Anne-Marie Desaulty

Clarisse Mariet

Philippe Dillmann

Jean-Louis Joron

Bernard Gratuze

Cecile Mahe-Le Carlier

Philippe Fluzin

TRACE ELEMENTS BEHAVIOUR IN DIRECT-AND INDIRECT-IRON METALLURGY: THE CASE OF PAYS DE BRAY (FRANCE)

Keywords: trace elements, bloomery process, indirect process, slag inclusions, provenance

In order to determine the trace element chemical signature from the ore to the slag inclusion of the ferrous artefacts in the Pays de Bray (France), 143 samples were analysed by INAA, ICP-MS and LA-ICP-MS. Results show that, for geochemical reasons, some elements must be avoided to determine the chemical signature. Nevertheless, a main part of elements initially present in the ore allows to determine the chemical signature of the Pays de Bray. This signature is significantly different from the one of other regions and can be use for further studies about the commercialisation of Pays de bray ferrous artefacts in the neighbouring regions.

INTRODUCTION

Major and trace element analysis of non metallic inclusions in iron archaeological objects are for about 10 years the topic of numerous studies dedicated to the distinguishing of ironmaking processes [START_REF] Dillmann | Diffraction X, Microdiffraction X et Microfluorescence X sous Rayonnement Synchrotron et analyses comparées pour la caractérisation des inclusions[END_REF] but also to the determining of the artefact provenance [START_REF] Paynter | Regional variations in bloomery smelting slag of the iron age and romano-british periods[END_REF][START_REF] Schwab | The provenance of iron artefacts from Manching: a multitechnique approach[END_REF][START_REF] Buchwald | Slag analysis as a method for the characterization and provenancing of ancient iron objects[END_REF][START_REF] Buchwald | The characterisation and provenancing of ore, slag and iron from the Iron Age settlement in Snorup[END_REF][START_REF] Coustures | The use of trace element analysis of entrapped slag inclusions to establish ore-bar iron links: examples from two gallo-roman iron-making sites in France (Les Martys, Montagne Noire, and Les Ferrys, Loiret)[END_REF][START_REF] Hedges | Source determination of iron currency bars through the analysis of slag inclusions[END_REF][START_REF] Devos | Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) for spatially resolved trace analysis of early-medieval archaeological iron finds[END_REF][START_REF] Chouinard | Archéologie et archéométallurgie de la forge et des forgerons de l'habitation Loyola en Guyane[END_REF]. These latter studies mainly focus on the link between objects and ores or archaeological sites. However, to our knowledge, despite of the fact that the behaviour of major elements initially present in the ores is relatively well known, none of published studies describes in detail the one from ore to the final artefact of the trace elements in the two ironmaking chains. Yet, the comprehension of trace element behaviour is crucial before any provenance studies. Indeed, it will be shown in the following that, for different reasons, some elements could not be chosen for determining a chemical signature. The Pays de Bray region is very interesting for historical, archaeological and archaeometrical studies linked to ironmaking. Indeed, iron production is attested in this area on a wide temporal range (from the protohistoric to the modern age). Moreover, since the end of the 15 th c., indirect ironmaking took place in this region [START_REF] Arribet-Deroin | Fondre le fer en gueuses au XVIe siècle. Le haut fourneau de Glinet en Pays de Bray (Normandie)[END_REF]. Several ironmaking archaeological sites in the Middle Ages, covering the transition from the bloomery to the indirect process were excavated, providing many slag and products to be analysed: le Prés de Montadet (direct, from the 1 st century BC to the 1 st century AD), le Chemin des Flots (direct, 14 th century) Glinet (indirect, 15 th -16 th century). Last but not least, the diffusion of the iron products from this area is documented by some historical sources: iron from the Pays de Bray was commercialised in the neighbouring city as Rouen and Beauvais. For this reason, with the final aim to precise the commercialisation of iron products from the Pays de Bray at different periods, a trace element study in order to reveal the chemical signature of the area and follow it in the slag inclusions of the metal was undertaken. Thus, in a first part, trace element behaviours are discussed from the geochemical point of view and a first set of trace elements will be eliminated. In the second part, the specific element behaviour in the two iron-making processes will be described. These two parts will allow to choose pairs of elements that are able to characterize the chemical signature of the Pays de Bray and discriminate among those of other areas in France (Lorraine, Basse Normandie) in slag, and slag inclusions.

METHODOLOGY AND EXPERIMENTAL TECHNIQUES

The slag, laitier and ore sample for INAA (Neutron Activation) and ICP-MS (Inductively Coupled Plasma-Mass Spectrometry) analyses (see table 4) have been crushed into powder in an agate shatter box, and sieved at 125 µm. After crushing the laitiers, the eventual cast iron prills were removed using a magnet. Iron samples for LA-ICP-MS and EDS-SEM analyses (see below) have been mounted in epoxy resin and successively polished using SiC abrasive paper (grade 80 to 4000). Microscopic observation have been performed by using an OLYMPUS optical microscope under reflected light. Bulk major element composition has been measured by Energy Dispersive Spectrometry (EDS) coupled with a Scanning Electron Microscope (SEM) at Laboratoire Pierre Süe (CEA/CNRS, Saclay, France). The use of a Si(Li) detector with a beryllium thin window and a numerical acquisition chain allows to detect and quantify relatively light elements as Oxygen with a good accuracy (several relative percents on iron oxides and glass standards). The Idefix setup from the SAMX Company was used for acquisition and quantification. This analytical system is mounted on a Cambridge Stereoscan 120 SEM. Measurements were performed with a 15kV accelerating voltage. Minor elements can be detected and quantified for content over 0.5% mass . For these content range (<1% mass ), it was considered that the relative quantification error is about 10%. For the other elements (>1 % mass ) the error is overevaluated by considering a relative error of 2%. It has to be specified that the measurement can be considered as quantitative only when the sample does not contain elements lighter than Oxygen. This is the case for all samples in this study except some ore that are mainly constituted of iron carbonates. Bulk trace element composition has been determined by INAA and ICP-MS in the Pierre Süe Laboratory.

INAA analyses have been done according to the following procedure: aliquots of about 120 mg (for ore and slag) and 50 mg (for cast iron and iron object) are wrapped in high purity aluminium foils. To monitor the neutron flux an international geochemical standard is placed together with the samples in an aluminium shuttle. The irradiation is filtered by a Cdcover to keep out thermal neutrons. All samples irradiations have been carried out at the nuclear reactor Osiris of the CEA Saclay Center (France) directly connected to Laboratoire Pierre Sue (LPS) by a closed circuit water pipe. They are irradiated under an epithermal neutron flux of 1.1•10 13 n.cm 2 .s -1 for 12 hours. After one week cooling, the samples are transferred into plastic tubes. The tubes are weighted before and after the transfer to avoid errors produced by possible losses during sample handling. Two counting sequences are carried out: (1) after one-week cooling: La, Sm, U are determined by 50000 s counting; (2) after one month cooling: Sc, Co, Ni, Rb, Cs, Ba, Hf, Ce, Eu, Yb, Th are determined by 20 000 to 60 000 s counting. The counting system used for the acquisition and the processing of the spectra consists of several high resolution γ-spectrometers. Each γ-spectrometer includes a Canberra Ge(Li) detector and a Nuclear Data ADC analyser coupled to a PC computer. Concentrations are calculated using a comparative method with international geochemical standards (GSN, BEN, ACE, Centre de Recherches Pétrographiques et Géochimiques, France; GXR4, United States Geology Survey, USA) and standard made in LPS: silica doped with trace elements. Except for Zr, it was considered that the relative quantification error is inferior to 5%. ICP-MS samples have been prepared by acid decomposition in open vessel. Measurements have been performed using a quadrupole ICP-MS spectrometer Thermo Electron X7 ICP-MS (Thermo Fisher Scientific) equipped with a concentric nebuliser. Signal optimization is obtained by using a 10 µg.g -1 solution of Be, Mg, Co, Ni, In, Ce, Ba, Pb, Bi and U. The spectrometer is optimised to provide minimal values of the ratios CeO + /Ce + and Ba 2+ /Ba + and optimum intensity of the analytes. The methods used for calibration consists of spiking aliquots of a sample with four different quantities of a mixed standard solution (method of standard additions). For all elements, it was considered that the relative quantification error is about 7%. The major elements composition of the slag inclusions entrapped in the metallic artefacts have been analysed by EDS coupled to SEM with the same conditions described above. Trace elements in slag inclusions were measured by using laser ablation in combination with Inductively Coupled Plasma-Mass Spectrometry (LA-ICP-MS) at the Centre Ernest Babelon (CNRS, Orléans).LA-ICP-MS analyses were made using a VG Plasma Quad PQXS in conjunction with Nd:YAG laser, operating at 266nm. Samples were ablated for 50 s and the diameter of the ablation crater was about 200 µm. For the calibration the international geostandard Nist 610 was used in addition to references glasses with high iron content synthesized starting from crushed slag and analyzed by INAA.

CORPUS

Different types of samples were gathered for this study: ores, slag and metallic artefacts. Hereafter will be described the different sampling contexts:

ORES FROM THE PAYS DE BRAY

Geological background

Across the end of the tertiary and beginning of the quaternary era, compressional tectonic caused the folding of the continental shield forming the Pays de Bray anticline. Consecutively, during the quaternary era, the morphological high was subjected to an intense erosion which led to the ablation of the capping limestone hard layers down to the tender layers at the core of the Pays de Bray. Such an erosional event exposed the inner geological formation (upper cretaceous, lower cretaceous and upper Jurassic). The exposed geological section on the flanks of the syncline provides several ore deposits different by enrichment and age. The geological ore source localization, together with historical documents, toponyms and traces of extractions suggest the wide exploitation of the wealdien ore (lower cretaceous) [START_REF] Arribet-Deroin | Fondre le fer en gueuses au XVIe siècle. Le haut fourneau de Glinet en Pays de Bray (Normandie)[END_REF]. Ore samples were collected in different zones of the Pays de Bray corresponding to the wealdien period on the geological map.

The morphology of the samples corresponds rather well to the description of the wealdien ores made by previous studies [START_REF] Lapparent | Mémoire pour servir à l'explication de la carte géologique détaillée de la France[END_REF][START_REF] Barbier | Une campagne de recherches minière en Seine-Inférieure[END_REF][START_REF] Mary | Etude sur l'Urgonien du Bray à Rainvilliers (Oise). ompte rendu du Congrès national des sociétés savantes[END_REF].Three types of ore can be distinguished:

-Type I: nodule surrounded by a laminated crust of a few minimeters to a few centimetres -Type II: ferruginous sandstone characterized by centimetric vacuoles, filled with sandstone of clearer colour.

-Type III: ferruginous sandstone X-rays diffraction allowed to identify the different iron phases which compose the ores (Table1).

Table1 Archaeological sites Three archaeological sites were selected for this study : two sites where the bloomery process was performed (Prés de Montadet, dated from the Proto-historical and Gallo-roman periods, and Chemin des flots, dated by 14 C between 1300 and 1445 AD excavated by Christophe Colliou [START_REF] Colliou | Le Chemin du Flot, parcelle[END_REF]) and one associated to the indirect process (Glinet, excavated by Danielle Arribet [START_REF] Arribet-Deroin | Fondre le fer en gueuses au XVIe siècle. Le haut fourneau de Glinet en Pays de Bray (Normandie)[END_REF]). At Glinet and le Prés de Montadet, ore fragments with comparable morphologies to the three geological ore types were found, sometimes in important quantities (more than 50 kg at le Prés de Montadet). The major composition of the ore have been analysed by EDS coupled to SEM (table 2).

ORES FROM OTHER GEOGRAPHICAL AREAS

In order to compare the Pays de Bray chemical signature to the one of other regions, another phosphorus ore was chosen to be analysed. This ore is the minette de Lorraine provided by Cecile Mahe-Le Carlier and Marc Leroy and containing about 2% mass phosphorus. These kinds of ore have been described in details in many publications [START_REF] Leroy | La sidérurgie ancienne en Lorraine avant le haut-fourneau. Etude du développement historique et des conditions techniques de l'utilisation du minerai oolithique lorrain (la minette) en métallurgie de réduction directe[END_REF].

SLAG FROM THE PAYS DE BRAY

Archaeological slag

As for the ore, slag have been collected in the three archaeological reference sites (i.e. Glinet, le Prés de Montadet and Chemin des flots)

Indirect process

The site of Glinet, on which a blast furnace and a refinery were excavated allow to collect typical slag of the indirect process: the laitier, a slag coming from the ore reduction stage, but also refining cakes produced during the refining of cast iron. Laitier are constituted of low iron glasses containing sometimes an important number of cast iron prills. The average major composition of these laitier is presented in table 3. Refining slag are produced during the oxidation of cast iron to remove elements as carbon, silicone ore phosphorus; this stage takes place in an open hearth and typical plano-convex cakes are produced during the operation; several tenth of these cakes were found on the site of Glinet. They are constituted of iron oxides (wüstite, FeO), silicates (fayalite, Fe 2 SiO 4 ) and phosphates and of glassy phases. In some cases metallic iron can be embedded in the slag. The average major element content of the Glinet refining cakes is presented in table 3.

Bloomery process Typical slag of the bloomery process were also collected at Prés de Montadet and Chemin des Flots. On the proto-historical zone of le Prés de Montadet, slag entrapped at the bottom of the shaft furnace was found. Moreover, in the Gallo-roman zone of this site and at Chemin des flots, typical tapped slag heaps were excavated and sampled. These types of slag are mainly constituted of iron silicates and oxides with more or less glassy phases. The average major element content of the Prés de Montadet and Chemin des Flots slag is presented in table 3. Seven slag collected in various slag heaps of Pays de Bray were also analysed.

Experimental restitution of the bloomery process

In order to obtain wastes (i.e. slag) and metallic products obtained from well known ore and conditions, experimental reductions were conducted in a furnace based on the archaeological data of le Prés de Montadet. Two experiments (XP03 and 09) used a type I geological ore found near this site. Another one (XP05) used the archaeological type I ore. Tapped and entrapped slag were collected and analysed for the XP03 and XP05.

SLAGS FROM OTHER AREAS Bloomery process

As for the ore, samples from other areas were collected to compare the chemical signatures. slag issued from experimental smelting using the minette de Lorraine. Moreover tapped and entrapped slag coming from a site situated at several 10 km from the Pays de Bray, the Merovingian site of Guichainville (Basse Normandie) were also collected and analysed. Lastly, it was also interesting to analyse some slag coming from a totally different geological context, the French Pyrénées, and coming from the site of Castel-Minier (medieval period) excavated by Florian Tereygeol [START_REF] Leroy | First examinations of ore, slag and iron artefacts from iron-making site of castel-minier (Aulus -les-Bains,09[END_REF]. This will allow us to verify that some trace element ratios are constant whatever the origin of the sample.

FERROUS ARTEFACTS FROM THE PAYS DE BRAY

In order to follow the trace element chemical signature in the slag inclusions entrapped in the metallic artefacts and formed during the ironmaking process, several objects were selected and analysed. Unfortunately, none of the archaeological excavated sites from the bloomery process gives ferrous artefacts. Thus, we chose to analyse the product of the experiments described in the precedent paragraph. Slag inclusions of the XP03 and XP09 blooms were analysed. Indirect process Cast iron samples from the Glinet site were collected and analysed. This ferrous alloy is a grey phosphorus cast iron with about 1-2 % mass Si and P.

FERROUS ARTEFACTS FROM OTHER AREAS

Slag inclusions of the bloom issued from experimental smelting using the minette de Lorraine were analysed.

The whole of the analyzed samples are presented in table 4.

RESULTS AND DISCUSSION

Goldschmidt stated the rules of substitution (camouflage, captures, admission) which govern the localization of the elements in traces in the mineral phases [START_REF] Vidal | Géochimie[END_REF]. These rules of substitution show us that mainly the ionic radius and the valence define the chemical behaviour of an element [START_REF] Vidal | Géochimie[END_REF][START_REF] Hagemann | Introduction à la géochimie et ses applications[END_REF]. Thus elements which have very close ionic radius and valence behave like "quasi-isotopes", their ratios remain constant. It will be thus useless to choose these couples to trace any regional chemical signature, since these ratios will be constant for any ores and the same than the one of the upper continental crust. This specific "quasi-isotope" behaviour is clearly observed on Figure 1. The Hf /Zr ratio (respectively valence: +IV, ionic radius: 0.78Å and valence: +IV, ionic radius: 0.79Å) and the Ce/La ratio (respectively valence: +III, ionic radius: 1.09Å and valence: +III, ionic radius: 1.13Å) are constant for samples coming from different regions (Pyrénées, Lorraine, Basse Normandie). Geochemistry gives other helpful indications: elements which are very sensitive to alteration, i.e. highly hydrophile (Na, Br…) and Cs, Rb, K, Sr, Ba, must be used with caution because their initial contents can be modified during the burying [START_REF] Rollinson | Using Geochemical Data: Evaluation, Presentation, Interpretation[END_REF]. The second point that has to be taken into account for studying trace elements as far as ironmaking is concerned is the influence of the thermodynamical conditions during the different processes on the element behaviour. The analysis of ores, slag and slag inclusions of experimental bloom (see table 4 (corpus)) allow to precise this behaviour in function of the two processes (i.e. the direct and the indirect ones).

(1) The bloomery process

Hereafter elements which have a strong affinity for the silicated phases are defined as lithophile. The direct process takes place at relatively low temperature (about 1300-1400°C) and the melting point of iron is not reached. Thus, the resulting bloom will still contain part of the slag in the form of inclusion. It has been observed, that the content ratio of lithophile elements remains constant from the ore to these slag inclusion, including the slag itself, such an example for Th/U ratio is given on figure 2. This is the proof that, as summarised on figure 3, the lithophile elements present in the ore are not partitioned between slag and iron, they are completely oxidized in the bloomery furnace; such elements will be concentrated in the silicate-rich waste of the operation: the slag. Elements which totally remain in the iron.

Elements which are partitioned between slag and iron.

Elements no analysed.

Fig. 3 The elements distribution after direct reduction

(2) The indirect method

The reduction stage of the indirect process takes place in a blast furnace in which temperatures are much higher than for the direct process and the melting point of the metal is attempted (i.e. T > 1534°C). Because of that, elements which totally remained in the slag during the direct process appear to be partially reduced in the blast furnace and are partitioned between the cast iron and the slag. This can be observed on figure 4, for example for Cr, V, Zn, Rb, As, Sb, Mo and Nb that are detectable in the laitier but also in the cast iron samples. Moreover, some of them as S, Mn, Cr, Ti and V form specific mineralogical phases: carbonitride for Ti, V and Cr; carbides and sulphides for Mn [START_REF] Dillmann | Diffraction X, Microdiffraction X et Microfluorescence X sous Rayonnement Synchrotron et analyses comparées pour la caractérisation des inclusions[END_REF]. Nevertheless, despite of the high temperature taking place in the blast furnace it seems that the most of the lithophile elements are not reduced during the indirect process and are not detectable (i.e. are present in quantity lower than 0.5 ppm) in the analysed cast iron samples. Figure 5 sums up all these observations. Elements which totally remain in the cast iron.

Elements which are partitioned between laitier and cast iron.

Elements no analysed.

Fig. 5 The elements distribution after indirect reduction At this stage, it appears that, for the indirect process, past all the lithophile elements coming from the ore are eliminated in the laitier during the reduction. Thus, slag and inclusions formed during refining from an oxidation of the elements constituting cast iron will contain none of these elements initially present in the ore. It implies that the reduction behaves as a filter which tends to erase the chemical signature of this ore. As a result it appears difficult to trace the indirect process from the ore to the finished object resulting from refining using trace element signature. At the opposite the filter effect of the indirect reduction looks as a powerful tool to distinguish the two processes. In fact slags obtained from direct reduction, and thus inclusions, are rich in lithophile trace elements initially present in the ore, whereas slag and inclusions resulting from the indirect refining are strongly depleted in lithophile trace elements. Therefore as it was already observed for some major elements [START_REF] Dillmann | Diffraction X, Microdiffraction X et Microfluorescence X sous Rayonnement Synchrotron et analyses comparées pour la caractérisation des inclusions[END_REF][START_REF] Dillmann | Slag inclusion analyses for studying ferrous alloys employed in French medieval buildings: supply of materials and diffusion of smelting processes[END_REF] lithophile elements enable to discriminate the two processes. Such behaviour clearly appears in refined slag from Glinet compared to other direct reduction slags from the Pays the Bray (i.e. obtained with the same type of ore) figure 6. The content differences for Y, La Sm, Yb, Th, and U, between the two types of slag are sufficient to discriminate between the processes. Recognizing the refining process on the base of trace elements is an important archaeometric task. These results open the way to further statistical studies. Figure 6 also shows the important among of the elements coming from pollution by charcoal, addings or refining hearth lining. Indeed whereas elements like Ba and Sr are completely absent in the cast iron, they are present in quantity higher than 50 ppm in the refining slag. Based on the trace element behaviour discussed in the previous paragraphs, is now possible to characterize the chemical signature of the Pays de Bray region. This signature is defined by using several element pairs selected according to the following characters: -Avoiding element pairs with a "quasi-isotope" behaviour (i.e.; which ratios are always constant). -Exclusive use of elements which pass completely into the slag during the reduction process in order to avoid misleading due to partial distribution of an element between slag and metal.

-Avoiding hydrophile elements which contents can be modified by weathering.

-Avoiding elements which could be present in calcic or sand addings during the ironmaking process (Sr, Zr.), in charcoal ashes (Sr, Rb, Ba…) or driven by pollution of the clay lining of the hearths (Ti, V…) [START_REF] Thomas | The determination of bloomery furnace mass balance and efficiency[END_REF][START_REF] Serneels | Analyse chimique des matières premières et des produits de l'opéraion de reduction ds le four basque d'agorregi[END_REF].

We thus selected the following ratios: Yb/Ce, Eu/Sm, Ce/Th, La/Sc, U/Th , Nb/Y. Yb/Ce, Eu/Sm, Ce/Th and La/Sc have good linear correlations (R=0.96, 0.99, 0.96 and 0.94 respectively). It appears that the chemical signature of the Pays de Bray can be defined using these couples, from the ore to the slag inclusions for the direct process and to the laitier for the indirect one. This result open the way to further provenance studies if the signature of the Pays the Bray can be distinguished from the one of ores coming from other regions. This point will be considered below. Nevertheless, some specific couple seems to distinguish different population groups depending on the sample location. This is the case for the U/Th ratio that shows three different well separated populations within the Pays de Bray: A first population represents ores found on the archaeological sites as well as slag and slag inclusions of the experimental blooms, but also geological ores. For this population, U and Th are linearly well correlated (y=0.42696x; R=0.9453). A second population groups the Glinet laitiers, the Prés de Montadet slag, and the various other slag collected in the Pays de Bray. For this population, U, Th are also well linearly correlated (y=1.0584x; R=0.9053). A third population groups the Chemin des flots slags. These different correlations have not been elucidated yet but can be probably explained by an initial dispersion in the ore composition. In the same way, the Nb/Y correlation also highlights different compositional groups: (i) group with the archaeological and geological ores, the slags and the slag inclusions of the experimental blooms, the Prés de Montadet slags, and the various slags collected in the Pays de Bray (y=0.1287x, R=0.9307) (ii) A group containing the Chemin des flots slags, (iii) a strongly dispersed group pertaining to the Glinet laitiers. This latter one can be explained by the fact that, as said before, Nb is an element that partly reduced during the indirect process. Despite of this fact this Nb/Y ratio was already considered because it is able to sign the Pays the Bray comparing to other regions (see below). The other groups are not elucidated yet but can be due to a slight variation of the Pays de Bray ore signature. In a last step, the Pays de Bray chemical signature was compared with other regions. First another region using a phosphorus ore as for the Pays de Bray was considered: in Lorraine, the main used ore is the "minette de Lorraine". It could be interesting to compare the trace element signature. Another interesting region is the Pays d'Evreux less than 50 km of the Pays de Bray, with the archaeological site of Guichainville. Figure 8 shows different trace elements ratios that defined well the Pays the Bray compared to the other areas. It appears that some ratios are not able (Sm/Eu) to make any distinctions and are the same for the three regions. Some other ratios (Yb/Ce, Th/U) are efficient to distinguish ores coming from the Pays the Bray from the Minette de Lorraine. Some elements pair ratios allow to distinguish well ores coming from the three areas; this is the case for Ce/Th, La/Sc and, despite of its relative variability in the pays de Bray (see before), the Nb/Y ratio.

Fig. 1

 1 Fig. 1 Trace element variation diagrams for the pays de Bray and other regions: (a) Hf versus Zr contents; (b) Ce versus La contents

Fig. 2

 2 Fig. 2 Th versus U contents for XP03 ores, slag and slag inclusions

Fig. 4

 4 Fig. 4 Distribution of trace elements between the "laitier" and cast iron (Site of Glinet).

Fig. 6

 6 Fig. 6 Distribution of trace elements between the refining slag and reduction slag

  Figure 7 shows (a) Yb versus Ce contents; (b) Eu versus Sm contents; (c) Ce versus Th contents ; (d) La versus Sc contents; (e) U versus Th contents; (f) Nb versus Y for various samples of the Pays de Bray.

Fig. 7

 7 Fig. 7 Trace element content diagrams for the pays de Bray area

Fig. 8

 8 Fig. 8 Trace elements variation diagrams for the Pays de Bray (a) Yb versus Ce contents; (b) Eu versus Sm contents; (c) Ce versus Th contents; (d) La versus Sc contents; (e) U versus Th contents; (f) Nb versus Y contents

  : Constitutive phases of the ores

	Type	Type I		Type II	Type III
	Localization Nodule	Crust	Phase 1	Phase 2
	Iron oxides Siderite FeCO 3 Goethite FeOOH Quartz SiO 2	Quartz SiO 2	Quartz SiO 2
				Goethite FeOOH	Goethite FeOOH	Goethite FeOOH

Table 2 :

 2 Average major element content of the different ores

	Average	Type I	Type II	Type III	Type I	Type II	Type III
	content	Glinet	Glinet	Glinet	Prés de	Prés de	Prés de
	% mass				Montadet	Montadet	Montadet
	O	47	50	46	22	28	26
	Mg	0.6	0.1	0.1	0.8	0.1	0.1
	Al	2.2	1.2	1.1	1.2	1.8	3.4
	Si	5.4	13	9.6	3.2	7.2	31
	P	0.5	0.8	1.0	0.1	0.1	0.3
	S	0.4	0.3	0.1	0.2	0.2	0.2
	K	0.4	0.4	0.9	0.2	0.2	0.5
	Ca	1.1	0.5	0.7	0.8	0.1	0.1
	Mn	0.6	0.4	1.0	1.0	0.2	0.1
	Fe	42	33	40	70	62	38

Table 3 :

 3 Average major element content of the different archaeological wastes (EDS coupled to SEM)

	Average	Laitiers	Refining	Slag	Slag
	content	Glinet	slag	Prés de	Chemin
	% mass		Glinet	Montadet	des Flots
	MgO	0.9	0.3	0.1	0.1
	Al 2 O 3	7.7	0.5	3.5	10
	SiO 2	63	9.8	26	29
	P 2 O 5	0.8	10	0.6	2.3
	SO 3	0.7	1.1	0.2	0.1
	K 2 O	1.7	0.7	0.6	1.8
	CaO	17	3.6	0.9	1.0
	MnO	0.8	0.7	1.0	0.9
	FeO	7	72	66	54

Table 4 :

 4 Samples collected for the present study

	Type	Site	Number of samples	Remark
	Geological ores	Pays de Bray	3 *	Wealdien
				type I
	Archaeological ores	Prés de Montadet	12	Pays de Bray
		(Pays de Bray)
	Archaeological ores	Glinet	23	Pays de Bray
		(Pays de Bray)
	Archaeological ores	Lorraine	5	Minette de Lorraine
	Laitiers	Glinet	25	Indirect process
		(Pays de Bray)
	Refining slag	Glinet	6	Indirect process
		(Pays de Bray)
	Entrapped slag	Prés de Montadet	15	Bloomery process
		(Pays de Bray)		Proto-historical
				period
	Tapped slag	Prés de Montadet	9	Bloomery process
		(Pays de Bray)		Gallo-roman period
	Tapped slag	Chemin des Flots	13	Bloomery process
		(Pays de Bray)		Medieval period
	Entrapped slag	Lorraine	3	From minette de
				Lorraine

ACKNOLEDGEMENTS

The authors are extremely grateful to the following people for their help with this paper by providing samples: Marc Leroy, Paul Merluzzo, Christophe Colliou, Danielle Arribet-Deroin, Nolwen Zaour and Florian Tereygeol. We thank Pr Michel Treuil for its helpful discussions. Thanks also to Pr Daniele Brunelli for his help concerning the fabrication of synthesized glasses.

CONCLUSION

The aim of this study was to unravel the behaviour of the trace elements in the two ironmaking processes: direct and indirect. To address this goal, 143 representative samples from the Pays de Bray as well as other areas (the Pyrenees, Lorraine, Basse Normandie) were analyzed. Trace element analyses of ores, slag, laitiers, cast irons and iron products allow stressing the following conclusions:

Several elements ratio can not be used for trace element chemical signature, on the one hand because they are "quasi-isotopes" and have the same behaviour, on the other hand because they content in the ore are influenced by alteration.

During the indirect process only Co, Ni and Cu completely pass into cast iron. All other elements partition between laitier and cast iron. As a consequence the inclusions formed after cast iron refining are strongly depleted in lithophile trace elements. This confirms same conclusions already observed for major elements by other studies [START_REF] Dillmann | Slag inclusion analyses for studying ferrous alloys employed in French medieval buildings: supply of materials and diffusion of smelting processes[END_REF]. An important consequence is that slag inclusions, therefore, forgot the ore chemical signature. On the contrary, in the direct process, a large spectrum of trace elements strongly passes quasi exclusively from ore to slag. They are hosted in trapped slag inclusions in the final iron. The content ratio of lithophile elements remains constant from the ore to slag inclusions. For this process it is thus possible to trace the chemical signature from the ore to the final object. Moreover, the different behaviour between the two processes allows discriminating between them.

Selected pairs of trace elements (Yb/Ce, Ce/Th, La/Sc, U/Th, Nb/Y) allowed us to characterize the chemical signature of the Pays de Bray. These couples of element allow discriminating the chemical signature of different geological and archaeological regions (Pays de Bray, Lorraine, Basse Normandie).The possibility to discriminate the provenance regions opens new perspectives in the traceability of archaeological materials as, for instance, the source of the construction irons of important historical buildings in the north of France (Amiens and Rouen).


David Costantini
email: david.costantini@mnhn.fr

Paco Bustamante

Maud Brault-Favrou

Giacomo Dell'omo

Patterns of mercury

Keywords: Antioxidant, Methylmercury, Oxidative damage, Shearwater, Trophic level

Introduction

Mercury (Hg) is a highly toxic non-essential metal of major concern for wildlife and human health. Hg is released into the environment from both natural and anthropogenic sources [START_REF] Eagles-Smith | Modulators of mercury risk to wildlife and humans in the context of rapid global change[END_REF]. The UNEP estimated that the largest sources of Hg emissions from human activities are artisanal and small-scale gold mining, and coal burning (UNEP, 2018).

Global Hg emissions and concentrations in the environment have grown slightly in the period 2010-2015 because declines of commercial Hg use in the developed world have been matched by increases in Hg release into the environment by developing countries (Science for Environment Policy, 2017; [START_REF] Streets | Five hundred years of anthropogenic mercury: spatial and temporal release profiles[END_REF]. This scenario is further worsened by the capacity of Hg to spread easily across environments because it can travel long distances far from emission sources and remains in the atmosphere for up to a year (Science for Environment Policy, 2017;[START_REF] Streets | Five hundred years of anthropogenic mercury: spatial and temporal release profiles[END_REF]. In aquatic ecosystems, Hg is converted into methylmercury by microorganisms, allowing its efficient bioaccumulation and its biomagnification up the food chain. Once accumulated into the body, Hg may cause a range of adverse effects on neurobehavioural, endocrine, immune or reproductive functions [START_REF] Tan | The endocrine effects of mercury in humans and wildlife[END_REF]Whitney & Cristol, 2018). These evidences have urged the adoption of new regulations to protect human health and the environment from anthropogenic emissions and releases of Hg into the environment (e.g., the Minamata Convention on Hg, signed in 2013 and entered into force in 2017, http://www.mercuryconvention.org/). The Mediterranean basin is significantly affected by Hg contamination because geochemical processes related to volcanic activity and cinnabar deposits together with industrial and agricultural activities release large amounts of Hg into the environment [START_REF] Rajar | Mass balance of mercury in the Mediterranean sea[END_REF][START_REF] Žagar | Modelling of mercury transport and transformations in the water compartment of the Mediterranean Sea[END_REF][START_REF] Sunderland | Future trends in environmental mercury concentrations: implications for prevention strategies[END_REF][START_REF] Salvagio Manta | Fluxes and the mass balance of mercury in Augusta Bay (Sicily, southern Italy)[END_REF] Science for Environmental Policy, 2017; [START_REF] Streets | Five hundred years of anthropogenic mercury: spatial and temporal release profiles[END_REF]. Furthermore, oligotrophic conditions of the Mediterranean Sea favour the biomagnification of Hg in food webs [START_REF] Chouvelon | Oligotrophy as a major driver of mercury bioaccumulation in medium-to high-trophic level consumers: A marine ecosystemcomparative study[END_REF]. Mediterranean marine organisms are therefore exposed to high risks of Hg contamination. Piscivorous birds are particularly prone to accumulate Hg because of the biomagnification [START_REF] Eagles-Smith | Modulators of mercury risk to wildlife and humans in the context of rapid global change[END_REF]Whitney & Cristol, 2018). Levels of Hg in seabirds breeding in Mediterranean islands are actually significantly higher than those recorded in seabirds from Atlantic islands (e.g., [START_REF] Renzoni | Comparison between concentrations of mercury and other contaminants in eggs and tissues of Cory's shearwater Calonectris diomedea collected on Atlantic and Mediterranean islands[END_REF][START_REF] Ramos | The influence of breeding colony and sex on mercury, selenium and lead levels and carbon and nitrogen stable isotope signatures in summer and winter feathers of Calonectris shearwaters[END_REF].

The Environmental Quality Standards Directive (2008/108/EC modified by Directive 2013/39/EU) of the European Union determined quality standards for Hg levels in both water and fish in order to protect piscivorous animals from its toxic effects. In piscivorous animals, a long-term accumulation may lead to chronic effects. Once accumulated in the body, Hg may affect fundamental fitness traits, such as reproduction and lifespan (e.g. [START_REF] Tartu | To breed or not to breed: endocrine response to mercury contamination by an Arctic seabird[END_REF][START_REF] Tartu | Age-related mercury contamination and relationship with luteinizing hormone in a longlived antarctic bird[END_REF][START_REF] Tartu | Corticosterone, prolactin and egg neglect behavior in relation to mercury and legacy POPs in a long-lived Antarctic bird[END_REF][START_REF] Tartu | Mercury exposure, stress and prolactin secretion in an Arctic seabird: an experimental study[END_REF] with negative consequences at the population level [START_REF] Evers | Adverse effects from environmental mercury loads on breeding common loons[END_REF]Goutte et al., 2014a,b). The number of studies that addresses the mechanistic underpinnings of Hg effects on marine animals is, however, still limited (Whitney and Cristol, 2018). There is a need for more studies that assess the effects of Hg on wildlife, both at individual and population level, in order to understand fully its impact on ecosystems. A growing body of evidence indicates that Hg exposure induces molecular oxidative damages and changes in antioxidant molecules in a number of seabird species [START_REF] Henny | Nineteenth century mercury: hazard to wading birds and cormorants of the Carson River, Nevada[END_REF][START_REF] Hoffman | Subchronic effects of methylmercury on plasma and organ biochemistries in great egret nestlings[END_REF][START_REF] Hoffman | Mercury and drought along the lower Carson River, Nevada: III. Effects on blood and organ biochemistry and histopathology of snowy egrets and black-crowned night-herons on Lahontan reservoir, 2002-2006[END_REF][START_REF] Costantini | Oxidative stress in relation to reproduction, contaminants, gender and age in a long-lived seabird[END_REF][START_REF] Gibson | A positive correlation between mercury and oxidative stress-related gene expression (GPX3 and GSTM3) is measured in female double-crested cormorant blood[END_REF]. The hypothesis that exposure to Hg is linked to increased oxidative stress is also supported by direct biochemical evidences. The underlying mechanisms of action of Hg involve (i) toxic effects via bonding to sulfhydryl groups of proteins and impairment of the glutathione system and (ii) formation of reactive oxygen species mainly via Fenton reaction, finally producing mutagenic and carcinogenic end-products of oxidative stress cascade [START_REF] Koivula | Metal-related oxidative stress in birds[END_REF][START_REF] Jomova | Advances in metal-induced oxidative stress and human disease[END_REF].

In this study, our aims were to (i) describe the individual and temporal variation in blood concentration of total Hg (THg), (ii) identify the factors that affect Hg exposure (parental identity, trophic level and foraging area) and (iii) determine the individual-and populationlevel connections between Hg and blood-based markers of oxidative status in chicks of Scopoli's shearwaters (Calonectris diomedea) breeding on the island of Linosa in the southern Mediterranean. The island lies in the Strait of Sicily at ca. 250 km from Augusta bay, which is one of the most contaminated areas with Hg in the Mediterranean Sea [START_REF] Salvagio Manta | Fluxes and the mass balance of mercury in Augusta Bay (Sicily, southern Italy)[END_REF]. In addition, we also analysed the correlation between blood Hg and body condition index of chicks because Hg may cause loss of energetic stores (e.g., fat) in young birds [START_REF] Scheuhammer | The chronic toxicity of aluminium cadmium, mercury, and lead in birds: A review[END_REF]. Prior work in Linosa carried out in 1982 showed that the Hg concentration in eggs (mean ± SD) was 5.90 ± 1.45 µg g -1 dw and in tissues collected from adults varied from 0.87 ± 0.17 µg g -1 dw in fat to 86.16 ± 99.16 µg g -1 dw in liver [START_REF] Renzoni | Comparison between concentrations of mercury and other contaminants in eggs and tissues of Cory's shearwater Calonectris diomedea collected on Atlantic and Mediterranean islands[END_REF]. These concentrations were high compared to those recorded in Atlantic shearwaters [START_REF] Renzoni | Comparison between concentrations of mercury and other contaminants in eggs and tissues of Cory's shearwater Calonectris diomedea collected on Atlantic and Mediterranean islands[END_REF]. However, there are no recent data on Hg exposure, nor on the potential effects on the physiological conditions in birds. We carried out the study on chicks at fledging age because (i) the impact of Hg on the organism may be especially relevant when contamination occurs in early life, (ii) they provide a valuable snapshot of the local contamination because they are fed by parents that forage in the vicinity of the breeding colony (e.g., [START_REF] Carravieri | Trace elements and persistent organic pollutants in chicks of 13 seabird species from Antarctica to the subtropics[END_REF] without any interference of Hg accumulated by adult shearwaters in the wintering areas of West Africa [START_REF] Müller | Individual consistency and sex differences in migration strategies of Scopoli's shearwaters Calonectris diomedea despite year differences[END_REF] and (iii) the influence of Hg exposure within the egg is negligible owing to the growth dilution effect [START_REF] Ackerman | Bird mercury concentrations change rapidly as chicks age: toxicological risk is highest at hatching and fledging[END_REF].

Materials and Methods

Study species and study area

The Scopoli's shearwater is a pelagic seabird that breeds in the Mediterranean. Scopoli's shearwaters consume a wide range of prey including pelagic fish (e.g., sardines), planktonic crustaceans, fish larvae and discards from fisheries [START_REF] Grémillet | Irreplaceable area extends marine conservation hotspot off Tunisia: insights from GPS-tracking Scopoli's shearwaters from the largest seabird colony in the Mediterranean[END_REF][START_REF] Cianchetti-Benedetti | Interactions between commercial fishing vessels and a pelagic seabird in the southern Mediterranean Sea[END_REF]. In Linosa, the fishing areas of adults are located near the island [START_REF] Cianchetti-Benedetti | Interactions between commercial fishing vessels and a pelagic seabird in the southern Mediterranean Sea[END_REF]. Recent work on the Linosa population showed little concern of exposure to several persistent organic pollutants for the conservation status of this shearwater population [START_REF] Costantini | Individual variation of persistent organic pollutants in relation to stable isotope ratios, sex, reproductive phase and oxidative status in Scopoli's shearwaters (Calonectris diomedea) from the Southern Mediterranean[END_REF]. However, populations of the Scopoli's shearwater are estimated to be in slow decline (www.iucnredlist.org). BirdLife International (2015) provided an estimate of 30,500-48,100 pairs for the European population. Defos du [START_REF] Defos Du Rau | Reassessment of the size of the Scopoli's Shearwater population at its main breeding site resulted in a tenfold increase: implications for the species conservation[END_REF] estimated the presence of 141,000-223,000 breeding pairs in the whole Mediterranean basin. Scopoli's shearwaters have an estimated lifespan of ca. 25 years [START_REF] Fransson | EURING list of longevity records for European birds[END_REF] and start breeding when they are around 5 years old [START_REF] Thibault | Cory's shearwater[END_REF]. Shearwaters lay one egg in May and chicks leave the colony at around the end of October. We carried out the fieldwork on the island of Linosa (35°52' N, 12°52' E) in the Sicily Channel (Figure 1). All adults and chicks have been tagged with permanent metal rings since 2007. Each nest was also tagged with a permanent number and georeferenced; the maximum linear distance between nests included in the present study was ca. 750 m (Figure 1).

Sampling

Blood samples were collected from chicks near fledging in the period 27 September to 10 October 2016, 4 to 14 October 2017, and 30 September to 12 October 2018. Upon capture, we first collected a sample of venous blood (0.5 ml) from the tarsal vein and then we measured the body mass, and both the beak length and beak width of each chick. When back to the field station, within 1-2 h from blood collection, we centrifuged (4'000 rpm for 4 minutes) the tubes containing the whole blood in order to separate the serum from the erythrocytes. Both serum and erythrocytes were stored in separate tubes at -20 °C in the field station and at -80 °C in the laboratory.

Mercury

We quantified the THg (expressed as µg g -1 dw dry weight) in freeze-dried erythrocytes (aliquots ranging from 0.33 to 2.7 mg) following the protocol described in Chouvelon et al. (2009). We measured the THg in blood because prior work on several vertebrates showed that (i) it is highly and positively correlated with the blood concentration of methylmercury, (ii) most of the Hg occurs in the methylated form within the body tissues, and (iii) blood THg reflects short-term exposure and is a valuable nonlethal predictor of Hg concentrations in internal tissues (e.g., [START_REF] Skerfving | Mercury in women exposed to methylmercury through fish consumption, and in their newborn babies and breast milk[END_REF][START_REF] Thompson | The chemical form of mercury stored in south Atlantic seabirds[END_REF][START_REF] Monteiro | Seabirds as monitors of mercury contamination in the Portuguese Atlantic Ph[END_REF][START_REF] Sakamoto | Declining risk of methylmercury exposure to infants during lactation[END_REF][START_REF] Eagles-Smith | Mercury correlations among six tissues for four waterbird species breeding in San Francisco Bay, California, USA[END_REF][START_REF] Yates | Mercury in bats from the northeastern United States[END_REF]. Briefly, we used blanks and the certified reference material (CRM) TORT-3 Lobster Hepatopancreas (NRC, Canada; certified Hg concentration: 0.292 ± 0.022 µg g -1 dw) to check the analytical quality. The average value (± SD) of CRM was 0.285 ± 0.002 µg g -1 dw (n = 15) with a recovery of 98 %.

The quantification limit of the method was 0.5 ng. The average variation coefficient of samples analysed in duplicate was 0.76 %.

Stable isotopes

The isotopic niche of seabirds was used as a proxy of their ecological niche: δ 13 C values indicate the foraging habitat (inshore/benthic vs. offshore/pelagic), while δ 15 N values indicate the trophic level [START_REF] Newsome | A niche for isotopic ecology[END_REF]. An amount of 0.30 ± 0.05 mg of dried erythrocytes were weighed in tin cups for stable isotope analyses. Carbon and nitrogen stable isotope ratios were measured using an elemental analyzer (Flash 2000, Thermo Scientific, Milan, Italy) coupled with a Delta V Plus with a Conflo IV interface (Thermo Scientific, Bremen, Germany).

Values were expressed in the δ unit notation as parts per mille (‰) deviation from standards (Vienna Pee Dee Belemnite for δ 13 C and N2 in air for δ 15 N) following the formula: δ 13 C or δ 15 N = [(Rsample/Rstandard) -1] × 10 3 , where R is 13 C/ 12 C or 15 N/ 14 N, respectively. The analytical imprecisions were < 0.10 ‰ for carbon and < 0.15 ‰ for nitrogen.

Oxidative status markers

We measured four markers of oxidative status following previous protocols (e.g., [START_REF] Costantini | Loss of integration is associated with reduced resistance to oxidative stress[END_REF]. Briefly, we measured the concentration of protein carbonyls (PCs, marker of protein oxidative damage) in serum using the Protein Carbonyl Colorimetric assay (Cayman Chemical Company, Ann Arbor, MI, USA); the activity of the antioxidant enzyme glutathione peroxidase (GPx) in erythrocytes using the Ransel assay (RANDOX Laboratories, Crumlin, UK); the activity of the antioxidant enzyme superoxide dismutase (SOD) in erythrocytes using the Ransel assay (RANDOX Laboratories, Crumlin, UK); the serum non-enzymatic antioxidant capacity (OXY) against the hypochlorous acid (HOCl, oxidant naturally generated in cells) using the OXY-Adsorbent test (Diacron International, Grosseto, Italy). We used the Bradford protein assay (Bio-Rad Laboratories, Hercules, CA, USA) with albumin as a reference standard to quantify the concentration of proteins in serum and erythrocytes. Quality controls were included in all assays performed. Values of markers were expressed as nmol/mg proteins for PCs, units/mg proteins for both GPx and SOD, mmol/l of HOCl neutralized for OXY.

Statistical analyses

All analyses were carried out using SPSS Version 23 (Armonk, NY, USA). First, we carried out a model selection to identify the best-fitting linear mixed model to Hg concentration data from a set of candidate models using the Akaike Information Criterion (AIC). We used AIC values unadjusted for sample size because these provided very similar results to AIC values adjusted for sample size (data not shown). Models were first ranked according to AIC values.

We then calculated ΔAIC and Akaike weights. The ΔAIC is the difference of each model AIC value relative to the AIC of the best model. A ΔAIC < 2 suggests substantial evidence for the model [START_REF] Burnham | Model selection and multimodel inference: a practical information-theoretic approach[END_REF]. Akaike weights are obtained by transformation of AIC values and indicate the probability that the model is the best among all candidate models [START_REF] Burnham | Model selection and multimodel inference: a practical information-theoretic approach[END_REF]. THg values were square-root transformed to achieve a normal distribution of model residuals. As fixed factors, we considered the sampling year and the sampling date to control for any among and within year variation in environmental parameters that might have affected the condition of chicks, δ 13 C and δ 15 N. The female identity was included as a random factor in all tested models (similar results were obtained if the male or the nest identify was included instead of female identity). In preliminary analyses, we also entered into the models the factors longitude and latitude of the nest or quadratic terms for latitude and longitude to account for nonlinear relationships and spatial autocorrelation [START_REF] Legendre | Spatial autocorrelation: trouble or new paradigm?[END_REF]. All factors were not significant, thus they were not further considered in the final analyses. We then tested correlations between Hg and all markers of oxidative status by using Pearson correlation coefficient (similar results are obtained using square-root transformed Hg values). Finally, we tested the correlation between THg and body condition index (body mass normalised by body size index obtained from principal components analysis, explained below) using partial correlation. To do so, body mass was included in the partial correlation model as dependent variable, and both THg concentration and body size index as independent variables. This approach enabled to measure the strength and direction of the relationship between Hg and body mass whilst controlling for the effect of body size. The body size index was estimated using the PC1 from a PCA (60.5 and 56.7 % of variance for males and females, respectively) on beak length and beak width for males (loadings: 0.75) and females (loadings: 0.78), separately. These analyses on body condition index were carried out for males and females, separately, because they differed in both body mass and body size metrics.

Results

The concentration of THg in erythrocytes averaged 0.81 µg g -1 dw and varied from 0.23 to 4.29 µg g -1 dw (median = 0.72 µg g -1 dw, Table 1). One chick was excluded from the following statistical analyses because it was an influential outlier owing to a very high concentration of Hg (4.29 µg g -1 dw). The best-fitting model included sampling year and δ 15 N as main predictors (Table 2); no other models were within 2 ΔAIC of the top model. The THg concentration was significantly higher in 2018 than in both 2016 and 2017, while it was similar between 2016 and 2017 (Table 3). Chicks that were fed upon higher trophic level prey (i.e., higher δ 15 N values) had higher THg levels (Table 3, Figure 2). The maternal contribution was not significant (Wald = 0.01, P = 0.99), indicating that the variation in THg among related nestlings born in different years from the same mother was similar to that among unrelated nestlings. We found a similar result when the model was restricted to only those females that contributed a nestling in each of the three study years (data not shown). Neither the δ 13 C values nor the sex explained the among chick variation in blood THg because models including these two factors were not supported (Table 2).

We found significant correlations between THg and protein carbonyls (r = 0.18, p = 0.004), GPx (r = -0.17, p = 0.005) or OXY (r = -0.13, p = 0.021) using individuals sampled in all three years. Blood THg and SOD were not significantly correlated (r = 0.06, p = 0.19). All correlations were no longer significant (p ≥ 0.21) when we used z-score transformed data to remove any effect owing to among year variation in both THg and marker values. Finally, we found that males with higher values of body condition index had also higher blood THg (partial correlation, r = 0.18, p = 0.04), while we did not find any correlation between blood THg and body condition in females (partial correlation, r = -0.03, p = 0.78). When we used z-score transformed values of Hg, body mass, and body size, the correlation between body condition index and THg in males was even stronger (partial correlation, r = 0.28, p = 0.004), while we did not find any correlation between blood THg and body condition in females (partial correlation, r = 0.06, p = 0.56).

Discussion

THg variation and comparison with other studies on seabirds

We found large variation among chicks and sampling years in the blood concentration of THg.

We sampled the chicks around three months after hatching (phase of asymptotic growth), thus we can reasonably exclude a significant contribution of Hg excreted by mothers into the single egg they lay because prior work on the related Cory's shearwater (Calonectris borealis)

showed that chicks have a fast excretion rate mainly owing to body and plumage growth (growth dilution effect, [START_REF] Monteiro | Seabirds as monitors of mercury in the marine environment[END_REF]. Cory's shearwater chicks may, for example, excrete around 40-60 % of the Hg intake through the plumage (Monteiro and Furness, 2001b). Also, Hg has a half-time in blood of around six days owing to storage in internal tissues and excretion from the body (Monteiro and Furness, 2001b). The exponential phase of growth in chick Scopoli's shearwaters occurs in the first two months of life, thus it was ended a few weeks before we collected the blood samples. This suggests that the tHg concentrations reflected mainly the current exposure through the food owing to a modest excretion capacity.

Prior work showed that levels of Hg in eggs of Scopoli's shearwaters collected in three Mediterranean islands (Crete, Linosa, and Majorca) in the period 1982-1984 were 2.5-3.5 times higher than those recorded in eggs of Cory's shearwaters collected from Selvagen island in the Atlantic Ocean [START_REF] Renzoni | Comparison between concentrations of mercury and other contaminants in eggs and tissues of Cory's shearwater Calonectris diomedea collected on Atlantic and Mediterranean islands[END_REF]. Hg values in the three Mediterranean stations were quite homogeneous. Hg concentrations of all body tissues analysed were also higher in the Mediterranean stations than in the Atlantic. In particular, the liver Hg levels in Scopoli's shearwaters were four to six times higher than those in Cory's shearwaters [START_REF] Renzoni | Comparison between concentrations of mercury and other contaminants in eggs and tissues of Cory's shearwater Calonectris diomedea collected on Atlantic and Mediterranean islands[END_REF]. Similarly, [START_REF] Ramos | The influence of breeding colony and sex on mercury, selenium and lead levels and carbon and nitrogen stable isotope signatures in summer and winter feathers of Calonectris shearwaters[END_REF] found that the concentration of Hg in feathers collected from Mediterranean shearwaters in 2001 was significantly higher than in those collected from Atlantic shearwaters in the same year.

THg blood levels in shearwater chicks may be considered as moderate to high if compared to levels detected in other seabirds, some of which living in areas highly contaminated with Hg (e.g. [START_REF] Sebastiano | High levels of mercury and low levels of persistent organic pollutants in a tropical seabird in French Guiana, the Magnificent frigatebird, Fregata magnificens[END_REF][START_REF] Sebastiano | Trophic ecology drives contaminant concentrations within a tropical seabird community[END_REF][START_REF] Carravieri | From Antarctica to the subtropics: contrasted geographical concentrations of selenium, mercury, and persistent organic pollutants in skua chicks (Catharacta spp.)[END_REF][START_REF] Carravieri | Trace elements and persistent organic pollutants in chicks of 13 seabird species from Antarctica to the subtropics[END_REF]. Blood THg concentrations in Scopoli's shearwaters were (i) higher than those recorded in blood of chicks of Larus canus in north Germany [START_REF] Kahle | Bird blood as bioindicator for mercury in the environment[END_REF], Catharacta skua on Shetland and Western Isles [START_REF] Bearhop | Bioamplification of mercury in great Skua Catharacta skua Chicks: the influence of trophic status as determined by stable isotope signatures of blood and feathers[END_REF], Hydropogne caspia in San Francisco bay, California [START_REF] Eagles-Smith | Mercury correlations among six tissues for four waterbird species breeding in San Francisco Bay, California, USA[END_REF], seven seabird species on Machias Seal Island in Canada (e.g., Sterna paradisaea, Fratercula arctica, Somateria mollissima; [START_REF] Bond | Mercury concentrations in seabird tissues from Machias Seal Island, New Brunswick, Canada[END_REF], Sula nebouxii on Sinaloa in the Gulf of California [START_REF] Lerma | Lead, cadmium and mercury in the blood of the blue-footed booby (Sula nebouxii) from the coast of Sinaloa, Gulf of California, Mexico[END_REF], four tern species and Leucophaeus atricilla in French Guiana [START_REF] Sebastiano | Trophic ecology drives contaminant concentrations within a tropical seabird community[END_REF], Catharacta maccormicki on Adélie land [START_REF] Carravieri | From Antarctica to the subtropics: contrasted geographical concentrations of selenium, mercury, and persistent organic pollutants in skua chicks (Catharacta spp.)[END_REF] and seven seabird species on Adélie land, Mayes island, Possession island and Amsterdam island (e.g., Aptenodytes forsteri, Pagodroma nivea, Halobaena caerulea, Eudyptes chrysocome; [START_REF] Carravieri | Trace elements and persistent organic pollutants in chicks of 13 seabird species from Antarctica to the subtropics[END_REF], (ii) similar to those in blood of chicks of Himantopus mexicanus in San Francisco bay, California [START_REF] Eagles-Smith | Mercury correlations among six tissues for four waterbird species breeding in San Francisco Bay, California, USA[END_REF], Fregata magnificens in French Guiana [START_REF] Sebastiano | High levels of mercury and low levels of persistent organic pollutants in a tropical seabird in French Guiana, the Magnificent frigatebird, Fregata magnificens[END_REF][START_REF] Sebastiano | Trophic ecology drives contaminant concentrations within a tropical seabird community[END_REF] and five seabird species on Possession island and Amsterdam island (e.g., Aptenodytes patagonicus, Thalassarche carteri; [START_REF] Carravieri | Trace elements and persistent organic pollutants in chicks of 13 seabird species from Antarctica to the subtropics[END_REF] and (iii) lower than those in blood of chicks of Catharacta lonnbergi on Kerguelen, Crozet and Amsterdam island [START_REF] Carravieri | From Antarctica to the subtropics: contrasted geographical concentrations of selenium, mercury, and persistent organic pollutants in skua chicks (Catharacta spp.)[END_REF] and Diomedea amsterdamensis on Amsterdam island [START_REF] Carravieri | Trace elements and persistent organic pollutants in chicks of 13 seabird species from Antarctica to the subtropics[END_REF]. Finally, levels of blood THg of shearwater chicks were also comparable to those recorded in adults of two seabird species from Arctic and Antarctica that were likely high enough to interfere with major endocrine mechanisms regulating reproductive behaviour (black-legged kittiwake Rissa tridactyla in Svalbard: [START_REF] Tartu | To breed or not to breed: endocrine response to mercury contamination by an Arctic seabird[END_REF][START_REF] Tartu | Mercury exposure, stress and prolactin secretion in an Arctic seabird: an experimental study[END_REF]; snow petrel Pagodroma nivea in Antarctica: [START_REF] Tartu | Age-related mercury contamination and relationship with luteinizing hormone in a longlived antarctic bird[END_REF][START_REF] Tartu | Corticosterone, prolactin and egg neglect behavior in relation to mercury and legacy POPs in a long-lived Antarctic bird[END_REF]. This large among species variation in blood THg appears to be mainly explained by geographic/spatial factors rather than trophic niche and also by unidentified characteristics of the species themselves (e.g., capacity of excretion, life-history), which deserve comparative models to be unraveled [START_REF] Carravieri | Trace elements and persistent organic pollutants in chicks of 13 seabird species from Antarctica to the subtropics[END_REF].

Sources of Hg contamination

Scopoli's shearwaters consume a wide range of prey [START_REF] Grémillet | Irreplaceable area extends marine conservation hotspot off Tunisia: insights from GPS-tracking Scopoli's shearwaters from the largest seabird colony in the Mediterranean[END_REF] including discards from fisheries [START_REF] Cianchetti-Benedetti | Interactions between commercial fishing vessels and a pelagic seabird in the southern Mediterranean Sea[END_REF]. Accordingly, we found moderate variation in δ 15 N among chicks; values ranged from 7.4 to 9.6 ‰ and the chick excluded from statistical analyses because of its very high THg concentration (i.e., outlier) also had the highest δ 15 N value. Chicks had values of δ 15 N similar to those recorded particularly in sardines, but also in squids and anchovies in Mediterranean [START_REF] Cardona | The role of sardine as prey for pelagic predators in the western Mediterranean Sea assessed using stable isotopes and fatty acids[END_REF][START_REF] Albo-Puigserver | Trophic structure of pelagic species in the northwestern Mediterranean Sea[END_REF], suggesting that shearwaters were feeding on these particular prey. Prior work showed that sardines from the Sicily channel had Hg levels significantly higher than in sardines from other areas of the Mediterranean [START_REF] Copat | Heavy metals concentrations in fish from sicily (Mediterranean Sea) and evaluation of possible health risks to consumers[END_REF][START_REF] Brambilla | Mercury occurrence in Italia seafood from the Mediterranean Sea and possible intake scenarios of the Italian coastal population[END_REF][START_REF] Llull | Mercury concentrations in lean fish from the Western Mediterranean Sea: dietary exposure and risk assessment in the population of the Balearic Islands[END_REF]. Our results show that the trophic level of prey estimated by δ 15 N values was significantly related to blood THg concentrations of chicks. This finding is in agreement with prior work, which showed that the blood concentration of THg is significantly linearly related to its intake in both chick and adult Cory's shearwaters (Monteiro and Furness, 2001a,b). Our results are also in line with work on other seabirds. [START_REF] Bearhop | Bioamplification of mercury in great Skua Catharacta skua Chicks: the influence of trophic status as determined by stable isotope signatures of blood and feathers[END_REF] found that some variation in blood THg of chick great skuas was explained by dietary specialisation because chicks that were fed upon higher trophic level prey had higher levels of THg in blood (correlation between blood THg and δ 15 N: Shetland, r = 0.48; Western Isles, r = 0.46). Similarly, chicks with higher δ 15 N values had higher blood THg in six seabird species in French Guiana (r ≥ 0.44, [START_REF] Sebastiano | Trophic ecology drives contaminant concentrations within a tropical seabird community[END_REF]. In the Kerguelen islands community, species-related Hg variations in chick feathers of 21 seabird species were highly and positively linked to δ 15 N values [START_REF] Blévin | Wide range of mercury contamination in chicks of Southern Ocean seabirds[END_REF]. This highlights the occurrence of efficient Hg biomagnification processes within marine trophic webs.

δ 13 C values (range: -18.5 to -19.6 ‰) showed little variation, indicating that the foraging range of adult shearwaters was rather small. It is therefore not surprising that the levels of THg were not explained by the foraging area, as values of δ 13 C were not associated with blood THg concentrations. In a previous GPS-tracking study of the same population, we actually found that adults go fishing mostly in the south of the island with a maximum linear distance of 75 ± 44 km (mean ± SD; [START_REF] Cianchetti-Benedetti | Interactions between commercial fishing vessels and a pelagic seabird in the southern Mediterranean Sea[END_REF]. This result further supports the assumption that our data reflect local exposure.

Finally, we did not detect any differences in THg levels between male and female chicks. Similar results were obtained in previous work on adult Cory's shearwaters (Monteiro and Furness, 2001a). However, while males in better body condition had higher blood THg concentrations, the body condition of females was not correlated to their blood THg concentrations. We do not know the reasons for this difference between sexes. For example, if males receive more food than females, we might expect them to be more exposed to THg. Prior work on chick Cory's shearwaters (Monteiro and Furness, 2001b) did not find any effect of Hg administration on body condition; however, males and females were pooled together in the statistical models. This sex effect deserves further investigation.

Hg and blood oxidative status

We found little evidence for any possible short-term effects of Hg exposure on blood-based oxidative status markers. We found a positive correlation between the blood concentration of THg and protein oxidative damage and negative correlations between blood THg and two antioxidant markers (GPx and OXY). These results would indicate that chicks suffer higher oxidative stress when being exposed to higher Hg. However, these correlations were affected by variation among sampling years in the mean values of these metrics. This result suggests little evidence for any possible short-term effects of Hg on the oxidative status at individual level. However, we cannot rule out the possibility that the among-year variation would indicate an effect of Hg on oxidative status at population level. This might be possible if (i) the withinyear variation in THg was not large enough to induce visible effects or (ii) other environmental factors that also affect the oxidative status varied among years. These hypotheses deserve further work to be elucidated.

The correlation between THg and markers at population level are in agreement with the literature. Prior work on other seabirds found that the activity of GPx decreased with increased Hg in double-crested cormorants Phalacrocorax auritus [START_REF] Henny | Nineteenth century mercury: hazard to wading birds and cormorants of the Carson River, Nevada[END_REF], great egrets Ardea alba [START_REF] Hoffman | Subchronic effects of methylmercury on plasma and organ biochemistries in great egret nestlings[END_REF], and snowy egrets Egretta thula [START_REF] Hoffman | Mercury and drought along the lower Carson River, Nevada: III. Effects on blood and organ biochemistry and histopathology of snowy egrets and black-crowned night-herons on Lahontan reservoir, 2002-2006[END_REF]; the expression of the antioxidant genes GPx-3 and glutathione S-transferase µ3 increased with Hg exposure in female double-crested cormorants [START_REF] Gibson | A positive correlation between mercury and oxidative stress-related gene expression (GPX3 and GSTM3) is measured in female double-crested cormorant blood[END_REF]; an increase of lipid oxidative damage was also related to high blood concentrations of Hg in wandering albatrosses Diomedea exulans [START_REF] Costantini | Oxidative stress in relation to reproduction, contaminants, gender and age in a long-lived seabird[END_REF].

Conclusions

We found large within-and among-year variation in the blood concentration of THg of chicks, but not between male and female chicks. THg levels may be considered as moderate to high if we compare them to those detected in other seabirds. The correlations between blood THg and markers of oxidative status or individual body conditions indicate little concern for short-term harmful effects. In our population, most hatched chicks (> 90 %) survive until fledging, thus they appear to tolerate these levels of Hg. However, we cannot exclude that any possible effects might emerge later in life.

Prior work on other seabirds showed that adults had levels of blood THg from 3 to 14 times higher than those recorded in their nestlings (e.g., [START_REF] Bearhop | Bioamplification of mercury in great Skua Catharacta skua Chicks: the influence of trophic status as determined by stable isotope signatures of blood and feathers[END_REF][START_REF] Sebastiano | Trophic ecology drives contaminant concentrations within a tropical seabird community[END_REF]. If this is also the case for Scopoli's shearwaters in our study area, taking the average of 0.81 µg g -1 dw as a reference for chicks, we might expect adults to have levels of blood THg ranging from ca. 2.43 to a very high concentration of 11.34 µg g -1 dw. This indicates that it will be important in future work to determine the THg levels of adults and if these are harmful to their physiological or reproductive functions.

Finally, it has been predicted that the new EU regulations would have caused a reduction of the total mass of Hg in the Mediterranean in 2020 by about 3-12 % compared to the levels measured in 2005 [START_REF] Rajar | Mass balance of mercury in the Mediterranean sea[END_REF]. Although in vicinity of our study area, there are still active significant sources of anthropogenic Hg [START_REF] Salvagio Manta | Fluxes and the mass balance of mercury in Augusta Bay (Sicily, southern Italy)[END_REF], it will be of interest to verify if Hg exposure in shearwaters follows such expectations. Given the widespread diffusion of the Scopoli's shearwater in the Mediterranean, easy access to nests, fidelity of the birds to the breeding site and its habits of wintering in West Africa [START_REF] Müller | Individual consistency and sex differences in migration strategies of Scopoli's shearwaters Calonectris diomedea despite year differences[END_REF], it could be used for a prolonged biomonitoring of Hg exposure along a geographic and latitudinal gradient.

supported the fieldwork and the costs for laboratory analyses. The research was carried out with moral responsibility for the animals, trying to minimize the disturbance to the nest and the birds. No nest desertions were observed due to our operations. We also thank C. Churlaud from the Plateforme Analyses Elémentaires of LIENSs laboratory for her support in Hg analyses and G. Guillou from the Plateforme Analyses Isotopiques of LIENSs laboratory for running the stable isotope analyses. Thanks are due to the CPER (Contrat de Projet Etat-Région) and the FEDER (Fonds Européen de Développement Régional) for funding the IRMS and the AMA of LIENSs laboratory. The Institut Universitaire de France (IUF) is acknowledged for its support to PB as a Senior Member.

Figure 1 .

 1 Figure 1. Distribution of nests on Linosa island. Birds breed inside crevices in the lava

Figure 2 .

 2 Figure 2. Relationships between blood Hg concentrations and the stable isotope ratios δ 13 C and δ 15 N. The regression line is shown only when the relationship was statistically significant.

Figure 3 .

 3 Figure 3. Relationships between population-level blood Hg concentrations and three markers

Table 1 .

 1 Descriptive statistics of mercury, stable isotopes and markers of oxidative status reported for each sampling year, separately. Values are shown as mean ± standard error; sample sizes are reported between brackets.

	2016	2017	2018

Acknowledgements

Fieldwork was carried out under the authorizations issued by the Regione Siciliana, Assessorato Agricoltura e Foreste with letter Prot. 17233 dated 1 December 2010 and subsequent communication Prot. 2452 dated 01 February 2018. We thank two anonymous reviewers for providing comments on our manuscript, Prof. B. Massa from Palermo University for institutional and personal support and V. Roatti for assistance in the fieldwork. Ornis italica