E Vandenberghe

S Rouesne

A Urvoy

R Ambard

Chosson

M Le Saux

J C Brachet

V Vandenberghe

E Rouesne

S Urvoy

A Ambard

R Chosson

Effect of a pre-oxide on the high temperature steam oxidation of Zircaloy-4 andM5 alloys

Keywords: Zircaloy-4, M5Framatome, oxidation in steam at high temperature, pre-oxide, LOCA

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

Introduction

In some hypothetical accidental scenarios in nuclear Pressurized Water Reactors (PWR), such as LOss of Coolant Accidents (LOCA), fuel cladding tubes made of zirconium alloys can be exposed for a few minutes to steam at High Temperature (HT, up to 1200°C) before being cooled and then quenched in water by the emergency core cooling systems. During in-service operation, an oxide layer appears on the outer surface of the cladding due to corrosion by the water environment (155 bar, ~300-340°C at the cladding surface, with Li, B and H2). The thickness of this outer pre-oxide layer can reach several tens of microns after several years depending on the alloy and the in-reactor exposure conditions. An oxide layer may also form on the cladding inner surface due to chemical-physical interaction with the fuel.

A review of the data available in the literature has shown that a pre-existing oxide layer may have an influence on the subsequent oxidation of the material under steam at HT. Most of these data are summarized in Table 1 for Zircaloy-4 (Zy-4) and M5Framatome † alloys, pre-oxidized outof-pile or in PWR. The results show that the oxidation in steam at HT is delayed in the presence of a pre-existing oxide layer. The effect of the pre-oxide seems to depend on the alloy and the conditions of both pre-oxidation and oxidation at HT. In most cases, the thicker the pre-oxide, the larger the delay in the oxidation at HT. However, some results show that the protective effect of the pre-oxide in terms of weight gains deteriorates beyond a given pre-oxide thickness, varying from one study to another (10 µm according to [START_REF] Leistikow | Kinetics and morphology of isothermal steam oxidation of Zircaloy-4 at 700-1300 C, Report KFK 2587[END_REF], between 30 and 80 µm according to [START_REF] Baek | Steam oxidation of Zr-1.5Nb-0.4Sn-0.2Fe-0.1Cr and Zircaloy-4 at 900-1200°C[END_REF] or 40 µm according to [START_REF] Guilbert | Influence of a pre-oxide layer on oxygen diffusion and on postquench mechanical properties of Zircaloy-4 after steam oxidation at 900[END_REF] [START_REF] Guilbert | Effect of pre-oxide on Zircaloy-4 high temperature steam oxidation and post-quench mechanical properties[END_REF]), probably due to the differences in pre-oxidation conditions. Nevertheless, the weight gain at HT of the pre-oxidized material never exceeds (or only slightly) that of the non-pre-oxidized material.

With the exception of a few specific conditions (post-breakaway oxidation at temperatures typically lower than 1050°C [START_REF] Leistikow | Oxidation Kinetics and Related Phenomena of Zircaloy-4 Fuel Cladding Exposed to High Temperature Steam and Hydrogen-Steam Mixtures under PWR Accident Conditions[END_REF] [START_REF] Billone | Cladding Embrittlement during Postulated Loss-of-Coolant Accidents[END_REF][16] [START_REF] Portier | Influence of Long Service Exposures on the Thermal-Mechanical Behaviour of Zy-4 and M5M5™ Alloys in LOCA Conditions[END_REF] [START_REF] Vandenberghe | Sensitivity to Chemical Composition Variations and Heating/Oxidation Mode of the Breakaway Oxidation in M5 Cladding Steam Oxidized at 1000°C (LOCA Conditions), TopFuel 2012[END_REF], oxidation under high steam pressures [START_REF] Park | Pressure effects on high temperature steam oxidation of Zircaloy-4[END_REF] [START_REF] Saux | Influence of Steam Pressure on the High Temperature Oxidation and Post-Cooling Mechanical Properties of Zircaloy-4 and M5™ Cladding (LOCA Conditions)[END_REF] or secondary hydriding due to steam starvation inside the cladding after ballooning and burst [START_REF] Stuckert | QUENCH-LOCA program at KIT on secondary hydriding and results of the commissioning bundle test QUENCH-L0[END_REF] [START_REF] Brachet | Study of secondary hydriding at high temperature in zirconium based nuclear fuel cladding tubes by coupling information from neutron radiography/tomography, electron probe micro analysis, micro elastic recoil detection analysis and laser induced breakdown spectroscopy microprobe[END_REF]), as-received zirconium alloys do not absorb a significant amount of hydrogen when oxidized in steam at HT [26]. Hydrogen pickup during oxidation in steam at HT is not provided in most papers dealing with the oxidation of pre-oxidized zirconium alloys. The scarce data available (see Table 1) show that the pre-oxidized materials may † M5Framatome is a registered trademark of Framatome or its affiliates, in the USA or other countries.

Preprint submitted to Journal of Nuclear Materials 3 absorb hydrogen during HT oxidation while the non-pre-oxidized materials oxidized under the same conditions do not.

The mechanical behaviour of the cladding during and after cooling from HT can be modified by oxidation and hydrogen [START_REF] Billone | Cladding Embrittlement during Postulated Loss-of-Coolant Accidents[END_REF] [START_REF] Portier | Influence of Long Service Exposures on the Thermal-Mechanical Behaviour of Zy-4 and M5M5™ Alloys in LOCA Conditions[END_REF][27] [START_REF] Yueh | Loss of Coolant Accident Testing Round Robin, TopFuel 2013[END_REF][29] [START_REF] Turque | Mechanical Behavior at High Temperatures of Highly Oxygen-or Hydrogen-Enriched α and Prior-β Phases of Zirconium Alloys[END_REF]. Thus, the effect of a pre-existing oxide layer on the oxidation kinetics and the hydrogen pickup of the cladding material during subsequent exposure under steam at HT is an important issue.

This work aims at improving both experimental databases and knowledge on the effect of a preexisting oxide layer on the oxidation of Zr alloys under steam at HT. The influences of the alloy, the pre-oxidation condition, the pre-oxide thickness and the condition of oxidation at HT are investigated. The materials and the experimental procedures used for this study are described in Section 2. The results are presented in Section 3 and further discussed in Section 4. Part of the results has already been reported elsewhere [START_REF] Brachet | Hydrogen Content, Preoxidation, and Cooling Scenario Effects on Post-Quench Microstructure and Mechanical Properties of Zircaloy-4 and M5™ Alloys in LOCA Conditions[END_REF] [START_REF] Saux | Influence of Pre-Transient Oxide on LOCA High Temperature Steam Oxidation and Post-Quench Mechanical Properties of Zircaloy-4 and M5™ cladding, 2011 Water Reactor Fuel Performance Meeting[END_REF].

Materials and experimental procedures

Materials and conditions of pre-oxidation

The experiments were made on stress-relieved annealed low tin Zy-4 and fully recrystallized M5Framatome cladding tube samples, with an outer diameter and a thickness of 9.5 and 0.57 mm, respectively. The nominal chemical compositions of the materials are given in weight % (wt%) in Table 2. Samples (2 to 7.5 mm-long) were pre-oxidized up to 2160 days in various conditions: ambient air at 550°C, steam at 415°C, static water at 340-360°C with a typical PWR primary water chemistry, flowing water at 350°C with typical PWR primary water thermal-hydraulic and chemical environment. The purpose of this parametric study was to investigate the sensitivity to pre-oxidation conditions and thus to pre-oxide microstructure, in order to improve the understanding of the effect of a pre-oxide layer on HT oxidation and eventually to provide insights for assessing the effect of a pre-oxide formed in reactor. Preoxide thicknesses were deduced from the measured weigh gain values by considering that one micron of zirconia corresponded to a surface weight gain per unit surface area of 0.15 mg/cm 2 . Pre-oxide thicknesses ranged from 3 to 60 µm. The samples absorbed more or less hydrogen during pre-oxidation depending on the conditions. Hydrogen contents at the end of pre-oxidation were either measured by using an inert gas fusion thermal conductivity (hot extraction) technique (on the samples then oxidized at HT or on equivalent samples, including the oxide layers) or estimated from known correlations between weight gain and hydrogen Preprint submitted to Journal of Nuclear Materials 4 pickup established from data obtained on other Zy-4 and M5Framatome samples pre-oxidized in the same conditions. Pre-oxidation conditions and hydrogen contents of the tested samples are given in Table 3 for Zy-4 and Table 4 for M5Framatome. Hydrogen contents are related to the total mass of the samples. The slight hydrogen pickup measured for samples pre-oxidized in air at 550°C may be due to humidity in air.

Optical microscopy observations of cross-sections (perpendicular to the tube sample axis) of a selected number of samples confirmed that the pre-oxide layers were homogeneous, similar on the inner and outer surfaces (with the exception of the samples pre-oxidized in flowing water in the Reggae loop, as anticipated) and their thicknesses were consistent with those expected. These observations also showed that the metal/pre-oxide interface was scalloped (undulations with microscale amplitude and wavelength). The microstructure of the pre-oxide layers depended on their thickness and the conditions under which they were formed. The preoxide layers of a few microns thick appeared dense (i.e. low porosity) and adherent to the metal on optical micrographs. Networks of cracks parallel to the pre-oxide surface (called circumferential cracks) were observed within thicker pre-oxides (Fig. 1). In pre-oxide layers of several tens of microns thick (> 30 µm typically), cracks perpendicular to the pre-oxide surface (called radial cracks) were evidenced in the outer part of the layer (they did not seem to reach the metal/oxide interface). These circumferential and radial cracks were more numerous and open in pre-oxides formed in air at 550°C than in layers formed in water at 340-360°C (Fig. 1).

Oxidation in steam at high temperature

Oxidation tests were performed in pure steam (no carrier gas) at atmospheric pressure at 850, 900, 1000 and 1200°C, with holding times between 55 and 9500s depending on the oxidation temperature. The conditions of oxidation in steam at HT tested for each pre-oxidation condition are given in Table 3 for Zy-4 and Table 4 for M5Framatome. Some of the samples were oxidized on both their outer and inner surfaces (two-side oxidation); in that case, 20 to 40 mm-long samples were used. Other samples, 32 to 40 mm-long, were oxidized on their outer surface only (one-side oxidation). To prevent steam from coming into contact with the cladding inner surface, non-pre-oxidized end caps in Zircaloy-4 were welded under secondary vacuum at both extremities. The oxidation of end-caps was included in the weight gain measurements (the surface of end caps represented approximately 35% of the total surface of the specimen exposed to steam). In the tested conditions, end-caps are expected to oxidize at a Preprint submitted to Journal of Nuclear Materials 5 faster rate than the cladding sample. As a consequence, the weight gains reported for the onesided oxidized specimens probably overestimate the actual weight gain of the cladding samples, in particular for the longest oxidation times. The oxidation behaviour was probably affected in the immediate vicinity of the welds but no detrimental effect was expected on the overall oxidation of the sample. Post-oxidation observations have shown that oxidation was uniform along the cladding sample (maximal variation of the oxide thickness of a few percent typically), including near the end-caps. It cannot be excluded that hydrogen pickup was promoted in the vicinity of the welds. However, it is supposed that the hydrogen potentially absorbed at sample ends during oxidation at HT did not have time to diffuse very far, excepting eventually for the longest oxidation times tested. Thus, the results of one-side oxidation tests are potentially less accurate for the longest oxidation times tested, nonetheless without questioning the main conclusions.

Oxidation at HT was carried out by using two facilities respectively called DEZIROX 1 and CINOG HP [START_REF] Portier | Influence of Long Service Exposures on the Thermal-Mechanical Behaviour of Zy-4 and M5M5™ Alloys in LOCA Conditions[END_REF][21] [23][27]. Both one-side and two-side oxidations were performed in DEZIROX 1. Only two-side oxidation tests were carried out in CINOG HP. For one-side oxidation, the sample hanged from an alumina holder via a hole in the sample upper end-cap.

For two-side oxidation, the bottom end of the sample was positioned on an alumina holder allowing free access of steam to both inner and outer surfaces of the sample. Steam was produced from an electrode-type steam generator in DEZIROX 1 and from demineralized water in CINOG HP. Steam flow was directed down through an alumina chamber in DEZIROX 1 and a quartz-tube chamber in CINOG HP. The steam flow rate normalized to the cross-sectional area of the steam chamber was about 70 mg/(cm 2 •s) in DEZIROX 1 and 140 mg/(cm 2 •s) in CINOG HP, which was sufficient to avoid steam starvation [START_REF] Kawasaki | Oxidation of Zircaloy-4 under High Temperature Steam Atmosphere and Its Effect on Ductility of Cladding[END_REF] [START_REF] Leistikow | Oxidation Kinetics and Related Phenomena of Zircaloy-4 Fuel Cladding Exposed to High Temperature Steam and Hydrogen-Steam Mixtures under PWR Accident Conditions[END_REF]. The samples were heated to the desired temperatures by using a vertical resistive furnace in DEZIROX 1 and an induction coil in CINOG HP.

In DEZIROX 1, the samples were inserted into the furnace once it was tuned and stabilized at the target isothermal temperature and flushed with steam for at least 1 hour. The power to the furnace was controlled via a built-in thermocouple. The set-point temperature was fixed before testing using a rod equipped with type S (PtRh-Pt) thermocouples, inserted at the same position as the sample into the furnace. Furthermore, temperature benchmarks are regularly performed on specimens equipped with type S thermocouples spot-welded on their outer surface. The samples reached the target temperature approximately 30s after their introduction into the furnace, for all temperatures tested. The oxidation time commenced when the sample Preprint submitted to Journal of Nuclear Materials 6 was placed into the furnace. After isothermal holding, the samples were directly quenched in water down to room temperature.

In the CINOG HP facility, the specimens were heated at 20°C/s up to the set-point oxidation temperature without any temperature overshoot. The outer temperature of the samples was measured in the region near their mid-length using two optical pyrometers. One pyrometer was used to monitor the temperature (feedback to the furnace power) and the other one was used to check the temperature. The oxidation time commenced when the oxidation temperature was reached. After the HT oxidation, the specimens were cooled down by shutting off the induction heating. The achieved cooling was quite fast (cooling rate between 100 and 150°C/s down to 500°C) but significantly slower than during a direct water quench.

The absence of temperature overshoot was verified for both facilities. Temperature variation around the target temperature was lower than ±5°C. Temperature gradient was lower than 10°C along the samples oxidized in DEZIROX 1 and along the 5 mm-long central region of the samples oxidized in CINOG HP.

As partly shown in [START_REF] Saux | Influence of Steam Pressure on the High Temperature Oxidation and Post-Cooling Mechanical Properties of Zircaloy-4 and M5™ Cladding (LOCA Conditions)[END_REF], it was checked beforehand, by performing comparative tests on nonpre-oxidized materials, that the results obtained in the two facilities are comparable.

Sample dimensions and weight were measured before and after testing with an accuracy of 10 -3 mm for diameter and thickness, 10 -2 mm for length and 0.1 mg for weight.

Characterizations after oxidation at high temperature

Hydrogen content measurements were performed by an inert gas fusion thermal conductivity technique (HORIBA EMGA-821 and LECO RH404 analysers) on samples extracted from a select number of samples oxidized at HT. Two to four hydrogen content measurements were performed per sample, on pieces generally cut from the middle part of the sample. The oxide layers were not removed before hydrogen analysis so that the reported hydrogen contents are related to the total mass of the samples. The hydrogen content absorbed during oxidation at HT, , was deduced from the difference between average hydrogen contents measured after and before oxidation, in weight ppm (wppm). The hydrogen content of the samples two-side oxidized was divided by a factor two approximately (ratio between the cladding outer diameter and the sum of its inner and outer diameters) to be compared to the hydrogen content of one-sided oxidized samples, assuming that hydrogen absorption is the same through the outer surface and the inner surface. Indeed, the outer and inner oxide layers showed rather similar aspect and thickness. In a few cases, negative hydrogen pickup values were obtained.

Preprint submitted to Journal of Nuclear Materials 7

They did not reveal desorption of hydrogen during the treatment at HT a priori; they were rather due to the uncertainties on the hydrogen contents before oxidation at HT: these contents were sometimes estimated from measurements performed on other dedicated samples, and besides it is possible that hydrogen was not uniformly distributed within some of the samples.

The hydrogen pickup fraction, , has been defined as the ratio of the hydrogen absorbed by the material over the total hydrogen generated during the oxidation. It was calculated as follows:

=

where and are the atomic masses of oxygen and hydrogen respectively, is the total mass of the oxidized sample and is its mass before oxidation. This is a mean (integrated) value of the fraction of hydrogen absorbed from the beginning of oxidation, not an "instantaneous" value.

Several layers can be observed after cooling in the samples pre-oxidized then oxidized at HT: a ZrO2 oxide layer (remaining pre-oxide and/or oxide formed at HT), a metallic layer of oxygen-stabilized αZr phase (hexagonal close-packed structure) called αZr(O) containing 2 to 7 wt% of oxygen, and a prior-βZr layer (with a typical Widmanstätten or parallel-lath structure morphology resulting from the transformation of the βZr phase, with a body-centered cubic structure, into the αZr phase, during quenching) or a two-phase αZr + prior-βZr layer depending on the oxidation temperature. The microstructure and the thickness of the different layers were observed by microscopy on polished transverse cross-sections obtained from the middle part of selected samples that had been oxidized at HT (Fig. 2). One should keep in mind that the observations were made after cooling down to room temperature. They were not necessarily representative of the state of the oxide at HT, before cooling: the oxide can be damaged during cooling, under the effect of internal stresses generated from phase transformations of the metal and the oxide, and from differences between the thermal coefficients of the different phases/layers constituting the oxidized cladding material [START_REF] Saux | An approach to study oxidation-induced stresses in Zr alloys oxidized at high temperature[END_REF].

Optical microscopy was used to measure the oxide thickness (measurement uncertainty of a few microns). When the thickness of the oxide layer was expected to be low, a nickel coating was applied before imbedding and polishing the sample on its inner and outer surfaces in order to stabilize the oxide layer and minimize the end effects during polishing. As already Preprint submitted to Journal of Nuclear Materials 8 done in [START_REF] Saux | Influence of Steam Pressure on the High Temperature Oxidation and Post-Cooling Mechanical Properties of Zircaloy-4 and M5™ Cladding (LOCA Conditions)[END_REF] and [START_REF] Brachet | Hydrogen Content, Preoxidation, and Cooling Scenario Effects on Post-Quench Microstructure and Mechanical Properties of Zircaloy-4 and M5™ Alloys in LOCA Conditions[END_REF], Scanning Electron Microscopy (SEM) (mainly in back scattered electron mode) and a specific image analysis procedure were used to measure the fractions of αZr(O) and prior-βZr across the cladding wall thickness. In the following, the so-called αZr(O) phase layer corresponds to the "continuous" αZr(O) phase layer containing more than 90% of αZr(O). The measurements of oxide and αZr(O) phase thicknesses were performed at two to four azimuthal locations per sample.

The oxygen concentration profile across the wall thickness of a selected number of samples was quantified by Electron-Probe Micro Analysis (EPMA) (CAMECA SX100 electron microprobe) with a typical accuracy of ±0.1 wt%. The measured data were corrected from surface contamination.

Results

Results obtained in the same conditions on the non-pre-oxidized materials have already been partly presented in previous papers [20][21][23][25]

[27] [START_REF] Saux | Influence of Pre-Transient Oxide on LOCA High Temperature Steam Oxidation and Post-Quench Mechanical Properties of Zircaloy-4 and M5™ cladding, 2011 Water Reactor Fuel Performance Meeting[END_REF]. The facility used and the type of oxidation applied (one-side or two-side) are specified in Table 3 for Zy-4 and Table 4 for M5Framatome. The oxidation conditions applied on the preoxidized materials were similarly applied on the non-pre-oxidized materials.

Oxidation at 850°C

After 600s at 850°C, the samples with 3-8 µm-thick pre-oxides formed in static water at 360°C or 5 µm-thick pre-oxides formed in air at 550°C have lower weight gains than the nonpre-oxidized materials (Fig. 3). The thicker the pre-oxide layer the lower the weight gain. In the case of Zy-4, the weight gains of samples with 5 µm-thick pre-oxides formed in air at 550°C were higher than those expected for the same pre-oxide thickness formed in water at 360°C, and a significant hydrogen pickup was observed (about 100 wppm, ≈ 30% while it was negligible or very low for the as-received material or the material with 8 µm-thick preoxides formed in water at 360°C. In the case of M5Framatome, weight gains were close, whether the pre-oxide have been formed under air at 550°C or under water at 360°C, and no hydrogen was measured.

Oxidation at 900°C

Fig. 4 shows the results obtained after 6000s at 900°C together with those reported in [10][11]. It must be recalled that for this study hydrogen analyses were performed on samples including the oxide layers while the oxide layers had been sandblasted in [10][11]. After

Preprint submitted to Journal of Nuclear Materials 9 oxidation at HT and quenching, hydrogen is expected to be mainly located in the prior-βZr layer; αZr(O) and oxide layers contain only a small amount of hydrogen [START_REF] Brachet | Study of secondary hydriding at high temperature in zirconium based nuclear fuel cladding tubes by coupling information from neutron radiography/tomography, electron probe micro analysis, micro elastic recoil detection analysis and laser induced breakdown spectroscopy microprobe[END_REF]. Thus, the hydrogen contents measured by keeping the oxide layers may slightly underestimate the hydrogen content within the metal. The weight gain decreases with increasing the pre-oxide thickness, up to about 40 µm (Fig. 4). The weight gains obtained for 4-28 µm-thick pre-oxide layers formed under water at 340-360°C and under O2 at 425°C were close to one another.

However, the material with 7 µm-thick pre-oxides formed under O2 at 425°C showed a slightly higher weight gain than the one with 8 µm-thick pre-oxides formed under water at 360°C.

While no significant hydrogen pickup was measured for as-received Zy-4 after 6000s at 900°C, the pre-oxidized material absorbed hydrogen, more or less depending on the pre-oxide thickness and the conditions in which it was formed (Fig. 4). According to Guilbert et al.'

results [START_REF] Guilbert | Effect of pre-oxide on Zircaloy-4 high temperature steam oxidation and post-quench mechanical properties[END_REF], the hydrogen pickup reduced to the outer surface (i.e. hydrogen pickup oxidation divided by approximately a factor two in the case of two-side oxidation) was about 230 wppm for the material with 7 µm-thick pre-oxides formed under O2 at 425°C, then it decreased with increasing the pre-oxide thickness, up to about 40 µm. However, the hydrogen pickup fraction remained almost the same (17-18%). This suggests that the decrease in hydrogen pickup as a function of the pre-oxide thickness was related to the delay in the oxidation kinetics caused by the presence of a pre-oxide layer. The hydrogen pickup was larger when the pre-oxidation was carried out under O2 at 500°C [START_REF] Guilbert | Influence of a pre-oxide layer on oxygen diffusion and on postquench mechanical properties of Zircaloy-4 after steam oxidation at 900[END_REF] due to a faster oxidation rate, as has almost the same value.

The amount of hydrogen absorbed at 900°C was three times lower for samples with 8 µmthick pre-oxides formed under water at 360°C than for samples with 7 µm-thick pre-oxides formed under O2 at 425°C. Very low or negligible hydrogen pickup was observed after 6000s at 900°C in the case of Zy-4 with 18 and 28 µm-thick pre-oxides formed water at 350-360°C.

The results reported in [START_REF] Guilbert | Influence of a pre-oxide layer on oxygen diffusion and on postquench mechanical properties of Zircaloy-4 after steam oxidation at 900[END_REF] and [START_REF] Guilbert | Effect of pre-oxide on Zircaloy-4 high temperature steam oxidation and post-quench mechanical properties[END_REF] showed that the weight gains increase with increasing the pre-oxide layer thickness beyond 40 µm when it was formed under O2 at 425 or 500°C, and the material absorbed a large amount of hydrogen (for example, ≈ 70% for 64 µm-thick pre-oxides formed under O2 at 425°C).

Preprint submitted to Journal of Nuclear Materials 10

Oxidation at 1000°C

After oxidation shorter than about 5000s at 1000°C, weight gains at a given oxidation time were lower for samples pre-oxidized under water at 340-360°C than for the non-pre-oxidized samples, as shown in Fig. 5 for Zy-4 and Fig. 6 for M5Framatome. The thicker the pre-oxide (up to 59 µm for Zy-4 and to 10 µm for M5Framatome) the larger the benefit. The benefit was larger for Zy-4 than for M5Framatome, for pre-oxide thicknesses from 3 to 10 µm formed under water at 340-360°C: weight gains obtained at a given oxidation time were similar for the two alloys when pre-oxidized while Zy-4 oxidized faster than M5Framatome in the absence of a pre-oxide.

Like for the non-pre-oxidized materials, no significant hydrogen uptake was observed for the materials pre-oxidized in water at 340-360°C oxidized during less than 5000s at 1000°C, with the exception maybe of Zy-4 with 8 µm-thick pre-oxides which has absorbed a few tens of wppm of hydrogen after 1800s, corresponding to 8% of the total hydrogen generated by the oxidation reaction (Fig. 5). A larger hydrogen pickup (~200 wppm when reduced to the outer surface, ≈ 14%) was reported in [START_REF] Guilbert | Effect of pre-oxide on Zircaloy-4 high temperature steam oxidation and post-quench mechanical properties[END_REF] after 1800s at 1000°C for Zy-4 with 7 µm-thick pre-oxide layers formed in O2 at 425°C. No significant hydrogen pickup was evidenced after 400 or 1800s at 1000°C for M5Framatome with 3-5 µm-thick pre-oxides formed under water at 360°C (Fig. 6). Guilbert et al. [START_REF] Guilbert | Effect of pre-oxide on Zircaloy-4 high temperature steam oxidation and post-quench mechanical properties[END_REF] observed for Zy-4 pre-oxidized in O2 at 425°C an increase of weight gains and hydrogen uptakes after 900 or 1800s at 1000°C when increasing the preoxide thickness beyond 40 µm ( reached nearly 100% in the case of 6 µm-thick preoxides). However, the results presented herein did not show any significant hydrogen pickup after 400 or 1800s at 1000°C for Zy-4 with 59 µm-thick pre-oxide layers formed under water at 360°C.

After 200s at 1000°C, the weight gain was largely lower for Zy-4 with 50 µm-thick preoxides formed in air at 550°C than for the non-pre-oxidized material, but the first one has absorbed hydrogen (about 70 wppm, ≈ 44%) while the second one has not. After 400s at 1000°C, the presence on Zy-4 of a pre-oxide layer of 5, 25 or 50 µm formed under air at 550°C induced a decrease in weight gain comparable to that measured for pre-oxides formed under water at 340-360°C, but this was associated with a significant hydrogen pickup (> 100 wppm, corresponding to values of 18% for 5 µm-thick pre-oxides and 42-43% for 25 and 50 µm-thick pre-oxides).

Preprint submitted to Journal of Nuclear Materials 11 After 1800s at 1000°C, for both Zy-4 and M5Framatome, weight gains of the materials preoxidized under air at 550°C were slightly lower in the presence of a 25 µm-thick pre-oxide layer than without pre-oxide, but they were close from each other for 5 and 50 µm-thick preoxides, while weight gains were significantly lower when pre-oxides were formed under water at 340-360°C or O2 at 425°C. In the case of the materials pre-oxidized in air at 550°C, a hydrogen pickup of several hundreds of wppm was measured for Zy-4 after 1800s at 1000°C (Fig. 5), while the hydrogen pickup was significantly lower for M5Framatome (about 90 wppm i.e. ≈ 5% for 50 µm-thick pre-oxides; Fig. 6). The hydrogen content absorbed by Zy-4 pre-oxidized under air at 550°C increased in the course of oxidation. Hydrogen pickup fractions after 1800s at 1000°C were approximately 7, 27 and 17% for pre-oxide thicknesses of 5, 25 and 50 µm, respectively. They were nearly two times lower than after 200 or 400s at this temperature.

The lower weight gains measured in some cases when the material was pre-oxidized was due to a delayed growth of the new oxide layer at HT in the presence of a pre-existing oxide layer.

For example, in the presence of a 10 µm-thick pre-oxide, the fresh oxide has not yet begun to grow after 400s at 1000°C. It was observed that the new oxide layer did not grow uniformly at HT, in particular during the growth of the first microns: the metal/oxide interface appeared scalloped.

In the case of Zy-4 with 59 µm-thick pre-oxides formed in water at 360°C, circumferential cracks were observed within the pre-oxide remaining after oxidation during 400s at 1000°C then cooling, as well as a few radial cracks through the oxide layer. After 1800s at 1000°C, a network of circumferential cracks more or less interconnected was evidenced at the interface between the remaining pre-oxide and the oxide formed at HT, as well as radial cracks through the entire thickness of the pre-oxide and through at least a part of the oxide formed at HT (Fig. 7). In some cases, the oxide formed at HT was thicker beneath these radial cracks.

In the case of samples with 50 µm-thick pre-oxides formed under air at 550°C, circumferential cracks together with a few radial cracks were also observed within the preoxide remaining after 200 or 400s at 1000°C. After 1800s at this temperature, connected circumferential cracks appeared at the interface between the remaining pre-oxide and the oxide formed at HT, in addition to radial cracks crossing the whole pre-oxide thickness (Fig. 7). The oxide formed at HT also seemed to be damaged.

Preprint submitted to Journal of Nuclear Materials 12

In the case of samples with 10-13 µm-thick pre-oxides formed in water at 340-350°C, oxidized at 1000°C on their outer surface only, the inner pre-oxide, exposed to vacuum, has progressively reduced during the holding at HT, until it has completely disappeared after a holding time between 420 and 1290s. The thickness of the outer pre-oxide, exposed to steam, only slightly evolved at 1000°C.

After about 5000s at 1000°C, the oxidation kinetics (transition from a sub-parabolic to a nearly linear oxidation kinetics) sped up and the materials absorbed hydrogen (Fig. 5 and Fig. 6); this transition is commonly called breakaway oxidation. For oxidation times longer than 5000s, the weight gain and hydrogen uptake of samples with 10-13 µm-thick pre-oxides formed under water at 340-360°C were close of those of the samples without pre-oxides. The excess in weight gain measured for pre-oxidized M5Framatome samples was essentially due to the faster oxidation of the end-caps in Zircaloy-4 (non-pre-oxidized), included in the weight gain measurements. Thus it can be concluded that the breakaway oxidation resistance of Zy-4 and M5Framatome under steam at 1000°C was not significantly modified by 10-13 µm-thick preoxides formed under water at 340-360°C.

Oxidation at 1200°C

The weight gains after oxidation under steam at 1200°C tended to be lower for the preoxidized materials than for the as-received materials, for a given oxidation time (Fig. 8 for Zy-4 and Fig. 9 for M5Framatome). The benefit increased with increasing the pre-oxide thickness.

The oxidation at 1200°C was thus delayed in the presence of a pre-existing oxide layer formed at lower temperature. However, the weight gain of the pre-oxidized material progressively caught up with that of the non-pre-oxidized materials in the course of oxidation.

After 55-60s at 1200°C, the evolution of weight gains as a function of the pre-oxide thickness followed the same tendency for the materials with pre-oxides of 25 to 36 µm for Zy-4 (Fig. 8) and 13 to 35 µm for M5Framatome (Fig. 9) formed under static water at 360°C, pre-oxides of 15 µm formed under flowing water at 350°C, and pre-oxides of 60 µm for Zy-4 and 30 µm for M5Framatome formed under steam at 415°C.

After 600s at 1200°C, the weight gain progressively decreased with increasing the pre-oxide thickness, in similar ways for Zy-4 samples with 8 to 59 µm-thick pre-oxide layers formed

Preprint submitted to Journal of Nuclear Materials 13 under water at 360°C tested herein, samples with 9 to 64 µm-thick pre-oxides formed in O2 at 425°C tested by Guilbert et al. [START_REF] Guilbert | Effect of pre-oxide on Zircaloy-4 high temperature steam oxidation and post-quench mechanical properties[END_REF], and samples with 9 to 33 µm-thick pre-oxides formed in PWR tested by Ozawa et al. [START_REF] Ozawa | Behavior of Irradiated Zircaloy-4 Fuel Cladding Under Simulated LOCA Conditions[END_REF]. However, the samples with 25 and 50 µm-thick pre-oxides formed in air at 550°C or with 30 and 80 µm-thick pre-oxides formed in air at 500°C after pre-hydriding at 400°C [START_REF] Baek | Steam oxidation of Zr-1.5Nb-0.4Sn-0.2Fe-0.1Cr and Zircaloy-4 at 900-1200°C[END_REF] had weight gains close to those of the non-pre-oxidized material after 600s at 1200°C.

As-received Zy-4 did not absorb hydrogen when steam oxidized during 600s at 1200°C. On the one hand, for this oxidation condition, the material with pre-oxides of 8 µm formed under water at 360°C, of 25 and 50 µm formed in air at 550°C, or thinner than 40 µm formed under O2 at 425°C [START_REF] Guilbert | Effect of pre-oxide on Zircaloy-4 high temperature steam oxidation and post-quench mechanical properties[END_REF] did not absorb a significant amount of hydrogen. On the other hand, an important hydrogen pickup was reported in [START_REF] Guilbert | Effect of pre-oxide on Zircaloy-4 high temperature steam oxidation and post-quench mechanical properties[END_REF] for pre-oxides thicker than 40 µm formed in O2 at 425°C. This hydrogen pickup increased with increasing the pre-oxide thickness, due to an increase of the hydrogen pickup fraction (about 16% for 64 µm-thick pre-oxides). The material with 59 µm-thick pre-oxide layers formed under water at 360°C also absorbed some hydrogen when oxidized 600s at 1200°C but in a smaller quantity (about 90 wppm, ≈ 3%).

In some cases, radial cracks, present after pre-oxidation or formed at the beginning of the holding at HT, were observed in the pre-oxide layers. The new oxide started to form in the vicinity of these radial cracks at HT then tended to grow uniformly. The same observations were reported for samples corroded in PWR [START_REF] Nagase | Behavior of High Burn-up Fuel Cladding under LOCA Conditions[END_REF]. The interface between the prior-βZr and the αZr(O) layers, i.e. the diffusion depth of oxygen through the metallic substrate, was not significantly affected nonetheless by the sooner oxidation around the cracks in the pre-oxide.

Indeed, the diffusion of oxygen far from the metal/oxide interface is driven by the diffusion in volume of oxygen into the αZr(O) layer. Thus, the heterogeneous oxidation at the beginning of oxidation at HT did not seem to alter the later oxidation.

As illustrated in Fig. 8, and as previously shown for oxidation at 1000°C, the lower weight gains measured at 1200°C in the presence of a pre-oxide were due to a delayed oxide growth at HT. Nevertheless, the overall quantity of oxygen that diffused into the metallic substrate was the nearly same whether there was a pre-oxide layer or not. Indeed, the oxygen coming from the reduction of the pre-existing oxide at the ZrO2/αZr(O) interface has diffused into the metallic part, causing thickening of the αZr(O) phase layer (Fig. 10 The results illustrated in Fig. 10 (b) showed that the pre-oxide reduced at the beginning of oxidation whether the surface was exposed to vacuum or to steam. On the one hand, the preoxide whose surface was exposed to steam reduced from only a few microns. Its thickness then did not evolve anymore because the inward flux of oxygen in the oxide became higher than the inward flux in the αZr(O) phase [START_REF] Mazères | Experimental study and numerical simulation of high temperature (1100-1250 °C) oxidation of prior-oxidized zirconium alloy[END_REF], leading the growth of a new oxide layer beneath the remaining pre-oxide. On the other hand, reduction of the inner pre-oxide in contact with vacuum continued until it had completely disappeared. For example, complete reduction was achieved after a holding time between 187 and 520s at 1200°C for 24-26 µm-thick pre-oxides, and between 60 and 202s for 13-15 µm-thick pre-oxides. As long as the pre-oxide was not fully reduced, the growth kinetics of the αZr(O) phase layers were similar at the outer and inner peripheries of the samples one-sided oxidized at 1200°C: the αZr(O) phase layer grows at the same rate whether it is associated with the reduction of the pre-existing oxide or the growth of a fresh oxide at HT (Fig. 10). When the pre-oxide was completely reduced on the side non-exposed to steam, the thickness of the αZr(O) phase deriving from this reduction kept increasing in the case of Zy-4 with 24-26 µm-thick pre-oxides formed under water at 360°C until at least 1492s at 1200C°C. In the case of M5Framatome with 13-15 µm-thick pre-oxides formed under water at 360°C, the thickness of the inner "continuous" αZr(O) phase layer, not exposed to steam, slightly decreased during the holding at 1200°C. Indeed, when this relatively thin pre-oxide layer was entirely reduced, i.e. when there was no oxygen source anymore, the αZr(O) phase was progressively depleted in oxygen and transformed into the βZr phase, as predicted by diffusion calculations [START_REF] Leistikow | Oxidation Kinetics and Related Phenomena of Zircaloy-4 Fuel Cladding Exposed to High Temperature Steam and Hydrogen-Steam Mixtures under PWR Accident Conditions[END_REF].

Preprint submitted to Journal of Nuclear Materials 15

As illustrated in Fig. 10, the results obtained on Zy-4 and M5Framatome with 24-26 µm-thick and 13-15 µm-thick pre-oxides respectively, showed that when a new oxide layer began to grow at HT beneath the outer pre-oxide, exposed to steam, the αZr(O) phase layer grew faster than in the case of the non-pre-oxidized material. This may be due to a faster diffusion of oxygen through the αZr(O) phase when formed during the growth of this new oxide layer, which may be due for example to a particular stress state at the metal/oxide interface, to hydrogen pickup or to more heterogeneous thickness of the αZr(O) phase. Further investigation is needed to understand these effects.

Discussion

Oxidation and oxygen diffusion

The results have shown that the oxidation under steam at HT is delayed in most cases in the presence of a pre-oxide. The oxidation kinetics of the pre-oxidized material then catches up that of the non-pre-oxidized material: the fresh oxide tends to grow more quickly at HT depending on the pre-oxidation conditions.

The oxidation process under steam at HT of materials with pre-existing oxides involves two main steps, both associated with diffusion of oxygen through the metallic substrate: the preoxide layer first reduces at the beginning of the treatment at HT, then a new oxide grows at the metal/oxide interface (Fig. 12). This can be explained by the oxygen flux balance at the metal/oxide interface [START_REF] Mazères | Experimental study and numerical simulation of high temperature (1100-1250 °C) oxidation of prior-oxidized zirconium alloy[END_REF]: at the beginning of the holding at HT, the inward flux of oxygen in the pre-oxide # $% is lower than the inward flux in the αZr(O) phase # & ; later, the balance of the fluxes at the metal/oxide interface reverses, due to the decrease of the oxygen flux in the αZr(O) phase layer and the increase of the flux in the oxide layer, resulting from their respective thickening and thinning.

The pre-oxide and the oxide formed at HT have quite different (micro)structures. For example, the grains of the oxides formed at HT tend to be wider and more columnar than those formed at lower temperature and, with the exception of post-breakaway oxidation conditions, the oxides formed at HT usually do not show circumferential cracks. This may have an effect on the transport properties, of oxygen in particular. According to Mazères et al. [START_REF] Mazères | Experimental study and numerical simulation of high temperature (1100-1250 °C) oxidation of prior-oxidized zirconium alloy[END_REF], the effective coefficient of diffusion of oxygen at HT may be lower within the pre-oxide during its reduction than within the oxide formed at HT. The relatively fast growth kinetics of the new oxide at HT is still not clearly explained. As suggested in [START_REF] Mazères | Experimental study and numerical simulation of high temperature (1100-1250 °C) oxidation of prior-oxidized zirconium alloy[END_REF]¸ the pre-oxide may be Preprint submitted to Journal of Nuclear Materials 16 damaged and may lose, at least partially, its protective effect regarding oxygen diffusion when a fresh oxide layer starts to grow at HT. This may be associated with change in stoichiometry and phase transformation of zirconia, inducing volume changes and resulting in crack formation. It is also possible that oxygen diffusion in the αZr(O) phase is slower when this phase results from the reduction of the pre-oxide than when it forms during the growth of the new oxide layer at HT. Furthermore, diffusion of oxygen may be faster through the oxide formed at HT (beneath the remaining pre-oxide and the αZr(O) phase resulting from the partial reduction of the pre-oxide) than through the oxide formed at HT directly from the as-received metal, due to different microstructure (morphology of crystallites, …), porosity or stress state for example.

At HT, hydrogen mainly concentrates into the βZr phase layer [START_REF] Brachet | Study of secondary hydriding at high temperature in zirconium based nuclear fuel cladding tubes by coupling information from neutron radiography/tomography, electron probe micro analysis, micro elastic recoil detection analysis and laser induced breakdown spectroscopy microprobe[END_REF][27][37] [START_REF] Torres | Hydrogen motion in Zircaloy-4 cladding during a LOCA transient[END_REF]. The solubility limit of oxygen in the βZr phase is increased in the presence of hydrogen [START_REF] Brachet | Study of secondary hydriding at high temperature in zirconium based nuclear fuel cladding tubes by coupling information from neutron radiography/tomography, electron probe micro analysis, micro elastic recoil detection analysis and laser induced breakdown spectroscopy microprobe[END_REF][27] [START_REF] Guilbert-Banti | Influence of hydrogen on the oxygen solubility in Zircaloy-4[END_REF]. After oxidation at HT, an enrichment in hydrogen is generally observed at the interface between the αZr(O) layer and the prior-βZr layer [37][38]. This enrichment may have an effect on the solubility limit of oxygen at this interface. Moreover, the diffusion of oxygen may be accelerated in the presence of hydrogen [27][35]. Therefore it cannot be excluded that the hydrogen absorbed during pre-oxidation (when it was carried out under water or steam in particular) or during oxidation at HT, has an influence on the actual oxidation and hydrogen pickup at HT. However, no significant effect on these processes of a pre-hydriding up to 600 wppm was observed in [START_REF] Portier | Influence of Long Service Exposures on the Thermal-Mechanical Behaviour of Zy-4 and M5M5™ Alloys in LOCA Conditions[END_REF].

Although the growth of a fresh oxide at HT is in some cases delayed in the presence of a preoxide, the overall quantity of oxygen that diffused into the metallic substrate was nearly the same whether there was a pre-oxide layer or not. Indeed, the pre-oxide is a source of oxygen, as efficient as the outer steam. The mechanical behaviour of the material oxidized at HT is directly correlated to the amount of oxygen having diffused within the metal (excluding additional effect of hydrogen) [START_REF] Brachet | Hydrogen Content, Preoxidation, and Cooling Scenario Effects on Post-Quench Microstructure and Mechanical Properties of Zircaloy-4 and M5™ Alloys in LOCA Conditions[END_REF][40] [START_REF] Chung | Development of an Oxygen Embrittlement Criterion for Zircaloy Cladding Applicable to Loss-of-Coolant Accident Conditions in Light-Water Reactors, Zirconium in the Nuclear Industry[END_REF]. Thus, as shown by mechanical tests performed at 20 and 135°C [START_REF] Guilbert | Effect of pre-oxide on Zircaloy-4 high temperature steam oxidation and post-quench mechanical properties[END_REF][31], the residual ductility of pre-oxidized samples oxidised at HT and quenched is close to that of the non-pre-oxidized samples containing the same hydrogen content and oxidized under similar conditions.

Preprint submitted to Journal of Nuclear Materials 17

Hydrogen pickup

It was observed that in some conditions the pre-oxidized materials absorb hydrogen during oxidation at HT while the non-pre-oxidized materials do not. This supports the hypothesis that the oxide formed at HT after reduction of a pre-existing oxide does not have the same characteristics than the oxide directly formed from the bare metal.

Similar hydrogen pickup fractions were measured after 200 and 400s at 1000°C for Zy-4 with pre-oxides of 5, 25 and 50 µm formed under air at 550°C. The hydrogen pickup fraction (average value from the beginning of the oxidation) was lower after 1800s at this same temperature. To explain this decrease of the hydrogen pickup fraction, one can assume that the hydrogen pickup fraction has not evolve much until the fresh oxide had not yet begun to grow or remained relatively thin, then decreased (or even became negligible) from a given thickness of oxide formed at HT, and finally increased again when this thickness became important.

The hydrogen pickup fraction depends on the pre-oxidation conditions, the pre-oxide thickness, the alloy and the conditions of oxidation at HT: it varies from 0 up to several tens of percent. During in-service operations in PWR, the cladding material absorbs from 10 to 20% of the hydrogen generated from the corrosion reaction, depending on the alloy and the corrosion conditions [START_REF] Bossis | In PWR comprehensive study of high burn-up corrosion and growth behavior of M5® and recrystallized low-tin Zircaloy-4[END_REF]. The material can absorb higher hydrogen pickup fractions at HT partly because hydrogen (in solid solution) diffuses faster in the metal and the oxide.

As shown by Guilbert et al. [START_REF] Guilbert | Effect of pre-oxide on Zircaloy-4 high temperature steam oxidation and post-quench mechanical properties[END_REF], the hydrogen pickup of the pre-oxidized materials at HT can have consequences on their residual mechanical behaviour, since hydrogen has an embrittlement effect at low temperature [START_REF] Nagase | High burnup fuel behavior under LOCA conditions[END_REF][27] [START_REF] Yueh | Loss of Coolant Accident Testing Round Robin, TopFuel 2013[END_REF][29][30] [START_REF] Chung | Development of an Oxygen Embrittlement Criterion for Zircaloy Cladding Applicable to Loss-of-Coolant Accident Conditions in Light-Water Reactors, Zirconium in the Nuclear Industry[END_REF].

Corrosion and hydrogen pickup mechanisms

The oxidation kinetics and the hydrogen pickup are linked to the characteristics of the oxide, pre-existing or formed at HT: grains morphology, cohesion between grains, density, location of pores (resulting from clustering of vacancies for example) and cracks.

When the fraction of hydrogen absorbed is high (several tens of percent), it may be assumed that dissociation of water and reduction/recombination of hydrogen have mainly occurred at the vicinity of the metal/oxide interface. This presupposes that steam had access to the metal Preprint submitted to Journal of Nuclear Materials 18 through a network of open pores and/or interconnected cracks in the oxide (transport in the gas phase). This access is facilitated by the presence of radial cracks within the oxide, as observed for example before and after oxidation at HT in samples with 50 µm-thick preoxides formed under air at 550°C (Fig. 1 and Fig. 7). However, these cracks generally do not cross the whole thickness of the oxide layer. Pores and circumferential cracks within the oxide may therefore also contribute to the transport of steam to the metal. This network of interconnected defects may act as a "hydrogen pump" [START_REF] Große | Influence of oxide layer morphology on hydrogen concentration in tin and niobium containing zirconium alloys after high temperature steam oxidation[END_REF][44]: because of the oxidation of the metal, the partial pressure of gaseous hydrogen in pores and cracks near the metal/oxide interface would increase, leading to an additional hydrogen pickup, if it is assumed that the hydrogen absorbed by the metal comes from the dissolution of gaseous hydrogen (so that the solubility of hydrogen in the metal is proportional to the square root of the hydrogen partial pressure, following the Sieverts's law). Due to the resulting pressure decrease within these cracks, steam would be "sucked up" again. In theory, this process could be repeated until the hydrogen content in the metal is in equilibrium with the local hydrogen partial pressure in the atmosphere filling the cracks close to the metal-oxide interface (hydrogen pickup may be much larger than given by equilibrium with the ambient hydrogen concentration).

In cases where hydrogen pickup is lower (a few percent typically), dissociation of water and reduction/recombination of hydrogen may have taken place within the oxide layer, not far from the metal/oxide interface, at the interface between a "porous" oxide sublayer (presence of pores and interconnected cracks, weak intergranular cohesion due to disoriented grain boundaries for example) and a "dense" oxide sublayer. Indeed, a more or less continuous network of cracks or porosity nearly parallel to the metal/oxide interface have been observed in some cases near the interface between the remaining pre-oxide and the oxide formed at HT, or at the metal/oxide interface. Guilbert et al. [START_REF] Guilbert | Effect of pre-oxide on Zircaloy-4 high temperature steam oxidation and post-quench mechanical properties[END_REF] suggested that these interconnected cracks/porosity result from the clustering of the circumferential cracks initially present in the pre-oxide, during reduction of the pre-oxide (the cracks progressively move closer to the interface with the metal). The diffusion of hydrogen through the "dense" oxide sublayer, mainly via vacancies and interstitials a priori, would lead to a local enrichment in hydrogen at the metal/oxide interface, high enough for the metal to absorb hydrogen.

When there is no hydrogen pickup at HT, it may be assumed that dissociation of water and reduction/recombination of hydrogen have occurred at the outer surface or within the existing oxide but far from the interface with the metal, so that the potential access of hydrogen to the Preprint submitted to Journal of Nuclear Materials 19 metal (by solid state diffusion) would be relatively slow. The amount of hydrogen at the metal/oxide interface would remain too low for the metal to absorb hydrogen.

In the cases of pre-oxides formed under air at 550°C, the fraction of hydrogen absorbed by the material was lower after 600s at 1200°C than after 1800s at 1000°C, and even more than after 6000s at 900°C. As proposed in [START_REF] Guilbert | Effect of pre-oxide on Zircaloy-4 high temperature steam oxidation and post-quench mechanical properties[END_REF], this reduction of the hydrogen pickup fraction with increasing the oxidation temperature may be attributed to the fact that the solid state diffusion through the oxide is a thermally activated process (whose rate increases exponentially with temperature), while the transport in the gas phase shows a lower temperature dependence.

Therefore, it may be assumed that the relative contribution of the gas phase transport, expected to be responsible for high hydrogen pickup fractions, to the transport of oxygen and hydrogen species decreases with increasing the temperature. This may also be related to the influence of temperature on the oxide structure and microstructure [START_REF] Gosset | In-situ X-ray diffraction analysis of zirconia layer formed on zirconium alloys oxidized at high temperature[END_REF], which can have an effect on the transport of hydrogen species.

Conclusions

The effects of a pre-existing oxide layer on the subsequent oxidation under steam at HT was studied for Zy-4 and M5Framatome alloys. Oxidation tests were performed under steam (at atmospheric pressure) at 850, 900, 1000 and 1200°C, for holding times between 55 and 9500s, on samples with pre-oxides thicknesses between 3 and 60 µm, formed under various conditions (air at 550°C, steam at 415°C, static water at 340-360°C with a typical PWR primary water chemistry, flowing water at 350°C with typical PWR primary water thermalhydraulic and chemical environment).

It was shown that the HT steam oxidation is generally delayed in the presence of a pre-oxide.

The thicker the pre-oxide, the more pronounced the effect is. The pre-oxide reduces at the beginning of oxidation at HT then a fresh oxide grows when the oxygen flux balance at the metal/oxide interface reverses. Both processes are associated with diffusion of oxygen through the metallic substrate. The overall quantity of oxygen that diffuses into the metal is nearly the same whether it is associated with reduction of the pre-oxide or growth of a new oxide. The oxidation kinetics of the pre-oxidized material progressively catches up that of the non-pre-oxidized material in the course of oxidation at HT. In the conditions that have been tested, the oxidation kinetics of the pre-oxidized material never exceeds that of the non-preoxidized material, with the exception of 50 µm-thick pre-oxides formed under air at 550°C.

The difference between the oxidation rates of Zy-4 and M5Framatome at 1000°C are reduced in Preprint submitted to Journal of Nuclear Materials 20 the presence of a pre-oxide. The resistance to breakaway oxidation under steam at 1000°C is not significantly modified by pre-oxidation. At given pre-oxide thickness and HT oxidation conditions, the growth rate of the fresh oxide at HT depends on the pre-oxidation conditions.

Typically, HT oxidation is often faster after pre-oxidation under air at 550°C than with preoxides formed under water at 340-360°C.

In some cases, the pre-oxidized materials absorb hydrogen during oxidation under stream at HT while the non-pre-oxidized materials do not. The hydrogen pickup increases during oxidation, although the fraction of hydrogen absorbed does not. The hydrogen pickup fraction depends on the pre-oxide thickness, the conditions in which it was formed, the alloy, and the conditions of oxidation at HT: it varies from zero to several tens of percent. The pre-oxides may be ranked as follows, from the least to the most protective with respect to hydrogen pickup at HT: air at 550°C, O2 at 500°C, O2 at 425°C, water (PWR primary water chemistry)

at 340-360°C.

The results suggest that the oxygen and hydrogen transport mechanisms and kinetics, and the locations of water dissociation and hydrogen reduction/recombination at HT may be different whether there was a pre-oxide or not. Further investigations are still needed to improve understanding of the relationship between the characteristics of the pre-oxide, the corrosion mechanisms at HT and their effects on the oxidation and hydrogen pickup at HT, especially for real PWR environment. Table 3 Conditions of pre-oxidation, pre-oxide thicknesses and hydrogen contents after pre-oxidation, and conditions of oxidation under steam at HT, for Zy-4.

* content estimated from measurements on equivalent samples or from available correlations between weight gain and hydrogen content

  (a)) and associated oxygen Preprint submitted to Journal of Nuclear Materials 14 diffusion into the βZr layer. Fig.11compares oxygen concentration profiles measured by EPMA, after oxidation during approximately 60s at 1200°C, within the prior-βZr layer of samples with 13-15 µm or 36 µm-thick pre-oxide layers and of non-pre-oxidized samples, asreceived and pre-hydrided. For this oxidation condition, no fresh oxide has grown yet at HT in the sample with 36 µm-thick pre-oxides and only a few microns of fresh oxide have formed in the sample with 13-15 µm-thick pre-oxide layers. It was shown that the oxygen concentration profile within the prior-βZr layer was nearly the same for the pre-oxidized materials and the non-pre-oxidized materials containing a similar hydrogen content. The oxygen content is slightly larger than in the as-received material oxidized in the same conditions due to the effect of hydrogen on the solubility of oxygen in βZr, already reported in[START_REF] Brachet | Study of secondary hydriding at high temperature in zirconium based nuclear fuel cladding tubes by coupling information from neutron radiography/tomography, electron probe micro analysis, micro elastic recoil detection analysis and laser induced breakdown spectroscopy microprobe[END_REF][START_REF] Brachet | Hydrogen Content, Preoxidation, and Cooling Scenario Effects on Post-Quench Microstructure and Mechanical Properties of Zircaloy-4 and M5™ Alloys in LOCA Conditions[END_REF][31][START_REF] Guilbert-Banti | Influence of hydrogen on the oxygen solubility in Zircaloy-4[END_REF].

Fig. 1 . 25 Fig. 2 . 26 Fig. 3 .Fig. 4 . 28 Fig. 5 . 29 Fig. 6 . 30 Fig. 7 . 31 Fig. 8 . 32 Fig. 9 . 33 Fig. 10 . 34 Fig. 11 . 35 Fig. 12 .

 12522634285296307318329331034113512 Fig. 1. Optical micrographs (left) and field emission gun scanning electron microscopy micrographs (right) of transverse cross-sections of Zy-4 samples with pre-oxide layers (a) of 59 µm-thick formed under static water at 360°C and (b) 50 µm-thick formed in air at 550°C.

Table 1

 1 Data available in the literature relative to the effect of a pre-oxide layer on the steam HT oxidation of Zy-4 and M5Framatome (n/a: not available).

	Mate	Outer / inner	Pre-oxidation	HT steam oxidation	Weight gain and oxide	Hydrogen pick-	Ref.
	-rial	pre-ox. thick-	conditions	conditions	thickness compared to the	up at HT
		ness (µm)			non-pre-oxidized material
	Zy-4	60 / 60	Water 340°C to	1008-1240°C, 300-	Lower at the beginning of	n/a	[1]
			20 µm of pre-ox.	2400s	oxidation, then close
			then O2 400°C
		50 / 50	n/a	1100°C, 120-3600s	Lower for ox. times ≤ 1800s	n/a	[2]
					at 1100°C, then close
		few-50 / few-	Steam 350-600°C	1000-1200°C, 300s	Lower for pre-ox. < 10 µm,	n/a	[3]
		50			close or higher for thicker
					pre-oxides
		few-50 / few-	Steam 400-800°C	1000-1200°C, 300s	Lower at 1000 and 1100°C	n/a	[4]
		50			but not at 1200°C
		5-10 / 5-10	n/a 817°C	982-1316°C, 6-558s	Lower, the thicker the pre-	n/a	[5]
					ox. the larger the difference		[6]
		~1 / ~1	Water 300°C	900-1100°C, 60-5000s Slightly lower at 900 and	n/a	[7]
					950°C, similar at 1000 and
					1100°C
		4-7 / 4-7	Water + steam	700-850°C, 60-1500s	Lower, the thicker the pre-	n/a	[8]
			650°C		ox. and the lower the
					temperature the larger the
					difference
		6 / 6	Steam 450°C	900-1200°C, 2400s	Lower, the lower the	n/a	[9]
			103 bar		temperature the larger the
					difference
		30 and 80 /	Air 500°C (after	900-1200°C, 2400s	Lower, the lower the	n/a	[9]
		30 and 80	gaseous hydriding		temperature the larger the
			400°C)		difference, higher for 80 µm-
					thick than for 30 µm-thick
					pre-ox.
		10-70 / 10-70	O2 500°C	900°C, 6000s	Lower, increasing difference	Significant for	[10]
					with increasing pre-ox.	pre-ox. <
					thickness below 40 µm,	40 µm-thick,
					decreasing beyond	large for thicker
						pre-ox.
		7-64 / 7-64	O2 425°C	900-1200°C, 100-	Lower, increasing difference	Significant for	[11]
				6000s	with increasing pre-ox.	7-12 µm-thick
					thickness below 40 µm,	pre-ox., low for
					decreasing beyond at 900 and	18-32 µm-thick,
					1000°C	large for pre-ox.
						> 40 µm
		18 and 28 /	Water 350-360°C	900°C, 6000s	Lower	Not significant	[11]
		18 and 28		1000°C, 900s
		20 and 50 /	Steam 500°C	700-900°C, 1500s	Lower, no significant	n/a	[12]
		20 and 50			difference between 20 and
					50 µm-thick pre-ox.
		8 / 8	Steam 500°C	800°C, 1500s	Lower	n/a	[13]
		50-85 / 50-85	Flowing water	1000-1300°C, n/a	Close	n/a	[14]
			350°C
		71-95 / 11±4	PWR	1200°C, 62-206s	Lower	n/a	[15]
		68 / 11±4	PWR	1200°C, 174-534s	Slightly lower	Slightly	[15]
						negative
		40-44 / 11±4	PWR	1200°C, 174-323s	Slightly lower	Significant	[15]
		~30 / n/a	PWR	1204°C, 300-1200s	Close	n/a	[17]
		~30? / n/a	PWR	1000-1200°C, 120-	Slightly lower at the	n/a	[18]
				2760s	beginning of ox. at 1000 and
					1100°C, close at 1200°C
		7-56 / n/a	PWR	1030-1200°C, 300-	Lower, the thicker the pre-	n/a	[1]
				2400s	ox. and the lower the
					temperature the larger the
					difference

Acknowledgements

We thank C. Cobac, P. Crébier, D. Gilbon, D. Hamon, C. Hossepied, G. Nony, J. Rousselot and V. Rabeau from CEA, A. Cabrera, M. Blat-Yrieix, P. Jacques and B. Sebbari from EDF, and J.P. Mardon and V. Garat from Framatome, for their contributions to this work.


Isabelle Métais
email: imetais@uco.fr

Amélie Châtel

Mohammed Mouloud

Hanane Perrein-Ettajani

Mélanie Bruneau

Patrick Gillet

Nisrine Jrad

Catherine Mouneyrac

Is there a link between acetylcholinesterase, behaviour and density populations of the ragworm Hediste diversicolor?

Keywords: Hediste diversicolor, acetylcholinesterase, population density, behaviour, Adverse Outcome Pathway. Estuaries, as transitional zones, provide

The main objective of the present study was to explore the potential link between acetylcholinesterase (AChE) activity and burrowing behaviour of the ragworm Hediste diversicolor, which may have consequences at higher levels of biological organisation.

Two complementary studies were conducted. AChE activity, at the sub-individual level, and behavioural responses, at the individual level, were evaluated in worms from the Loire estuary (France), whereas density and biomass of H. diversicolor were determined at the population level. A Spearman positive correlation between both biomarkers (AChE and burrowing) suggested that inhibition of AChE activity was linked to behaviour impairments. At the population level, lower AChE and behaviour activities were detected in worms corresponding to lower population density and biomass.

These results provide direct empirical field evidence demonstrating the sensitivity of behaviour of H. diversicolor as a biomonitor of estuarine health status assessment.

they are affected by multiple anthropogenic activities, leading to the release of various types of contaminants. To estimate the health status of ecosystems, biomonitoring programs aimed to classically combine chemical and biological approaches (e.g. multibiomarker approach) and ecological indicators. Conventional monitoring techniques are mainly based on the determination of the contaminants' concentrations in sediments and water. Evaluation of chemical contamination is a complex task, as estuaries often present a mixture of contaminants and their metabolites, some of which are costly or not yet accessible to analyze. It is widely recognised that chemical data alone can only provide partial information; for example, the unpredictability of the processes affecting the speciation of pollutants may alter their bioavailability and therefore their toxicity [START_REF] Birch | A review of chemical-based sediment quality assessment methodologies for the marine environment[END_REF]. For this reason, the integration of biomarker responses with chemical and ecological approaches on sentinel species allows evaluation of the toxicity of mixtures of pollutants and sub-lethal effects even at low concentrations [START_REF] Amiard-Triquet | Aquatic Ecotoxicology: advancing tools for dealing with emerging risks[END_REF]. Biomarkers have been widely used to assess contaminant exposure and its effects in marine species such as the ragworm Hediste diversicolor, which plays an important role in the structure and functioning of marine ecosystems. However, biomarkers are subjected to the influence biotic and abiotic confounding factors (e.g. temperature, salinity, size/weight and reproductive status). Thus, to use these biomarkers in biomonitoring programs, it is necessary to previously determine baseline levels in species of interest, which is currently challenging. Recently, [START_REF] Barrick | A novel methodology for the determination of biomarker baseline levels in the marine polychaete Hediste diversicolor[END_REF][START_REF] Birch | A review of chemical-based sediment quality assessment methodologies for the marine environment[END_REF] determined baseline assessment criteria (BAC) and environmental assessment criteria (EAC) of biochemical biomarkers (energy reserves, catalase: CAT, glutathione S transferases: GST, acetylcholinesterase: AChE and thiobarbituric acid reactive substances: TBARs) in H. diversicolor from a reference site [START_REF] Durou | Biomonitoring in a clean and multi-contaminated estuary based on biomarkers and chemical analyses in the endobenthic worm Nereis diversicolor[END_REF]. Regarding burrowing behaviour, [START_REF] Kalman | Assessment of the influence of confounding factors (weight, salinity) on the response of biomarkers in the estuarine polychaete Nereis diversicolor[END_REF] showed no influence of confounding factors (weight, salinity and grain size) on burrowing behaviour in H. diversicolor.

Field studies aiming at establishing a link between biomarkers at a low level of organisation and possible effects at a higher level of organisation (e.g. population and community) remain to be conducted. The adverse outcome pathway (AOP) approach proposed by Ankley et al. (2010) aims to break through the gap linking direct molecular initiating events with adverse outcomes at a higher level of organisation. AChE, a core biomarker of neurotoxicity, is well recognised as a biomarker of ecological relevance, as its activity inhibition observed in laboratory or field studies may lead to physiological and behavioural impairments in organisms exposed to not only pesticides but also metals, polycyclic aromatic hydrocarbons (PAHs), pharmaceutical products or algal toxins [START_REF] Lionetto | Acetylcholinesterase as a biomarker in environmental and occupational medicine: New insights and future perspectives[END_REF][START_REF] Sturve | Environmental monitoring of pesticide exposure and effects on mangrove aquatic organisms of Mozambique[END_REF][START_REF] Lehtonen | Accumulation of nodularin-like compounds from the cyanobacterium Nodularia spumigena and changes in acetylcholinesterase activity in the clam Macoma balthica during short-term laboratory exposure[END_REF][START_REF] Kankaanpää | Accumulation and depuration of cyanobacterial toxin nodularin and biomarker responses in the mussel Mytilus edulis[END_REF][START_REF] Zhang | Respiratory toxicity of cyanobacterial aphantoxins from Aphanizomenon flos-aquae DC-1 in the zebrafish gill[END_REF].

Currently, the most robust of the developing pathway is the AOP linking AChE inhibition to acute mortality [START_REF] Russom | Development of an adverse outcome pathway for acetylcholinesterase inhibition leading to acute mortality[END_REF]. This AOP has been proposed to support integrated approaches for testing and assessment (https://aopwiki.org/aops/16). However, there are many biochemical, behavioural, and physiological processes associated with the link between AChE inhibition and acute mortality of organisms and, consequently, possible density depletion of the species considered. The Loire estuary is a multi-contaminated estuary displaying a diffusive pollution due to heavy metals, pesticides, PAHs and PCBs [START_REF] Blanchet-Letrouvé | Vg mRNA induction in an endangered fish species (Anguilla anguilla) from the Loire estuary (France)[END_REF], and AChE inhibition in organisms has been often linked to the presence of these contaminants. The main objective of the present study was to explore in the ragworm H. diversicolor from the Loire estuary (France), the potential link between AChE activity and burrowing behaviour that may have consequences at higher levels of biological organisation.

The sampling site was located on the largest mudflat of the polyhaline domain of the Loire estuary ('Les Brillantes') on the south shore downstream Paimboeuf (Fig. 1). Two zones were selected: the first one (Site 1: 47°16'55.70"N; 2° 3'47.60"W) was located in the upper intertidal area of the mudflat, 20 m from the shore, located close to an active eroded cliff (Thibault de [START_REF] Thibault De Chanvalon | Manganese, iron and phosphorus cycling in an estuarine mudflat sediment, Loire, France[END_REF]. The second one, (Site 2: 47°17'3.77"N; 2° 3'42.87"W) was located in the middle intertidal area. Two sampling campaigns were conducted in May 2013 (after the flood period) and February 2014 (during the flood period), respectively.

For AChE activity determinations, 10 worms (H. diversicolor) in the same range of wet weight (mean S1: 0.220±0.114 g; mean S2: 0.187±0.121 g) were gently collected by hand, kept in wet sediment from their site of origin, immediately frozen at the laboratory and then kept at -80°C until analysis. AChE activity was determined using the method described in [START_REF] Buffet | Behavioural and biochemical responses of two marine invertebrates Scrobicularia plana and Hediste diversicolor to copper oxide nanoparticles[END_REF]. Briefly, whole worm tissues were individually homogenised in 80 mM Tris-HCl pH 7.2, 200 mM NaCl, 1 mM Dithiothreitol supplemented with protease inhibitor cocktail according to the manufacturer's instructions. The homogenates were centrifuged at 9000 g for 30 min at 4°C, and supernatants were collected for protein [START_REF] Bradford | A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[END_REF] and AChE activity determinations. Ten microlitres of the supernatant was added in triplicate to 190 µl 10 mM DTNB and 100 µL 100 mM acetylthiocholine into a 96-well microplate. The reactions were read at 412 nm for 3 minutes. Results were expressed as nmoles of thiocholine produced per min and per mg protein (nmoles min -1 mg -1 protein).

Concerning behavioural experiments, burrowing tests were set up in a dark temperature-controlled room (15°C at the time of collection). Worms (H. diversicolor)

were submitted to burrowing tests as previously described by [START_REF] Bonnard | Effects of copper on the burrowing behavior of estuarine and coastal invertebrates, the polychaete Nereis diversicolor and the bivalve Scrobicularia plana[END_REF].

Sediments from each site were homogenised by hand one day before experimentation.

Briefly, burrowing behaviour was studied by placing individually 20 worms on sediments from their site of origin and recording their positions every 2 min for 30 min.

Automated behavioural tests were carried out with the Multifreshwater Biomonitor® (MFB) from LimCo International according to the methodology previously described and validated by [START_REF] Buffet | Mesocosm study of fate and effects of CuO nanoparticles on endobenthic species (Scrobicularia plana, Hediste diversicolor)[END_REF]. H. diversicolor movements inducing signals within the range of 0.5 Hz were identified as undulation, whereas signals induced between 1.0 and 2.0 Hz indicated head movements and feeding activity. One individual per chamber (n=7) was placed on sediment surface and allowed to acclimate 1 h before MFB recording during 7 h. One control chamber without any organisms was used for measuring background noise signals. Concerning density measurements, four sediment samples from both areas (Site 1 and Site 2) were randomly collected at low tide using plastic cores (10 cm x 35 cm) at a distance of 10 m from each other.

Sediment was sieved through a 1 mm mesh to collect and count worms. Fresh weight of H. diversicolor individuals was determined to establish biomass. An average of 4 biomass replicates was calculated as the sum of fresh weight (g FW) divided by the sediment surface sampled (m 2 ) and expressed as FWg m -2 . Statistical analysis for burrowing kinetics was performed on R (R Core Team 2014) using a generalised nonlinear model with α as burrowing rate (h -1 ). This generalised non-linear model with a conditional binomial stochastic part was fitted to each data set by using Bayesian inference as described in [START_REF] Buffet | Biochemical and behavioural responses of the marine polychaete Hediste diversicolor to cadmium sulfide quantum dots (CdS QDs): Waterborne and dietary exposure[END_REF]. For all the other parameters, results are presented as mean ± standard deviation, and statistical analyses were performed using XLSTAT 2012. Normality was assessed using the Shapiro-Wilk test. To define significant differences between sites for each sampling date, normally distributed data were analyzed by one-way ANOVA (multiple comparisons of mean values) followed by Tukey's HSD post hoc method. When normality was not established, the nonparametric Kruskal-Wallis and Mann-Whitney comparison tests were used. The level of significance was established at p < 0.05. A Spearman's correlation [START_REF] Daniel | Spearman Rank Correlation Coefficient[END_REF] was computed to quantify the relationship between AChE activity and behavioural toxicity parameter values. The Spearman's rank-order correlation is a non-parametric correlation coefficient ρ, measuring strength and direction of the monotonic relationship between AChE activity and MFB. The statistical significance test of the Spearman correlation was performed to investigate whether the null hypothesis 'H0:

There is no monotonic association between AChE and the behavioural toxicity parameter MFB' can be rejected. [START_REF] Amiard-Triquet | Aquatic Ecotoxicology: advancing tools for dealing with emerging risks[END_REF]. Mudflat sediments act as the final sink for various contaminants depending on their properties. Sorption of organic contaminants on sediments is controlled by grain size and organic matter content (Arslan-Alaton and Olmez-Hanci 2013). It has been demonstrated that bioavailable metals and organic contaminant contents were low in organic carbon enriched sites, tending to be adsorbed on fine-grained sediments [START_REF] Pelletier | Can sediment total organic carbon and grain size be used to diagnose organic enrichment in estuaries?[END_REF][START_REF] Morrison | Integrating Science and Resource Management in Tampa Bay, Florida[END_REF]. Site 1, compared to site 2, was composed of slightly fine sediment and was slightly enriched in total organic carbon known to have strong sorption capacities of contaminants [START_REF] Luthy | Organic contaminants in the environment: challenges for the water/environmental engineering community[END_REF][START_REF] Thibault De Chanvalon | Manganese, iron and phosphorus cycling in an estuarine mudflat sediment, Loire, France[END_REF]. Moreover, Site 1 was located in an active eroded cliff where vegetation and rhizosphere could develop synergies with microorganisms increasing pollutant removal rates such as PAHs (Thibault de [START_REF] Thibault De Chanvalon | Manganese, iron and phosphorus cycling in an estuarine mudflat sediment, Loire, France[END_REF]. At the individual level, burrowing kinetics of worms from Site 2 were significantly impaired compared to that in worms from Site 1. [START_REF] Schriks | Realtime automated measurement of Xenopus leavis tadpole behavior and behavioral responses following triphenyltin exposure using the multispecies freshwater biomonitor (MFB)[END_REF] used the MFB in Xenopus laevis as a sensitive and useful tool to quantify behavioural effects induced by toxic compound. In H. diversicolor, the use of MFB allows a more sensitive estimation of behavioural activities of worms, as differentiation between body undulation, head movements and feeding activity of worms can be assessed [START_REF] Buffet | Mesocosm study of fate and effects of CuO nanoparticles on endobenthic species (Scrobicularia plana, Hediste diversicolor)[END_REF]. In this work, results

showed lower body undulation of worms from Site 2 (vs Site 1). Statistical analysis In the invertebrate Gammarus fossarum, [START_REF] Xuereb | Acetylcholinesterase activity in Gammarus fossarum (Crustacea Amphipoda): Linking AChE inhibition and behavioural alteration[END_REF] showed that AChE inhibition levels greater than 50% led to population depletion. At the population level, worms exhibiting lower AChE activities and behaviour corresponded to those collected from the site (Site 2) where lower population densities were found. Indeed, densities of H. diversicolor showed a significantly contrasting status between both sites with significantly higher densities (280.61±325.69 to 634.57±294.45 ind.m -2 ) in the upper intertidal Site 1 and lower densities in the middle intertidal Site 2 (25.51±23.29 to 66.96±28.28 ind.m -2 ). These observations cannot be relied on a patchy distribution of worms on the mudflat, as the distribution of H. diversicolor is known to be rather relatively uniform regarding the regularly distributed openings of burrows on the sediment surface [START_REF] Kristensen | Distribution pattern of benthic invertebrates in Danish estuaries: The use of Taylor's power law as a species-specific indicator of dispersion and behavior[END_REF]. However, it has been demonstrated in H.

(
diversicolor a double-way relationship between reworking behaviour and density as a decrease in individual sediment reworking activity when density increase [START_REF] Duport | Effects of population density on the sediment mixing induced by the gallery-diffusor Hediste (Nereis) diversicolor O.F. Muller[END_REF]) and at high levels of algal enrichment [START_REF] Murray | Effects of algal enrichment and salinity on sediment particle reworking activity and associated nutrient generation mediated by the intertidal polychaete Hediste diversicolor[END_REF]. Moreover, at high abundances, it has been shown that territorialism by this ragworm species is a mechanism underplaying trophic competition, while both phenomena seem to disappear at low population density [START_REF] Kristensen | Distribution pattern of benthic invertebrates in Danish estuaries: The use of Taylor's power law as a species-specific indicator of dispersion and behavior[END_REF]. Site 1 was also enriched in living benthic foraminifera with a total density of 2199.35 individuals per 100 cm -3 compared to that at site 2, where a density of only 48 individuals per 100 cm -3 was found [START_REF] Mojtahid | Spatial distribution of living (Rose Bengal stained) benthic foraminifera in the Loire estuary (western France)[END_REF]. These authors correlated the microphytobenthic biofilm development on 'Les Brillantes' mudflat with the foraminifera species abundance, as it constitutes important quantities of food resources. [START_REF] Cardoso | Macroinvertebrate response to different species of macroalgal mats and the role of disturbance history[END_REF] showed that H. diversicolor had greater abundance in areas where algae were available, as a food source and a refuge from predators. [START_REF] Christensen | Carbon and nitrogen fluxes in sediment inhabited by suspension-feeding (Nereis diversicolor) and non-suspensionfeeding (N. virens) polychaetes[END_REF] showed that an abundant supply of nutritious food (phytoplankton) increased H. diversicolor metabolism considerably. To cope, chemical stress is metabolically costly leading to a shift in allocation of energy with implications for growth and reproduction processes [START_REF] Calow | How do physiological responses to stress translate into ecological and evolutionary processes?[END_REF]. Thus, in the present study, it could be suggested that lower food resources and energetic cost of defence mechanisms to cope with chemical stress in worms from Site 2 vs Site 1 could lead to a reduction of the most energetic expensive individual processes, growth and reproductive potential, resulting in detrimental effects observed at the population level.

In conclusion, the contrasted status of H. diversicolor densities between both sites could be partly explained by AChE activity decrease and behavioural impairments of worms from the middle intertidal mudflat (Site 2) compared with the upper intertidal site (Site 1), which could result in the detrimental population effects observed. This study brings direct empirical field evidence supporting impact of AChE inhibition and behaviour at the population level demonstrating the sensitivity of behaviour as a biomonitor of estuarine health status assessment, as previously suggested by [START_REF] Roast | Impairment of mysid (Neomysis integer) swimming ability: an environmentally realistic assessment of the impact of cadmium exposure[END_REF]. This integrated methodology should be included in biomonitoring surveys

to evaluate habitat vulnerability and potential risk on higher trophic levels.

  Spearman correlation) performed on the set results of AChE activities and behaviour (MFB) showing a positive correlation between both biomarkers (biochemical and behavioural) suggested that lower AChE activities were associated with lower behaviour of worms (and inversely). Feeding behaviour disturbances have been previously shown in H. diversicolor from the multipolluted Loire estuary as compared to controls from a reference site (Fossi-Tankoua et al., 2012). Recently, Harayashiki et al. (2016) demonstrated behavioural and AChE biochemical alterations in Penaeus monodon post larvae diet-exposed to inorganic mercury, reducing swimming activity.

Fig. 1 .

 1 Fig. 1. Brillantes mudflat is localized in the polyhaline domain of the Loire estuary

Fig. 2

 2 Fig. 2 BAC (baseline assessment criteria) and EAC (environmental assessment

Table 2 .

 2 Site 2) were expected. In May, AChE values for worms from Site 1 were in the yellow range expected for non-impacted site, whereas those for worms from Site 2 were in the range where sub-lethal effects were expected. Results of burrowing kinetics carried out with H. diversicolor from both areas (Sites 1 and 2), allowed to burrow in sediment from their site of origin, are shown in Table2. Burrowing kinetics of worms from Site 2 were significantly impaired in February 2014 compared to those in Site 1. Behavioural differences between worms from both sites measuring with the MFB are illustrated in Significant frequency decrease (p=0.021) corresponding to body undulation (0.5 Hz) was observed for worms from Site 2 compared to those from Site 1. Decreases of head movements and feeding activity (between 1.0 and 2.0 Hz) were also observed for worms from Site 2 vs those from Site 1 (p=0.066). The mean Spearman's

	Site 1) showed 40% and 79.5% of inhibition in May 2013 and February 2014,
	respectively (Table 1). Results showing the positions of AChE activity measurements
	in worms from both areas (Site 1 and Site 2) according to the four colour traffic light
	previously identified by Barrick et al. (2018) (red: lethal effects expected; orange:
	sublethal effects expected; yellow: range expected for non-impacted site; green:

Lower AChE activity was found in worms (H. diversicolor) sampled from Site 2 (10.07±5.80 nmole.min -1 .mg -1 proteins) than those in Site 1 (23.34±8.90 nmole.min - 1 .mg -1 proteins). AChE activities measured in H. diversicolor from Site 2 (compared to evidence of good health) are illustrated in Figure

2

. In February, AChE values were in the range where sub-lethal effects (worms from Site 1) and lethal effects (worms from correlation coefficient of 0.5404 revealed a positive correlation (p=0.0686) between AChE activities and MFB values. Data of densities and biomass of H. diversicolor at both sites are shown in Table 3. Densities and biomass of ragworms collected at Site 1 were significantly higher than those at Site 2 for both sampling dates (May 2013 and February 2014). (density and biomass) of worms. Results showed, at the biochemical level, a significant lower value of AChE activity in worms (H. diversicolor) from Site 2 (decrease of 40% in May 2013 and of 79% in February 2014 vs Site 1). Previously published studies reported that a reduction of 20% in AChE activity in fish and invertebrates indicates exposure to neurotoxic compounds (organophosphorus, pesticides), whereas depletion of AChE activity by 20-50% indicates sublethal impact (Amiard

Table 1 :

 1 AChE activity measured in H. diversicolor from site 1 and site 2 expressed in nmole.min -1 .mg -1 proteins. a significantly difference compared to b (p<0.05).

		AChE activity	AChE activity	P value
		(nmole.min -1 .mg -1 proteins)	(nmole.min -1 .mg -1 proteins)
		Site1	Site2
	May 2013	19.75±5.01 a	11.83±5.70 b	0.008
	February 2014	30.64±12.42 a	6.54±4.61 b	0.002

Table 3 .

 3 Intersite variations of H. diversicolor density (number of individuals N m -2 as mean±standard deviation) and biomass (Fresh weight FWg m -2 as mean±standard deviation) for each sampling period (May 2013 and February 2014). nd: not determined

		Density	Density	Biomass	Biomass
		(N.m -2 )	(N.m -2 )	(FWg.m -2 )	(FWg.m -2 )
		Site 1	Site 2	Site 1	Site 2
	May 2013	631.38±182.03	51.02±31.24	39.80±21.13	3.18±1.96
	February 2014	280.61±325.69	66.96±28.28	19.84±23.26	4.46±4.64

Acknowledgment. The research leading to these results has received funding from the RS2E-OSUNA project supported by the Région Pays de la Loire, France.