresorufin-O-deethylase activity (EROD) was determined following the hydroxylation of 7-ethoxyresorufin by the method of [START_REF] Flammarion | Statistical analysis of cyprinid ethoxyresorufin-O-deethylase data in a large french watershed[END_REF]. The reaction mixture consisted of a phosphate buffer (0.1 M, pH 6.5), 7-ethoxyresorufin (8 µM) and NADPH (0.5 mM). The change in fluorescence was recorded (excitation wavelength 530 nm, emission wavelength 585 nm) and enzyme activity calculated as pmol.min -1 .mg -1 protein using a Resorufin standard.

Glutathione S-transferase activity (GST) was determined following the conjugation of reduced glutathione with CDNB by the method of [START_REF] Habig | Glutathione S-transferases. The first enzymatic step in mercapturic acid formation[END_REF]. The reaction mixture consisted of a phosphate buffer (0.1 M, pH 6.5), reduced glutathione (1 mM) and CDNB (1 mM). The change in absorbance was recorded at 340 nm and enzyme activity calculated as µmol.min -1 .mg -1 protein using GST standard.

Catalase activity (CAT) activity was determined by the method of [START_REF] Babo | In vitro effects of thiram on liver antioxidant enzyme activities of rainbow trout (Oncorhynchus mykiss)[END_REF]. In brief, the assay mixture consisted of a phosphate buffer (100 mM pH 6.5) and H 2 O 2 (28 mM). Change in absorbance was recorded at 240 nm. CAT activity was calculated in terms of µmol.min -1 .mg -1 protein, using bovine erythrocyte Catalase as standard.

Physiological parameters 2.4.1. Growth index

Sea bass and turbot specific growth rates in weight (% per day) were estimated as : GW = 100(ln W 2ln W 1 )/(t 2t 1 ), where (W 1 ) and (W 2 ) are fish total body weight at times (t 1 ) (beginning of the experiment) and t 2 (time of collection). Similarly, the specific growth rate in length was estimated as : GL = 100(ln L 2ln L 1 )/(t 2t 1 ), where (L 1 ) and (L 2 ) are fish total length at times (t 1 ) and (t 2 ) respectively.

The recent growth index (RG) was determined by measuring the width of the peripheral daily increments of the otoliths from the previous 5 days before the end of the experiment. Sagittae were removed, cleaned and mounted on a glass side in thermoplastic cement (Crystal Bond). Sections of sagittae were obtained by polishing them in both side with grinding paper of decreasing grit sizes (5 to 0.1 µm) until increments at the outer edge were visible. Otoliths were etched for 30 sec with 0.1 M EDTA and analysed under transmitted light, using a video system fitted to a compound microscope (TNPC 5.0, NOESIS) [START_REF] Amara | Growth and condition of 0-group European flounder, Platichthys flesus as indicator of estuarine habitat quality[END_REF].

Condition Indices

We estimated three condition indices for the fish caged in the two stations and in the fish sacrificed at the beginning of the experiment (t 0 ): RNA:DNA ratio and TAG:ST ratio as indicators of nutritional status and Fulton's K condition index as an indicator of the fishes general well being. We calculated Fulton's K condition index with the formula :

K = 100(W/L 3 ),
where (W) is the body mass (mg) and (L) is the total length (mm).

The procedure used to determine RNA and DNA concentrations in individual fish was based on the Clemmesen method (1988), slightly modified as described by [START_REF] Amara | Growth and condition of 0-group European flounder, Platichthys flesus as indicator of estuarine habitat quality[END_REF]. The quantity of RNA and DNA was determined by the fluorescence-photometric technique using a specific nucleic acid fluorescent dye-ethidium bromide.

The amount of total lipids in each individual was measured on a sample of lyophilised muscle (0.07 g). Lipid extraction was conducted using the method of [START_REF] Bligh | A rapid method of total lipid extraction and purification[END_REF], slightly modified as described by [START_REF] Amara | Growth and condition indices in juvenile sole Solea solea measured to assess the quality of essential fish habitat[END_REF]. TAGs and sterols were separated from other lipids by performing thin layer chromatography (TLC).

Statistical analysis

Statistics were performed with Xlstat 2007. Mean comparisons of biomarker responses between t 0 and fish caged in station A and B were analysed using one-way ANOVA, followed by post-hoc Tukey tests. As biochemical and physiological biomarkers were analysed on the same fish, a Pearson product moment correlation matrix was computed using data from all the individuals collected from both stations, to examine the statistic links between parameters. A principal component analysis (PCA) was used to evaluate the relationships between the chemical sediment contaminants and fish biological responses.

Results

Environmental parameters

At the beginning of the experiment, bottom temperature (station A : 14.6°C ; station B : 14.9°C), salinity (station A : 34.1 PSU; station B : 33.5 PSU) and oxygen contents (station A : 7.86 mg.L -1 ; station B : 7.04 mg.L -1 ) were similar in the two caging stations. The studied area temperature had increased during June and July; the temperature was 18°C in both stations at the end of the caging exposure, which was also the highest temperature during the caging experiment.

The results of metal concentrations expressed as mg. kg -1 of dry sediment are reported for each station in Fig. 2, PAHs and PCBs concentrations in Table 1. Sediment samples indicated different levels of contamination in metallic and organic compounds. As expected, all metal concentrations analysed in sediment collected from station A, located in front of the harbour, were lower than station B and no PAHs were detected. A way to assess sediment toxicity is the use of contamination sediment quality guidelines. [START_REF] Long | Incidence of adverse biological effects within ranges of chemical concentrations in marine and estuarine sediments[END_REF] identified two guideline values: the effects range-low (ERL) and the effects range-median (ERM). Concentrations above the ERL, represent a possible-effects range within effects that would occasionally occur. The concentrations above the ERM values represent a probable-effects range within which effects would frequently occur. In our study, all chemical contaminants detected in station A were found to be lower than the toxic effect range ERL. Sediment from station B exhibited concentrations in Cd, Hg and Zn higher than ERL and lower than ERM.

The PAHs concentrations stayed below the guideline values and no PCBs was detected at any station (< 0.01 mg.kg -1 ).

Biochemical biomarkers

Because the cages were slightly damaged at station A, only 77% of the fish could be sampled (93% for sea bass and 59% for turbot). At station B, 94% of the fish were collected (100% for sea bass and 88% for turbot). No skeletal fish were found in any of the cages, or in the fish gut, from station A or B; we assumed that some fish had escaped when the cage was raised to the surface under difficult meteorological conditions. Therefore, the mortality rate was not assessed in the present study.

Several hepatic biomarkers were analysed, linked to biotransformation of xenobiotics (EROD and GST) and protection against oxidative stress (Catalase) (Fig. 3). Values of EROD activities were found lower in that of sea bass liver compared to turbot. Indeed, EROD activity measured in sea bass sacrificed at the beginning of the experiment (t 0 ) was below the detection limit, whereas some activities were detected in turbot (0.95 ± 0.22 pmol.min -1 .mg -1 of protein). For both species, a significant induction of EROD activities was observed in those fish caged in the two stations compared to t 0 . This induction was significantly higher in fish from station B (sea bass : 0.90 ± 0.53 pmol.min -1 .mg -1 protein; turbot : 2.88 ± 0.62 pmol.min - 1 .mg -1 protein) compared to station A (sea bass : 0.40 ± 0.30 pmol.min -1 .mg -1 protein; turbot :

1.59 ± 0.38 pmol.min -1 .mg -1 protein) (Anova, p<0.001).

For both species, no difference was observed in GST activities between those fish measured from t 0 and those caged in station A. However, GST activities were found significantly higher in fish caged in station B (sea bass : 0.62 ± 0.20 µmol.min -1 .mg -1 protein; turbot : 0.62 ± 0.14 µmol.min -1 .mg -1 protein) compared to reference station (A) (sea bass : 0.34 ± 0.16 µmol.min -1 .mg -1 protein; turbot: 0.44 ± 0.11 µmol.min -1 .mg -1 protein) (Anova, p<0.05).

Values of Catalase activities were found to be about ten times higher in turbot liver compared to sea bass. For CAT activities measured in fish caged in station A, a significant induction was observed in both species (sea bass : 1.40 ± 1.36 µmol.min -1 .mg -1 protein; turbot : 28.6 ± 7.2 µmol.min -1 .mg -1 protein) compared to t 0 (sea bass : 1.03 ± 0.22 µmol.min -1 .mg -1 protein; turbot : 23.1 ± 4.4 µmol.min -1 .mg -1 protein) (Anova, p<0.001). On the contrary, a significant decrease of CAT activities were measured in sea bass caged in station B (0.54 ± 0.18 µmol.min -1 .mg -1 protein) compared to station A (Anova, p<0.001). CAT activities were also lower in turbot from station B (23.1 ± 0.35 µmol.min -1 .mg -1 protein) compared to station A, but the differences were not significant (Anova, p=0.149).

Relationships with physiological biomarkers

Sea bass caged in station B presented a significant decrease of both specific growth rates, recent growth, Fulton's K condition index, RNA:DNA and TAG:ST ratios, compared to the reference station. For turbot, among all physiological parameters analysed, only the specific growth rates in weight, the Fulton's K condition and the TAG:ST ratio were significantly lower in station B compared to the reference station (Kerambrun et al., submitted for publication). It should be noted that comparisons of growth and condition indices between the two stations were not the purpose of this study. Consequently, in this paper we have focused only on their relationships with biomarker activities.

The relationships between biomarker activities, biological parameters and sediment chemical contaminants were analysed with a PCA (Fig. 4). The two first axes of the PCA explained 94.26 % of the global inertia in the data with mean explanations for the first axis (74.23 %). The first axis ordinates the stations along a pollution gradient, which appears to be positively correlated with GST activities. On the contrary, the PCA suggests a negative correlation between the contamination gradient and most of the physiological biomarkers.

EROD and Catalase activities appear to be more associated with the second axis which seems to differentiate sea bass and turbot among the values of parameters. Among the different biological indicators, the RNA:DNA ratio appears to be the most related with biomarkers activities.

Correlations between biomarkers and physiological parameters (growth and condition indices) were more precisely analysed for each species (Table 2). For each species, by using the data from all individuals collected from the two stations, a Pearson correlation matrix was computed to test the existence of significant links between biochemical and physiological parameters, and to evaluate the strength of these relationships. For sea bass, biochemical biomarkers were well related to physiological biomarkers. CAT activities were significantly and positively correlated with all growth and condition indices. For EROD and GST activities, most correlations were found to be significant and negatively correlated with physiological biomarkers. Among the 12 correlations analysed on sea bass, only 3 were not significant: GST activities with a TAG:ST ratio and EROD with the recent growth (RG) and the Fulton's K index. For turbot, biochemical biomarker activities were slightly related to growth and condition indices, with only significant correlation between EROD activities, the Fulton's K index and the lipid index (TAG:ST), and CAT activities with the lipid index.

Discussion

Biochemical biomarkers responses

Fish, display close physiological relationships to their environment as ectothermic organisms, and as such, are sensitive to environmental disruptions and in particular to chemical stress. As a consequence many authors have begun measuring fish cellular detoxification or defence mechanisms that occur in response to exposure select environmental xenobiotics [START_REF] Van Der Oost | Fish bioaccumulation and biomarkers in environmental risk assessment: a review[END_REF]. The first aim of this study was to analyse the response of three biochemical biomarkers in two marine fish species caged in a harbour polluted area. EROD activities measured in sea bass and turbot were in the same range of values than those measured on the same species by Teles et al. (2004) and [START_REF] Camus | Ethoxyresorufin-O-deethylase activity and fixed wavelength fluorescence detection of PAHs metabolites in bile in turbot (Scophthalmus maximus L.) Exposed to a Dispersed Topped Crude Oil in a Continuous Flow System[END_REF] respectively. [START_REF] Ahmad | Juvenile sea bass (Dicentrarchus labrax L.) enzymatic and non-enzymatic antioxidant responses following 17b-estradiol exposure[END_REF] also found comparable values of GST activities for sea bass, but no reference value of GST activities was found for turbot. These two biotransformation parameters (EROD phase I and GST phase II) activities were found to be higher in sea bass and turbot, caged in the inner part of the harbour, compared to the station located in front of it. Such induction of EROD and GST activity was also recorded in [START_REF] Stien | Evaluation of biomarkers in caged fishes and mussels to assess the quality of waters in a bay of the NW Mediterranean Sea[END_REF] in sea bass caged in a Mediterranean France harbour and for carp (Cyprinus carpio) caged in a Netherlander polluted site [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 biotransformation index (BTI)[END_REF]. EROD and GST are known to be induced by several organic contaminants such as PAHs and PCBs (Deviller et al., 2005;[START_REF] Castro | An In Situ Bioassay Integrating Individual and Biochemical Responses Using Small Fish Species[END_REF][START_REF] Fragoso | Evaluation of an exposure assay to measure uptake of sediment PAH by fish[END_REF]. As higher levels of PAHs were detected in station B compared to station A, the increase of these biotransformation enzymes could be explained by these organic compounds. However, other organic compounds not measured in this study such as dioxin could have enhanced these enzymes. Indeed, an EROD induction was observed in juvenile sea bream, Sparus aurata, exposed to a dioxin mixture [START_REF] Abalos | Effects on growth and biochemical responses in juvenile gilthead seabream (Sparus aurata) after long-term dietary exposure to low levels of dioxins[END_REF].

Complementary to these two biotransformation parameters, the catalase was measured to analyse the antioxidant status of juvenile fish. Catalase is an important enzyme in antioxidant defence system protecting animals from oxidative stress. Induction of biotransformation metabolism produces highly reactive oxygen species (ROS) as by-products leading to oxidative stress (Jifa et al., 2006). In this way, the antioxidant defence system may be induced in cells as response to ROS. In our study, a decrease of catalase activities was observed for both juvenile sea bass and turbot, even though the decrease was not significant for turbot. This inhibition was also observed in sea bass exposed to cadmium [START_REF] Romeo | Cadmium and copper display different responses towards oxidative stress in the kidney of the sea bass Dicentrarchus labrax[END_REF] and pacu (Piaractus mesopotamicus) exposed to copper [START_REF] Sampaio | Antioxidant defenses and biochemical changes in pacu (Piaractus mesopotamicus) in response to single and combined copper and hypoxia exposure[END_REF]. [START_REF] Palace | Interactions among antioxidant defences in liver of rainbow trout (Oncorhyncus mykiss) exposed to cadmium[END_REF] estimated that possible mechanisms by which metal produces lower catalase activity, may include direct metal-mediated structural alteration of the enzyme and depression of catalase synthesis. This phenomenon, could explain the decrease of catalase activities which occurred in our study, since sediment from station B showed concentrations of Cd, Hg and Zn higher than ERL guidelines.

In complex chemical mixtures, such as those found in the polluted sediments of the studied harbour, any biomarkers regulation was likely to be the net result of additive, synergistic or antagonistic chemical interactions. Indeed, while many studies have shown the induction of biotransformation enzymes by organics compounds, several studies have shown an inhibition of EROD activities by metals. EROD decrease was observed for sea bass, Dicentrarchus labrax [START_REF] Viarengo | Heavy Metal Inhibition of EROD Activity in Liver Microsomes from the Bass Dicentrarchus labrax Exposed to Organic Xenobiotics: Role of GSH in the Reduction of Heavy Metal Effects[END_REF] and leaping mullet, Liza saliens (Bozcaarmutlu and Arinç, 2004) following metal exposure. This inhibition was explained by binding of metal ions to the sulfhdryl group of the enzymes or generation of reactive oxygen species (ROS) (Bozcaarmutlu & Arinç, 2004). On the other side, recent studies have shown in vivo GST induction in zebra fish, Brachydanio rerio, exposed to heavy metals (Paris-Palacios et al., 2000) and a positive correlation was found between copper and GST induction in grey gold mullet, Liza aurata [START_REF] Pereira | Combined use of environmental data and biomarkers in fish (Liza aurata) inhabiting a eutrophic and metal-contaminated coastal system -Gills reflect environmental contamination[END_REF]. In the same way, various responses of CAT activity have been observed in fish exposed to organic or metallic contaminants in both field and laboratory experiments. Indeed, CAT has been shown to be either induced or inhibited by metals depending on the dose, the species or the route of exposure [START_REF] Romeo | Cadmium and copper display different responses towards oxidative stress in the kidney of the sea bass Dicentrarchus labrax[END_REF][START_REF] Sanchez | Copper-induced oxidative stress in three-spined stickleback: relationship with hepatic metal levels[END_REF].

Comparison between biochemical and physiological biomarkers

In the last years, the use of biochemical biomarker in fish has been enhanced by increased information about influencing factors [START_REF] Kammann | Genotoxic and teratogenic potential of marine sediment extracts investigated with comet assay and zebrafish test[END_REF]. However, the relationships between molecular responses and effects at higher levels of biological organisation have not been examined in great detail. In the second part of this study, we have compared the biochemical biomarkers with several growth and condition indices measured from the same fish. Examination of individual fish growth and condition is one method that has been successfully used to compare habitat quality among different juvenile nursery areas (e.g. [START_REF] Sogard | Variability in growth rates of juvenile fishes in different estuarine habitats[END_REF][START_REF] Meng | Using winter flounder growth rates to assess habitat quality in Rhode Island's coastal lagoons[END_REF][START_REF] Gilliers | Are growth and density quantitative indicators of essential fish habitat quality? An application to the common sole Solea solea nursery grounds[END_REF][START_REF] Searcy | Is growth a reliable indicator of habitat quality and essential habitat for a juvenile estuarine fish? Can[END_REF][START_REF] Amara | Growth and condition indices in juvenile sole Solea solea measured to assess the quality of essential fish habitat[END_REF].

The use of growth and condition as indices of habitat quality is based on the assumption that larger, faster-growing fish are healthier and hence experience more favourable environmental conditions than smaller, slower-growing fish. In a previous study, we analysed the relationships between chemical contamination and biological performance of juvenile marine fish using the caging method (Kerambrun et al., submitted for publication). We found a general decrease in growth and condition indices of juvenile fish caged in the contaminated station (B).

Sea bass

The results of comparison of parameter's means between the two stations, show that the inductions of EROD and GST, and the decrease of CAT activities found on sea bass caged in station B, are related to a significant decrease of both specific growth rates, recent growth, Fulton's K condition index, RNA:DNA and TAG:ST ratios. Using multiple correlations performed with the data from all the fish collected from both stations, no significant correlation was observed between EROD activities and the otolith's recent growth, or the Fulton's K index. This latter morphometric index is commonly used as indicators for an individual's general well-being, which represents a more longer-term indicator compared to some biochemical indices like RNA:DNA ratio [START_REF] Vasconcelos | Juvenile fish condition in estuarine nurseries along the Portuguese coast[END_REF]. Indeed, Ferron (2000) suggested that morphological indicators have lower sensitivity, longer latency and slower dynamics than nucleic acids and lipids which respond on a shorter scale. The same results were obtained for juvenile sole exposed for 15-days to copper [START_REF] Fonseca | Effect of copper exposure on growth, condition indices and biomarker response in juvenile sole Solea senegalensis[END_REF].

Biochemical biomarkers response represents short-term responses to contaminant and so they would be less related to longer-term physiological biomarkers. However, GST activities were significantly correlated with recent growth and the Fulton's condition index suggesting the potential higher relevance of this biotransformation enzyme than EROD.

High levels of both EROD and GST activities were significantly correlated with reductions of the somatic growth rates, RNA:DNA ratios and lipid index of sea bass caged in the contaminated station. This relation suggests that there are metabolic costs associated with the synthesis of these proteins or with detoxification processes [START_REF] Rose | Assessment of seasonal variability of biomarkers in three-spined stickleback (Gasterosteus aculeatus L.) from a low contaminated stream: Implication for environmental biomonitoring[END_REF]. Indeed, as growth is an integration of many processes [START_REF] Morales-Nin | The effect of Prestige oil ingestion on the growth and chemical composition of turbot otoliths[END_REF], such changes in fish metabolism could result, at a higher level, to growth impairments. Previous studies have analysed biochemical biomarkers and growth measurement relationships. [START_REF] Cao | Accumulation and oxidative stress biomarkers in Japanese flounder larvae and juveniles under chronic cadmium exposure[END_REF] reported that after cadmium exposure, the decrease in the growth of juvenile Japanese flounder (Paralichthys olivaceus), was related to inhibition of CAT and GST activities. The growth reduction of the greenback flounder (Rombosolea tapirina) exposed to contaminated sediment was also related to an induction of EROD activities [START_REF] Mondon | Histological, growth and 7-ethoxyresorufin Odeethylase (EROD) activity responses of greenback flounder Rhombosolea tapirina to contaminated marine sediment and diet[END_REF]. On the contrary, in [START_REF] Abalos | Effects on growth and biochemical responses in juvenile gilthead seabream (Sparus aurata) after long-term dietary exposure to low levels of dioxins[END_REF], the increase of EROD and GST activities in juvenile gilthead seabream (Sparus aurata) was not related to growth which showed no difference between dioxin-exposed and non-exposed fish.

The decrease of catalase activities measured in sea bass was associated with a decrease of all physiological biomarkers. Indeed, catalase activities showed the strongest correlations with growth and condition indices compared to the two biotransformation parameters measured. Bagnyukova et al. (2005) showed that a catalase inhibition in goldfish (Carassius auratus) was associated with lipid oxidative damage products due to an oxidative stress from the absence of cell protection by this antioxidant enzyme. They also showed that the response to catalase depletion involves compensatory changes in the activities of other antioxidant enzymes. This change in cell metabolism in response to the decrease of catalase activities could have played a role in the decrease of sea bass physiological status observed in station B.

Turbot

Despite high levels of biotransformation parameters found in turbot caged in station B, no difference in growth in length and RNA:DNA ratios was observed. These low responses of turbot growth parameters have been explained in our previous results by the non optimal environmental parameters for juvenile turbot growth (sediment grain size, turbidity and feeding behaviour) as well as for reference station (A). Cages are a valuable tool for comparing the relative growth of small juvenile fish in different habitats but are also known to have certain limitations [START_REF] Able | Habitat quality for shallow water fishes in an urban estuary: The effects of man-made structures on growth[END_REF][START_REF] Phelan | Estuarine and habitat-related differences in growth rates of young-of-the-year winter flounder (Pseudopleuronectes americanus) and tautog (Tautoga onitis) in the 3 north-eastern US estuaries[END_REF][START_REF] Oikari | Caging techniques for field exposures of fish to chemical contaminants[END_REF]. Cages confine fish to a small space, preventing movements to surrounding areas i.e. for food searching. The analysis of stomach of the two species indicated no food limitation as also observed in other caging studies [START_REF] Sogard | Variability in growth rates of juvenile fishes in different estuarine habitats[END_REF][START_REF] Kamermans | Importance of food availability for growth of juvenile southern flounder (Paralichthys lethostigma) in the Pamlico River estuary[END_REF]. Energetic studies have shown that the metabolic rates of flatfish can increase significantly when they are forced to bury in sediments with suboptimal grain sizes, or do not have access to sediments at all [START_REF] Howell | The influence of sand on the estimation of resting metabolic rate of juvenile sole Solea solea (L )[END_REF]. The non optimal grain size of the sediment, or the fact that the cage infrastructure may have limited the turbot burying, could explain the decrease of turbot condition in station A even if the sediment was slightly contaminated. Consequently, among the different physiological parameters analysed in turbot, only the specific growth rate in weight, the Fulton's K condition index and the index could be related with the significant EROD and GST induction. In particular, using multiple correlations performed with the data from all fish collected in both stations, the TAG:ST ratios were the ones significantly correlated with both EROD and CAT activities. Lipid depletion has been identified as a general metabolic response to stress [START_REF] Claireaux | Influence of oil exposure on the physiology and ecology of the common sole Solea solea: experimental and field approaches[END_REF]. TAGs are the principal reserve of energy in teleosts and the first components to be mobilised during periods of stress [START_REF] Galois | Change in biochemical composition and otolith microstructure of larval common sole Solea solea L. under experimental starvation[END_REF]. [START_REF] Hakanson | Analysis of lipid components for determining the condition ofanchovy larvae, Engraulis mordax[END_REF] reported that TAGs were used preferentially for short-term energy need.

Their decrease could be the direct consequence of the xenobiotic detoxification and regulation which involve both passive and active mechanisms and therefore require energy [START_REF] Alquezar | Effects of metals on condition and reproductive output of the smooth toadfish in Sydney estuaries, south-eastern Australia[END_REF]. Previous studies have reported similar depletion in lipid reserves of fish subjected to metal trace exposure [START_REF] Rowe | Growth responses of an estuarine fish exposed to mixed trace elements in sediments over a full life cycle[END_REF][START_REF] Fonseca | Effect of copper exposure on growth, condition indices and biomarker response in juvenile sole Solea senegalensis[END_REF].

Since turbot growth appears to have been influenced by other environmental factors with additional chemical contaminant effects, the relationships between biochemical biomarkers and growth parameters could not be established for this species. These differences in behaviour between a demersal fish like the sea bass and a benthic fish like the turbot, could explain the variations observed in our study to fish responses to chemical contamination.

These results show the importance in the choice of the fish species suitable for caging studies in order to minimise physiological stress in caged fish. Because of species specific differences in behaviour, feeding activities and physiological processes, fish biomarker responses and physiological parameters sensitivity, must be analysed carefully.

Conclusions

This multibiomarker approach allowed us to assess different levels of fish responses to chemical contamination. This study was a first approach of simultaneous comparison of both biochemical and physiological biomarkers in a caging study. For individual sea bass, biochemical biomarkers, growth and condition indices such as the Fulton's K condition index, the RNA:DNA ratio and the lipid storage index, showed numerous significant correlations.

Conversely, there were only a few significant correlations for turbot, suggesting a speciesspecific response. However, even if biochemical biomarkers responses and fitness are a direct measure of an individual fish's stress and health, they are not specific to fish stress responses to pollutants, as several other biotic and abiotic stressors can affect them. Environmental conditions must be considered when one is integrating both biochemical and physiological biomarkers responses with pollution stress. This can explain the species-specific response we observed.

Although based on the comparison of only two stations, our data support the use of caged juvenile sea bass as sentinel species due to some original ecological features of this species (e.g. large distribution, eurythermal and euryhaline species). We found that the combined use of several biochemical biomarkers and physiological responses such as growth and condition indices can improve fish health determination, and may provide a useful tool to monitor and assess habitat quality for juvenile fishes. ■ B (□ t 0 : reference ). Data represent mean ( SD) biochemical biomarkers activities and for each species, ( 1 ) represent significant difference (p<0.05) compared to "t 0 " and ( 2 ) compared to "station A".

Figure legends

Sea bass : n = 15 (t 0 ), n = 14 (A), n = 18 (B); Turbot : n = 15 (t 0 ), n = 10 (A), n = 14 (B).

Fig. 1 .

 1 Fig. 1. Locations of the two caging stations at the harbour of Boulogne sur Mer, France.

Fig. 2 .

 2 Fig.2. Mean ( SD) sediment metal concentrations (mg.kg -1 dry weight) in the two caging stations ( A and ■ B). * Concentration that exceeds the ERL (effects range-low) defined by[START_REF] Long | Incidence of adverse biological effects within ranges of chemical concentrations in marine and estuarine sediments[END_REF].

Fig. 3 .

 3 Fig. 3. Differences in Ethoxyresorufin-O-deethylase (EROD), Glutathione S-transferase (GST) and Catalase (CAT) activities of juvenile sea bass and turbot caged in station A and

Fig. 4 .

 4 Fig. 4. PCA based on biomarker responses (Ethoxyresorufin-O-deethylase -EROD-, Glutathione S-transferase -GST-and Catalase -CAT) of juvenile sea bass (□) and turbot (•) in relation with physiological biomarkers (specific growth rate in length -GL-and weight -GW-, recent growth -RG-, Fulton's condition index -K-, RNA:DNA and Triacylglycerol on sterol -TAG:ST-ratios) and chemical contamination (metals and total PAHs) measured in caging station A and B.

  Fig. 1

  correlations between biochemical biomarker responses (Ethoxyresorufin-O-deethylase -EROD-, Glutathione S-transferase -GST-and Catalase -CAT) of juvenile sea bass (□) and turbot (•) in relation with physiological biomarkers (specific growth rate in length -GL-and weight -GW-, recent growth -RG-, Fulton's condition index -K-, RNA:DNA and Triacylglycerol on sterol -TAG:ST-ratios) measured in juvenile sea bass (a) and turbot (b) caged in stations A and B. Significant correlation for p* < 0.05, p** < 0.01 and p*** < 0.001. Sea bass : n = 32 ; Turbot : n = 24

Table legends Table 1

 legends1 Mean ( SD) PAH and PCB concentrations (mg.kg -1 dry weight) in sediment from the two caging stations (A and B).

Acknowledgements

This work was supported by post Grenelle programme 189, DEVIL and the Franco-British INTERREG IVA European Programme, DIESE. We would like to thank Michel LAREAL for making the cages, and Gregory GERMAIN and François GEVAERT for their help during their placement at sea. We thank Peter MAGEE for revision of the English grammar and syntax (www.anglais.webs.com).


Brahim Selmaoui
email: brahim.selmaoui@ineris.fr

Jacques Lambrozo

Linda Sackett-Lundeen

Erhard Haus

Yvan Touitou

Acute exposure to 50-Hz magnetic fields increases interleukin-6 in young healthy men

Keywords:

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.

Introduction

It is well known that overhead transmission lines, transformers, and household appliances are major sources of electromagnetic field (EMF) exposure. For this reason a concern regarding the potential effect of extremely low frequency magnetic fields (ELF-EMF) on human health has been widely debated, the main concerns focusing on their carcinogenic potential and relationship with immune system functions. This debate was stimulated by some epidemiological studies suggesting that exposure to 50-60Hz MFs may be associated with an increased incidence of various types of cancer [START_REF] Wertheimer | Electrical wiring configurations and childhood cancer[END_REF][START_REF] Demers | Occupational exposure to electromagnetic fields and breast cancer in men[END_REF][START_REF] Olsen | Residence near high voltage facilities and risk of cancer in children[END_REF][START_REF] Savitz | Magnetic field exposure in relation to leukemia and brain cancer mortality among electric utility workers[END_REF][START_REF] Kheifets | Occupational electric and magnetic field exposure and brain cancer: a meta-analysis[END_REF][START_REF] Fear | United Kingdom Childhood Cancer Study Investigators. Childhood cancer and social contact: the role of paternal occupation (United Kingdom)[END_REF][START_REF] Miller | Leukemia following occupational exposure to 60-Hz electric and magnetic fields among Ontario electric utility workers[END_REF][START_REF] Coogan | Occupational exposure to 60-hertz magnetic fields and risk of breast cancer in women[END_REF][START_REF] Thériault | Risks of leukaemia among residents close to high voltage transmission electric lines[END_REF][START_REF] Mcbride | Power-frequency electric and magnetic fields and risk of childhood leukemia in Canada[END_REF][START_REF]PART 1: Static and Extremely Low Frequency[END_REF].

As the immune system is involved in the control of cancer development and other diseases, it is important to know whether or not immunological functions are affected by the exposure to ELF-MF. Several studies have examined various immune cell parameters but with conflicting findings. ELF-MF were reported to produce changes in the activity or the number of circulating natural killer (NK) cells [START_REF] Mclean | Cancer promotion in a mouse-skin model by a 60-Hz magnetic field: II. Tumor development and immune response[END_REF][START_REF] Tremblay | Differential modulation of natural and adaptive immunity in Fischer rats exposed for 6 weeks to 60 Hz linear sinusoidal continuous-wave magnetic fields[END_REF][START_REF] House | Immune function and host defense in rodents exposed to 60-Hz magnetic fields[END_REF][START_REF] House | Modulation of natural killer cell function after exposure to 60 Hz magnetic fields: Confirmation of the effect in mature B6C3F1 mice[END_REF][START_REF] Bonhomme-Faivre | Effects of electromagnetic fields on the immune systems of occupationally exposed humans and mice[END_REF][START_REF] Gobba | Extremely low frequencymagnetic fields (ELF-EMF) occupational exposure and natural killer activity in peripheral blood lymphocytes[END_REF]. However, no changes were observed in other studies [START_REF] Ramoni | Human natural killer cytotoxic activity is not affected by in vitro exposure to 50-Hz sinusoidal magnetic fields[END_REF][START_REF] Selmaoui | Acute exposure to 50-Hz magnetic field does not affect haematological and immune functions in young healthy men. A circadian study[END_REF][START_REF] Thun-Battersby | Lymphocyte subset analyses in blood, spleen and lymph nodes of female Sprague-Dawley rats after short or prolonged exposure to a 50 Hz 100 microT magnetic field[END_REF](21)[START_REF] Ikeda | No effects of extremely low frequency magnetic fields found on cytotoxic activities and cytokine production of human peripheral blood mononuclear cells in vitro[END_REF].

Cytokines which are produced by a variety of cells were also investigated. Exposure to low frequency pulsed electromagnetic fields increased interleukin-1 and interleukin-6 production by human peripheral blood mononuclear cells [START_REF] Cossarizza | Exposure to low frequency pulsed electromagnetic fields increases interleukin-1 and interleukin-6 production by Human peripheral blood mononuclear cells[END_REF]. It has been shown that exposure to 50 Hz sinusoidal MFs decreased TNF-production, while no effect was found on IFN-production [START_REF] Petrini | Tumor necrosis factor a and interferon g production by human peripheral blood mononuclear cells exposed in vitro to sinusoidal 50 Hz magnetic fields[END_REF]. Another study has also reported that 50 Hz electromagnetic fields decreased TNF-and IFN-production and increased IL-1 production [START_REF] Jonai | Cytokine profile of human peripheral blood mononuclear cells exposed to 50 Hz EMF[END_REF]. It is to note that all these studies on cytokine production have been conducted in vitro. Overall, the possible effects of ELF magnetic fields have been investigated in different types of cell cultures. On the other hand, there are only a limited number of human studies that are dedicated to the investigation of effects of ELF on the immune system. The study presented here was designed to look for possible effects of acute exposure to 50-Hz magnetic fields (10 μT) on the IL-1 , IL-2, IL-6, IL-1RA, IL-2R production in humans.

Materials and Methods

Human subjects

Thirty-two healthy subjects volunteered for this study and provided written consent. All were men aged 20 to 30 years. They were selected after routine clinical and laboratory examinations. Selection criteria included: regular sleep habits, no medication, no chronic disease or disability, no recent acute illness, no smoking. Those selected were instructed to eat balanced meals and to abstain from consuming alcohol and coffee for 24 hours before and during each experimental session. They were synchronized with a diurnal activity from 08:00 to 23:00 and nocturnal rest.

Exposure facility

The experimental unit consisted of three independent rooms and a shared bathroom. The exposed subjects slept in the first room (2 beds), and the sham-exposed in the second (2 beds).

We did not inform subjects of their exposure status. The wooden beds were arranged parallel to each other in each room. Before setting up the exposure materials and beds, we used "Linda Wheel and EMDEX II dosimetry systems" to evaluate the noise of the background magnetic field. Its' level was less than 40 nanotesla in both bedrooms. In the area immediately surrounding the experimental site, the flux density of the geomagnetic field was approximately 46 µT. Since a minimum distance of 6 m between exposed and sham-exposed beds is required to prevent the generated field from affecting the sham-exposed group, we spaced them 7 m apart. The devices for generation, calibration, and continuous monitoring of the magnetic field were located in the third room [START_REF] Selmaoui | Magnetic fields and pineal function in human: Evaluation of nocturnal acute exposure to extremely low frequency magnetic fields on serum melatonin and urinary 6-sulfatoxymelatonin circadian rhythms[END_REF].

Magnetic field generation

A linearly polarized magnetic field (10 µT) was generated by a system based on Helmholtz coils. Three rectangular coils (120 x 140 cm) spaced 80 cm apart were used for each bed.

They were wrapped in wood and look like normal wooden stems attached to the bed and allowed subjects to feel comfortable towards the device. The coils were positioned at the level of the head, groin and ankles. When energized the coils did not produce any vibration.

The device was able to generate both continuous and intermittent exposure conditions. In the intermittent exposure, the device was turned "on" for one hour and "off" for the next hour.

Whenever the device was turned "on", the magnetic field was on a 15-second "on-off" cycle.

Since "on-off" switching operations induce high frequency transient electric fields that can have a biological effect, the signal generator was monitored by computer and synchronized with passages at zero of the current wave to avoid any transient fields. The magnetic field was generated by a Tollner generator and amplified by a Kepco amplifier, all under a computer controled system developed by Electricité de France, Research and Development Department engineers. A "Positron" dosimeter system was attached to the subject's body at the height of the pelvis, to control the exposure quality and to verify that the device was working. The 10 µT intensity was chosen as representative of the average level of exposure in occupational settings or near transmission lines for electrical workers after a dosimetry survey.

Experimental procedure

This experiment studied four subjects per day (2 exposed and 2 sham exposed), and each subject participated in three 24-hour sessions. They arrived at the laboratory at 09:30.

Catheters were placed in antecubital vein for the 24-hour period, and the first blood sample was obtained at 11:00. They slept in darkness between 23:00 and 08:00 and spent their waking time in the laboratory (diurnal activity from 08:00 to 23:00 and nocturnal rest). Blood samples (tubes without anticoagulant) were taken from 11:00 to 08:00 of the following day (11:00, 17:00, 22:00, 01:00, 04:00, 08:00). During the day, 15 minutes before each sampling, subjects were asked to lie down in semi recumbent position until sampling time. From 01:00 to 08:00, the samples were obtained under dim light (less than 50 Lux) without waking the subjects, who slept with eye-mask from 23:00 to 08:00. Exposure to magnetic field lasted from 23:00 until 08:00.

The first 24h session was performed before starting the experiment and leads subjects to be acclimated to the environment and devices.. We studied two groups: the first, sham-exposed, i.e., control (N = 12 -16), and the other, exposed (N = 12-16). During the second session, two weeks after the first, a continuous 50-Hz magnetic field of 10 µT was applied. In the third session, a month after the second, the same subjects previously exposed to a continuous magnetic field were exposed to an intermittent 50-Hz magnetic field of 10 µT. The same subjects served as sham contols in both sessions. No one has reported an auditory or tactile cue that could be produced by the coils when energized. This protocol was approved by the local ethics committee.

Cytokine assay

IL-1RA (R&D System, Minneapolis, MN, USA), IL-2R (Genzyme Corporation, Cambridge, MA, USA), IL-1 , IL-2, IL-6 (Biosource, Europe S.A., Fleurus Belgium), were assayed by ELISA in duplicate using serum samples drawn during the two 24h study days. Sampling occurred at 11:00, 17:00, 22:00, 01:00, 04:00, 06:00, and 08:00. For inter-assay coefficients of variation (see Table I).

Statistical analysis

The data are expressed as means ± SEM. Data were analyzed with 2-way ANOVA repeatedmeasures (GraphPad v5.02). The main effects of field, time factor and their interaction were tested (Table 2). Post-test Bonferroni was applied. The significance level was set at p < 0.05.

Results

Significant changes in the concentration of IL-6 were observed in the subjects exposed to intermittent MF. Bonferroni post-tests analysis showed an elevation in serum IL-6 was statistically significant at 06:00 and 08:00 with p < 0.01. However, exposure to continuous or intermittent magnetic fields of 10 µT did not affect or modify the cytokine production levels of IL-1 , IL-2, IL-1RA or IL-2R. No significant interaction between the field and the time of sampling was found (Table II). There was also no statistical significance of the main effects of the field (Table II). Data are presented in Figures 1 and2.

Discussion

Epidemiological studies have shown an association between ELF exposure above approximately 0.3-0.4 μT and an increased risk of childhood leukemia [START_REF] Greenland | A pooled analysis of magnetic fields, wire codes, and childhood leukemia. Childhood Leukemia-EMF Study Group[END_REF][START_REF] Ahlbom | A pooled analysis of magnetic fields and childhood leukaemia[END_REF]. However, despite several years of work, evidence from experimental studies to support a causal relationship between ELF exposure and leukemia is lacking and argues against causality.

Thus ELF-MF exposure continues to generate a debate about its possible effects on health. In addition, there is no identified mechanism by which ELF-MF exposure in doses below 5 μT might affect the incidence of cancer or any other disease in the human population [START_REF] Swanson | Biophysical mechanisms: a component in the weight of evidence for health effects of power-frequency electric and magnetic fields[END_REF]. As the immune system is involved in several types of cancers it is worthwhile to explore the effect of ELF-MF exposure on the immune system.

Our present controlled study was conducted in humans who were exposed for one night to 50Hz MF of 10 T from 23:00 to 08:00. Results showed that IL-6 production significantly increased in subjects exposed to intermittent but not with continuous exposure. Bonferroni post test showed that the significance is more observed in the last part of the night (06:00 and 08:00). Since IL-6 shows a circadian rhythm with peak in the morning hours [START_REF] Perry | Overnight variations in cortisol, interleukin-6, tumor necrosis factor alpha and other cytokines in people rheumatoid arthritis[END_REF], the data presented may suggest a circadian stage depending stimulation of IL-6 production by the intermittent MF. The increase of IL-6 production during that time was approximately 4 to 5 fold higher than control group. Even if non-significant, the upward trend for IL-6 was observed from 01:00 just after the onset of exposure time (23:00). IL-6 is a cytokine secreted predominantly by T-cells and macrophages with both pro-and anti-inflammatory effects. IL-6 stimulates the immune response to trauma and other tissue damage leading to inflammation [START_REF] Rs | Analytic review: interleukin-6 in surgery, trauma, and critical care: part I: basic science[END_REF]. Its role as an anti-inflammatory cytokine is mediated through its inhibitory effects on tumor necrosis factor alpha (TNFα) and IL-1 and activation of IL-1 RA and IL-10. IL-6 is a regulator of body temperature and fever and mediator of the acute phase response [START_REF] Mastorakus | Interleukin-6: a cytokine and/or a major modulator of the response to somatic stress[END_REF]. It crosses the blood brain barrier and plays a role in neuroinflammation and some types of dementia [START_REF] Spooren | Interleukin-6, a mental cytokine[END_REF]. IL-6 is relevant to many disease processes such as diabetes [START_REF] Op | Interleukin-6 and diabetes: the good, the bad, or the indifferent?[END_REF], atherosclerosis [START_REF] Dubiński | The role of interleukin-6 in development and progression of atherosclerosis] (in Polish)[END_REF], depression [START_REF] Dowlati | A meta-analysis of cytokines in major depression[END_REF], autoimmune diseases [START_REF] Tackey | Rationale for interleukin-6 blockade in systemic lupus erythematosus[END_REF][START_REF] Nishimoto | Interleukin-6 in rheumatoid arthritis[END_REF][START_REF] Patel | Interleukin-6 inhibition for treatment of rheumatoid arthritis: a review of tocilizumab therapy[END_REF], and some forms of cancer [START_REF] Smith | Interleukin-6 and prostate cancer progression[END_REF][START_REF]Cancer Patients typically have increased interleukin-6 levels[END_REF][START_REF] Berger | The interleukin-6 gene : a susceptibility factor that may contribute to racial and ethnic disparities in breast cancer mortality[END_REF][START_REF] Barton | Interleukin-6 and new strategies for the treatment of cancer, hyperproliferative diseases and paraneoplastic syndromes[END_REF]. High levels of IL-6 and IL-6R have been reported in several chronic inflammatory and autoimmune diseases as well as in cancer [START_REF] Rose-John | The IL-6/sIL-6R complex as a novel target for therapeutic approaches[END_REF][START_REF] Scheller | Interleukin-6 and its receptor: from bench to bedside[END_REF][START_REF] Barillé | The role of interleukin-6 and interleukin-6/interleukin-6 receptor-alpha complex in the pathogenesis of multiple myeloma[END_REF]. However, the cytokine production levels of IL-1 , IL-2, IL-1RA and IL-2R were not affected by the MF exposure neither in the continuous mode nor in the intermitten mode.

To date, and to our knowledge, information regarding the effects of extremely low frequency MF on human cytokines is scarce. Most studies were carried out in in vitro models. These studies on the peripheral blood mononuclear cells cultures (PBMC) have shown conflicting results. Some authors reported an increase of cytokine production such as IFN-and IL-4, IL-1, and IL-6 [START_REF] Cossarizza | Exposure to low frequency pulsed electromagnetic fields increases interleukin-1 and interleukin-6 production by Human peripheral blood mononuclear cells[END_REF][START_REF] Jonai | Cytokine profile of human peripheral blood mononuclear cells exposed to 50 Hz EMF[END_REF][START_REF] Salerno | Static magnetic fields generated by a 0.5 T MRI unit affects in vitro expression of activation markers and interleukin release in human peripheral blood mononuclear cells (PBMC)[END_REF] after exposure to ELF-MF or a decrease in TNF-and IFNproduction (25) while others did not find any effect on IL-1 , IL-8, IFN-, TNF-, IL-10, IL-6 and IL-2 production [START_REF] Petrini | Tumor necrosis factor a and interferon g production by human peripheral blood mononuclear cells exposed in vitro to sinusoidal 50 Hz magnetic fields[END_REF][START_REF] Pessina | Pulsed electromagnetic fields enhance the induction of cytokines by peripheral blood mononuclear cells challenged with phytohemagglutinin[END_REF][START_REF] Salerno | Static magnetic fields generated by a 0.5 T MRI unit affects in vitro expression of activation markers and interleukin release in human peripheral blood mononuclear cells (PBMC)[END_REF][START_REF] Ikeda | No effects of extremely low frequency magnetic fields found on cytotoxic activities and cytokine production of human peripheral blood mononuclear cells in vitro[END_REF][START_REF] Kleijn | Extremely low frequency electromagnetic field exposure does not modulate toll-like receptor signaling in human peripheral blood mononuclear cells[END_REF]. This inconsistency in the results might be due to the difference in the type of the magnetic field and/or the intensities used.

In our study, the effect observed on IL-6 was obtained with intermittent and not with continuous exposure. It is to note that a similar effect in vitro (an increase of IL-6) in the literature has been reported with pulsed magnetic field [START_REF] Cossarizza | Exposure to low frequency pulsed electromagnetic fields increases interleukin-1 and interleukin-6 production by Human peripheral blood mononuclear cells[END_REF], while no effect was found with static or continuous alternating magnetic field [START_REF] Salerno | Static magnetic fields generated by a 0.5 T MRI unit affects in vitro expression of activation markers and interleukin release in human peripheral blood mononuclear cells (PBMC)[END_REF][START_REF] Kleijn | Extremely low frequency electromagnetic field exposure does not modulate toll-like receptor signaling in human peripheral blood mononuclear cells[END_REF]. It is likely that the type of MF applied may be of importance and could have a different impact on the production of IL-6. Further confirmation of these findings in human subjects are desirable.

In animal studies, only a few studies have investigated cytokine production in immune cells after exposure to ELF EMFs. Some authors have reported that exposure of sheep to EMF had no effect on the activity of IL-1 or IL-2 [START_REF] Hefeneider | Long-term effects of 60-Hz electric vs. magnetic fields on IL-1 and IL-2 activity in sheep[END_REF]. A reduction in IL-1 activity was observed in their previous studies [START_REF] Hefeneider | Joint HVAC Transmission EMF Environmental Study: Final Report on Experiment 3[END_REF]. The authors concluded that the difference observed in the results might be attributed to magnetic field strength and/or the age of the animals used which could be important variables in determining whether EMF exposure will affect IL-1 activity.

Another study has reported that exposure to 1 mT ELF-MF increased IL-1 at transcriptional and translational level in rat cortical neurons [START_REF] Loreto | Fifty hertz extremely low-frequency magnetic field exposure elicits redox and trophic response in rat-cortical neurons[END_REF]. IL-1 release was also reported to increase in macrophages after 24 h exposure to 1 mT of 50Hz MF [START_REF] Frahm | Alteration in cellular functions in mouse macrophages after exposure to 50 Hz magnetic fields[END_REF] Conclusions:

The results obtained for IL-6 in the present study should be interpreted with caution and require further studies and confirmation to ascertain if the effect observed is due to MF exposure and reproducible. It is also important to stress the potential clinical significance of this increase of IL-6 concentration particularly since its receptor (IL-6R) had been found to be increased under exposure to MF [START_REF] Zhou | Gene expression of cytokine receptors in HL60 cells exposed to a 50 Hz magnetic field[END_REF].

Overall, the lack of effect observed in the present study on IL-1beta, IL-2, IL-1RA and IL-2R supports our previous findings in which acute exposure (9 hours) to 50-Hz magnetic fields (10 T) did not affect the immunologic and hematologic systems [START_REF] Selmaoui | Acute exposure to 50-Hz magnetic field does not affect haematological and immune functions in young healthy men. A circadian study[END_REF], melatonin secretion [START_REF] Selmaoui | Magnetic fields and pineal function in human: Evaluation of nocturnal acute exposure to extremely low frequency magnetic fields on serum melatonin and urinary 6-sulfatoxymelatonin circadian rhythms[END_REF], hormones of the hypothalamo-pituitary-thyroid and adrenal axis [START_REF] Selmaoui | Endocrine functions in humans exposed to 50-Hz magnetic field. A circadian study of pituitary, thyroid and adrenal hormones[END_REF], and a large number of clinical chemistry variables [START_REF] Selmaoui | Assessment of nocturnal exposure to 50-Hz magnetic fields on the human circadian system. A comprehensive study of biochemical variables[END_REF] including biogenic amines [START_REF] Selmaoui | Evaluation of the nocturnal levels of urinary biogenic amines in men exposed overnight to magnetic field[END_REF]. Even our chronic study on workers exposed daily to magnetic fields for a period of 1-20 yr in their workplace and at home showed that this exposure does not lead to alterations in the melatonin secretion [START_REF] Touitou | Magnetic fields and the melatonin hypothesis: a study of workers chronically exposed to 50-Hz magnetic fields[END_REF].

Table II :

 II Repeated-measures 2-way ANOVA: comparison between exposed and sham exposed by examining the main effects of the field (continuous or intermittent), the time factor and their interaction.

	Variables/factors	Continuous exposure	Intermittent exposure
		P	P
	IL-6
	Field	0.2878	0.0186
	Time	0.1156	< 0.0001
	Field X Time interaction	0.5635	0.0032
	IL-1RA
	Field	0.5198	0.3455
	Time	0.0012	0.0042
	Field X Time interaction	0.7975	0.3022
	IL-1 Beta
	Field	0.5556	0.6382
	Time	0.7566	0.6379
	Field X Time interaction	0.2267	0.4402
	IL-2
	Field	0.7220	0.3851
	Time	0.0962	0.5906
	Field X Time interaction	0.5663	0.1425
	IL-2R
	Field	0.0768	0.1441
	Time	0.0584	< 0.0001
	Field X Time interaction	0.9721	0.9587

Acknowledgements

We wish to thank the engineers from EDF for their precious help. This study was supported by a grant from le Ministère de l'Environnement (Ministry of the environment) and from Electricité de France (EDF).


Georges Giraud
email: georges.giraud@dijon.inra.fr

Cleo Tebby

Corinne Amblard

Measure e t of co su ers' wi e-related knowledge

Keywords: L66, D12 Wine, Knowledge-based economy, Consumer Clustering

It is stated, according to the paradigm of knowledge-based economy, that information asymmetry between consumers and producers is reduced thanks to information availability and dissemination through the Internet or other media channels. Conversely to this statement, several articles have pointed out that knowledge-based economy reinforces the information asymmetry between experts and novices among the consumers. Accordingly, we consider the heterogeneity of consumers by means of k-means clustering applied to a knowledge-oriented questionnaire. We then try to identify and qualify the differences between several groups of French respondents regarding their attitudes and behaviour towards wine.

Introduction

It is often stated, according to the paradigm of knowledge-based economy, that information asymmetry between consumers and producers is reduced thanks to information availability and dissemination through the Internet or other media channels. Conversely, several articles have pointed out that knowledge-based economy reinforces the information asymmetry between experts and novices among the consumers [START_REF] Hogg | Consumers in the Knowledge-Based Economy (KBE): What creates and/or constitutes consumer vulnerability in the KBE[END_REF][START_REF] Gregan-Paxton | Consumer Learning by Analogy: A Model of Internal Knowledge Transfer[END_REF][START_REF] Alba | Dimensions of Consumer Expertise[END_REF]. Accordingly, we consider the heterogeneity of consumers by means of Kmeans clustering applied to a knowledge-oriented questionnaire. Cluster analysis was used in order to obtain a reliable and significant distinction between respondents with respect to level of wine knowledge. Furthermore, we used different types of wine knowledge to capture both information availability and information asymmetry related to identified clusters of respondents. We then identified and qualified the differences between these groups of French respondents regarding their attitudes and behaviour towards wine and socio-demographic characteristics. The article presents previous results related to consumer knowledge and information processing (I), then it depicts survey methodology and data analysis (II) and it deciphers measurements and results obtained (III) before a synthesis discussion in the conclusive section.

Consumer knowledge with respect to wine

When choosing wine, consumers have to process several information regarding for example price, brand, vintage, or grape variety. Once at home those who wish to be reassured on the bottle they bought, can often find an overflow of information available on the Internet though plenty of websites belonging not only to companies but also to consumer associations or simply end-users clubs. However, most of the information available requires some skill in order to be intelligible. The main question may be: Are consumers able to interpret this overflow of information? In other words:

Are professional worlds still open for consumers? It was pointed out that the product class knowledge of respondents lowers the total search effort in view of a purchasing purpose [START_REF] Beatty | External Search Effort: An Investigation Across Several Product Categories[END_REF]. We consider that consumer search for information is not always provoked by immediate purchasing purpose, and may participate to a broader objective of building up knowledge-based expertise [START_REF] Bloch | Consumer Search: An Extended Framework[END_REF]). In the wine sector, it is generally considered that o su e s' k o ledge is suppl -chain driven as tasting is only possible after purchasing, and, when selling wine, most stakeholders are telling a story through labelling and wine guides.

From a sensory perspective, it was proven that the information provided on the label of a bottle allows consumers to discriminate Champagnes, while blind tests do not [START_REF] Lange | Impact of the information provided to consumers on their willingness to pay for Champagne : comparison with hedonic scores[END_REF]. It was pointed out that, for white wine, the context has a huge influence on the perception of wine, even for oenologists [START_REF] Brochet | Influence of the context on the perception of wine -Cognitive and methodological implications[END_REF]. More generally, wine appreciation is mainly based on semantic information [START_REF] Chrea | Semantic, Typicality and Odor Representation: A Cross-cultural Study[END_REF][START_REF] Fischer | The impact of geographic origin, vintage and wine estate on sensory properties of Vitis vinifera cV. Riesling wines[END_REF]. For instance, it was recently demonstrated that women may express a positive willingness to pay for men-recommended wines [START_REF] Brouard | Women and wine, an experiment with stated and real behaviour[END_REF]. It was also found that consumer knowledge of wine regions operates during the choice-making process for wine [START_REF] Barber | Wine region brand equity, A case for consumer values and location[END_REF].

It was recently proven that providing information does not lead to increased knowledge, as consumers are overwhelmed by warnings from consumer protection organizations, the media, government, and various scientific studies [START_REF] Conley | Consumer Responses to Food Safety Information from Print Media[END_REF]. They have often received conflicting information. The authors have showed that consumers are reasonably intelligent in their evaluation of information: they responded differently to information perceived as biased versus information perceived as objectively reported.

The phenomenon of cognitive overload due to limited information processing capabilities is well documented in the psychology literature [START_REF] Alba | Dimensions of Consumer Expertise[END_REF] and might be illustrated in the case of consumer attitude with respect to food. It was shown that the overload and complexity of information on food products results in misunderstanding and misinterpretation. Even when information is made sufficiently available and accessible to consumers, only a limited amount of this information is actually brought to o su e s' atte tio and raises interest for being processed in an environment characterised by information overload. Furthermore, there is a real potential danger of information overload. Interestingly, it was shown that consumers can decide to remain rationally ignorant due to the opportunity costs of information processing, related to time and allocation of cognitive capacity, exceed the expected marginal benefit of being fully informed [START_REF] Mccluskey | Political economy of the media and consumer perceptions of biotechnology[END_REF].

Survey and data collection

The paper presents the results of a consumer survey carried-out in France in 2006, focusing on knowledge on wine. French consumers are generally considered to frequently experience such beverage. The studied item was white wine, of which the consumption is less popular and more selective in France than red wine. We assume that there is information asymmetry among consumers, which means that close to the area of production, they may have become more familiar with a given wine, rather than those living far from this area. Hence, the survey was carried out in two different regions, namely Burgundy and Auvergne. The first is famous for its wines, while the second is not known for its wine production. The sampling was thus divided between a local region and a far-off region regarding the area of wine production. 300 consumers were recruited, half in Auvergne, half in Burgundy, on the basis of one criterion: to answer spontaneously white burgundy

wine when asked about their wine consumption. The final size of dataset was: 109 respondents in Auvergne and 113 in Burgundy at the end stage of the consumer survey.

The first step of the survey involved a household self-report of purchasing behaviour of white wine, indicating the items bought during the three months preceding the survey. This self-report indicated the quantity and the diversity of purchases, and the frequency of buying directly from the wine makers, which is a marker of high personal involvement in the choice making process, the use of other distribution channels was also documented. The respondents were then invited in the research i stitutes' p e ises to a s e a written questionnaire including twenty-two questions on key dimensions of product-oriented knowledge: processing, semantic and geography, all related to the relevant category of food product, i o de to assess espo de ts' a a e ess o the gi e i e. One additional section of the questionnaire was devoted to the usual socio-demographic descriptors of the respondents.

The main part of the questionnaire focused on time or spatial dimensions of the knowledge on processing, harvesting, wine-aki g, la elli g… Fo i sta e, o e of the p o essi g-oriented uestio s as Blending white and red wines is allowed for rosé wine-making, which one?

[Champagne rosé, osé d'A jou, rosé de Provence, do 't k o ] (correct response is underlined).

Semantic knowledge relating to general culture on wine, including wording and naming, was

Measurement and results

The distribution of ratings issued from the above-mentioned coding of knowledge level according to wine indicates that geography about wine is the dimension of knowledge most shared among the respondents, whereas the processing-related dimension for the studied products is more discriminating. General culture of products (semantic knowledge) is in medium position. As the respondents were wine consumers, being aware that the survey will focus on white wine, those interested in white wine consumption, are slightly more represented within the sampling, compared to the usual or casual consumers. This may imply that the proportion of respondents showing a high level of processing-related knowledge may be higher than expected. However, the targeted category Chardonnay, was never quoted by interviewers during the recruitment process, nor during the questionnaire stage, in order not to introduce bias in responses.

It is well-known that consumers feel charged with a mission and modify their responses when they know precisely the item for which they are being observed. According to the literature, we may call experts those respondents with a high level of knowledge and novices those showing a low level of knowledge. As the level of knowledge is divided into three dimensions, namely processing-related, semantic or geographical, the classification of respondents into experts versus novices will not be fully reliable per se and needs to be refined by means of clustering analysis.

K-means clustering was then used in order to better explain the diversity of knowledge displayed by the respondents according to the selected products. This method segments respondents into clusters according to their level in various types of knowledge. K-means clustering is a non-hierarchical clustering procedure: objects are assigned into a user-specified number of clusters. Four significant segments of respondents were identified by means of this method of classification. Each cluster is described by its relative positioning according to the level of knowledge in each dimension (see figure 1).

Figure 1. Percentage of right answers per cluster, see appendix for caption

The clusters are identified according to the level of knowledge and described according to purchase behaviour and socio-demographic characteristics of respondents. As expected, the distance between cluster Expert and cluster Novice is maximal as they are at the extreme positioning within the clustering (see figure 2). The two in-between clusters were named Knowledgeable and Connoisseur.

Processing-related knowledge and Geographical knowledge discriminate the clusters well, but Semantic knowledge acts to a lower extent (see table 1). Table 1 shows that the more complex the kind of knowledge, the higher is its discriminating power between respondents, and vice-versa, the more general the knowledge, the lower is the difference between respondents. In our case, processing-related knowledge is highly discriminating, whereas semantic one is less. An asymmetry effect is shown in figure 2, as the type of knowledge does not take away Expert and Novice luste s' responses with the same intensity. More complex is the knowledge of wine (processing-related and geographical knowledge), lower is the score of Novice cluster. While semantic knowledge, more available for everybody, is acting with symmetry between luste s' scores.

The clusters were cross-tabulated with the other data collected and some significant relationships were identified. For the cluster 1, so-called Expert, the main explaining factors are: self-statement of respondent as well aware about wine, using direct sale as a purchasing channel, region of residence (experts are more often from Burgundy), readings on oenology, level of stocks of wine from Burgundy, diversity of regions in the own wine cellar, number of bottles of white wine in the own The description of the clusters shows that the members of cluster Expert are older, are more often male and have higher income rather than the average of respondents. The intermediate clusters are well discriminated by means of the living region of the respondents: cluster 2, so-called Knowledgeable, counts respondents mainly from Auvergne, while those from cluster 3, so-called Connoisseur, are more frequent in Burgundy. The cluster 4, so-called Novice is at the opposite situation of class 1 Expert: younger, more female and low level of income.

Conclusion

The conclusion of this survey confirms the initial assumption: providing information increases information asymmetry between consumers. Providing more information fits well with information processing of those who are highly involved in wine consumption, here cluster Expert. On the other hand, it may lead to some cognitive overload for those who are not highly interested by wine consumption, here cluster Novice. The first cluster may be more cognitive-driven with respect to wine consumption, as wine is a complex and amazing story, while the last cluster may be more affect-oriented, as wine is simply pleasant, or not, to drink.

Efficient advertising and communication plans devoted to wine should take into account these refinements when targeting consumers. While knowledge-based economy states that information asymmetry between consumers and producers may be reduced by providing information available, it was shown in the present study that there are various types of consumers with different needs in quantity and type of information depending on their prior knowledge. Processing-related knowledge does not address the demand of information from usual or casual consumers, while it is worth to highlight for connoisseurs or experts. On the other hand semantic or geographical information, such as wording, naming, labelling or branding, would be better affordable for less involved and less aware consumers and will better address their expectations, which are not so focused but still worth considering. The worse would be to provide information without any clear target or focus, apart from providing information per se! This practice will lead for sure to fuelling the cognitive overload of consumers by means of an undifferentiated flow of information. This tendency would probably increase the information asymmetry between the consumers.

Interestingly, the results of the present study indicate that, among the respondents, the clusters Expert and Novice a e ope ati g a d f uitful atego ies he e plai i g o su e s' k o ledge related to wine. However these extreme categories do not fully document the wide spectrum of replies collected. The intermediate clusters Knowledgeable and Connoisseur have to be considered as promising medium categories in order to avoid a binary analysis with loss of variety. In the case of France, where the culture of wine is still vivid, the multidimensional aspects of wine knowledge should not be forgotten.

However, the present findings focus only on one country and one broad category of wine; they should be cross-compared with other countries and/or more precise wine categories in order to be enhanced. Self-estimation of wine knowledge could help to discriminate experts from novices but would not be reliable enough for solid clustering, although it fits well with the knowledge of the extreme classes of respondents. When the objective is to reach sufficient reliability of measurement for cluster analysis, self-reported purchases are useful as a validation dataset. Self-estimation of wine knowledge and self-reporting of wine purchases might be considered as an interesting trade-off for measurement of consumer knowledge with respect to wine, as the cost of this data collection is low.

  assessed ith uestio s su h as What is a vintage wine? A wine [older than 10 years, made from a single harvest, coming from a famous vineyard, do 't k o ] . To evaluate the geographical k o ledge, o e of the uestio s as What is the main grape variety used for making Côte Rotie wine? [Cabernet-Sauvignon, Syrah, Merlot, do 't k o ] . The complete list of questions is in appendix. Thus, each respondent obtained three scores on the basis of the level of knowledge shown through his/her responses among the three dimensions: processing, semantic and geography. For these dimensions an individual global score was given to each respondent.

Figure 2

 2 Figure 2. k-means clustering, final centres of clusters according to wine knowledge, N= 222

  are higher for experts.

Table 1 .

 1 ANOVA variable-cluster according to wine knowledge of respondents

	Zscore	F	Significance
	Processing-related knowledge	197.882	.000
	Semantic knowledge	80.566	.000
	Geographical knowledge	137.913	.000

This work was carried out with the financial support of the ANR -Agence Nationale de la Recherche-

The French National Research Agency under the Programme Agriculture and Sustainable Development, project ANR-05-PADD-012, Promotion du Développement Durable par les Indications Géographiques PRODDIG.

Appendix

List of questions (P: process-related knowledge, S: semantic, G: geographical) P 17: Which of these wines is made via carbonic maceration? P 1: From which grape variety is Bourgogne Passetoutgrain made? P 15: During wine-making, what is the first fermentation? P 3: What is spe ial a out i de paille ? P 19: Which of these rosé wines is a blend of white and red wines? P 2: How do you call the process of adding sugar to must during alcoholic fermentation? P 16: Is it possible to make white wine from black grapes? P 12: What is a varietal wine ? P 4: What are late harvest wines? S 8: What does VQPRD mean? S 7: What does PGI mean? S 6: What does PDO mean? S 18: Which of the following statements about varietal wine is correct? S 11: What is the shape of a Bordeaux wine bottle? S 10: What is a vintage wine? S 14: Which day is Beaujolais Nouveau released on the market? G 24: What is the particularity of appellation Château-Grillet? G 20: What is the main grape variety used for making Côte Rotie wine? G 22: Does a Bordeaux Supérieur wine label indicate a regional appellation? G 13: Does a Burgundy wine label indicate a regional appellation? G 9: What is the main grape variety used for making Burgundy white wines? G 23: From which vineyard does Châteauneuf-du-Pape come from?