Charles Romain
email: charles.romain@mines-albi.fr

Damien Texier

Clara Desgranges

Jonathan Cormier

Stéphane Knittel

Daniel Monceau

Denis Delagnes

Oxidation of Thin Nickel-Based Superalloy Specimens: Kinetics Study and Mechanical Integrity

Keywords: Nickel-based superalloys, High-temperature oxidation, Breakaway, Gradient of microstructure and properties, Microtensile testing

The oxidation behavior of a nickel-based superalloy was investigated from 650 to 1,000 °C for up to 1,000 h in air. Samples with thicknesses ranging from 20 to 500 µm were used to document the evolutions of microstructure and oxide scale. Oxidation products and subsurface evolution of the metal microstructure were characterized by XRD and EDS analyses. Local breakaway was observed after 600 h at 800 °C and after 100 h at 900 °C due to the full consumption of Cr from the alloy. Room temperature tensile tests were performed on aged and pre-oxidized specimens with thicknesses ranging from 20 µm to 500 µm, at 800 °C. The results were compared to tensile tests performed on the as-received metallurgical state. Both size effects due to sample thickness reduction and to sample thickness/pre-oxidation width ratio were examined onto the mechanical behavior. Interestingly, the formation of TCP phases, the oxide layer and the subsequent Cr-depleted subsurface region from either aging or oxidation treatments impairs the mechanical integrity. Bulk and subsurface regions are impacted, especially for tens-of-micrometer thin samples.

Introduction

Nickel-based superalloys are used as structural components of several generations of aircraft engines. The reduction in fuel consumption, for both environmental and economic matters, involves the manufacturers to design engines that can withstand ever-higher operating temperatures. The temperature range for turbine disk during the engine cycle is usually low enough for high-temperature oxidation not to be considered as a critical design criteria. Therefore, disk alloys do not require any additional protections (coatings, air cooling, etc.). Nevertheless, temperature tends to increase in turbines, and parts are also designed thinner for mass reduction, so that surface reactivity has to be now taken into account [START_REF] Bose | High temperature coatings[END_REF].

High-temperature oxidation alters both the surface and the subsurface region of the materials, leading to the formation of outer and inner oxidation products, metal recession but also a gradient in chemical composition, microstructure and physical properties [START_REF] Young | High Temperature Oxidation and Corrosion of Metals Second Edition[END_REF]. Evaluating and predicting the environmentassisted microstructure evolutions due to surface reactivity to further model the local deformation/damage evolution within the material is a challenging issue for engine manufacturers [START_REF] Tong | Creep, fatigue and oxidation in crack growth in advanced nickel base superalloys[END_REF][START_REF] Pineau | High temperature fatigue of nickel-base superalloys -A review with special emphasis on deformation modes and oxidation[END_REF]. Therefore, the high-temperature oxidation behavior of a chromia-forming polycrystalline nickel-based superalloy was examined in the present study using ultrathin specimens. This was done to investigate the changes in the type of oxides being formed, i.e., the breakaway oxidation due to Cr depletion [START_REF] Evans | Mechanisms of Breakaway Oxidation and Application to a Chromia-Forming Steel[END_REF]. The influence of the surface reactivity on the tensile properties of thin microtensile specimens is to be addressed [START_REF] Zupan | Development of high-temperature microsample testing[END_REF]. Scaling approach using ultrathin specimens aims to echo the long-term exposure conditions needed to activate breakaway on conventional size specimens for very short exposures times, thanks to the limited amount of alloying elements [START_REF] Texier | Size effect on the plastic behaviour of polycrystalline materials: grain size, free surface and precipitation state[END_REF][START_REF] Texier | Screening breakaway in MCrAlY coatings for shortterm oxidation at high temperature[END_REF][START_REF] Young | A Simple Expression for Predicting the Oxidation Limited Life of Thin Components Manufactured from FCC High Temperature Alloys[END_REF].

The use of thin samples for oxidation and for mechanical testing results from the desire to exacerbate the effect of the oxidation-affected region up to breakaway phenomenon activation on the mechanical integrity. However, size reduction in mechanical samples to dimensions comparable with the microstructure scale could arise with technical and scientific issues. For a sample to be representative of the materials mechanical behavior, the threshold is set as the representative volume element (RVE) with a sufficient size to provide an appropriate homogenized behavior. This point leads to investigate the number of grains contained in the sample's thickness for a given polycrystalline material. The activation of breakaway mechanisms for reasonable oxidation durations requires samples as thin as technologically possible. A 20-µm-thin sample provides a 0.04-mm 2 section with 1 to 3 grains in thickness for an ASTM 10 grain-sized alloy but hundreds of thousands of grains in the 2-mm-wide and 5-mm-long gage region. As RVE is questionable in the section of samples and animates the scientific community disagreements on the use of micromechanics, the gage volume of the thinnest sample fulfills RVE requirements in terms of materials considerations and elastic behavior; the Young's modulus was found constant and independent of the sample thickness on all tensile tests. Disparities in the plastic behavior between surface grains and core grains might come from the presence of free surface compared to grain boundaries. A change in mechanical behavior is supposed from polycrystalline behavior to multicrystalline behavior for thin samples as demonstrated by Texier et al. on Inconel 718 and Keller et al. on pure nickel [START_REF] Texier | Size effect on the plastic behaviour of polycrystalline materials: grain size, free surface and precipitation state[END_REF][START_REF] Keller | Microstructural size effects on mechanical properties of high purity nickel[END_REF]. The transition of behavior is expected between 30 and 60 µm according to the average grain size of ASTM of 8-9.

The present paper intends to explore the microstructure evolutions and oxidation kinetics up to the breakaway using ultrathin samples. Their consequences onto the mechanical properties and lifespan of materials are also exposed. The experimental results were obtained after oxidation holding times up to 1,000 h between 650 and 1,000 °C for samples of different thicknesses ranging from 500 µm down to 20 µm with t/d ratio (thickness/grain size) of 2 to 50.

Experimental Procedures

The material used in the present study was a γ/γ' polycrystalline nickel-based superalloy with a grain size index of 8-9 (ASTM), i.e., 5-15 µm, and a typical composition presented in Table 1. The samples for oxidation test with a 10 × 15 mm size were taken from a forged piece via electrical discharge machining and further drilled (Ø 1.5 mm) for holding during oxidation tests. Samples with thicknesses ranging from 15 to 500 μm with thickness deviations less than 2 μm were obtained by mechanical polishing using a precision polishing jig following the method detailed in previous work by Texier et al. [START_REF] Texier | Micromechanical testing of ultrathin layered material specimens at elevated temperature[END_REF]. The samples were ground then polished using the jig on SiC paper down to a P2400/800 grit with sufficient material removal for each SiC paper to minimize residual stresses at the surface of the samples and produce a repeatable surface finishing. All sides were manually polished to the same surface finish prior to grinding. The final dimensions (after polishing, primary ultrasonic ethanol and drying cleaning) were measured via optical analysis using a LEICA DMS 300 calibrated microscope for length and width to allow surface computation through edge detection algorithms (OpenCV python library [START_REF] Bradski | The OpenCV library[END_REF]). Thickness was measured via a 1 µm precision micrometer on a 5-point grid. Specimens were finally cleaned following a sequence constituted by: ultrasonic acetone immersion (10 min), drying, ultrasonic ethanol (20 min) and final drying, followed by the initial mass measurement. Isothermal oxidation was performed in static laboratory air using a Nabertherm furnace. Oxidation samples were suspended using platinum wires during oxidation tests at 650, 700, 800, 900 and 1,000 °C. For each temperature, different holding times were investigated: 10, 30, 100, 200, 600 and 1,000 h. Samples were introduced in the hot furnace and air-quenched after oxidation interruption. The temperature was controlled via two K-type thermocouples positioned at different locations inside the furnace, one being in contact with the samples.

Mass gain measurements were evaluated using a Sartorius MC5 with 2 µg accuracy.

X-ray diffraction (XRD) analyses were carried out using a X'PERT-PANALYTI-CAL from PHILIPS with a Cu-Kα radiation (λ = 1.54 Å), covering a range of angles (2θ) from 15° to 100° with a step size of 0.033°. Cross-sectional (supported by metallic rings in cold resin) and surface observations were performed using a field-emission gun scanning electron microscope (FEG-SEM) Nova NanoSEM 450 from THERMOFISHER SCIENTIFIC in a backscattered electron mode to document the oxide scale and the in-depth microstructure evolution of the alloy. Chemical analyses using energy-dispersive X-ray spectroscopy (EDS) were conducted using a FEG-SEM JEOL JSM 7800F Prime equipped with an EDS SDD X-Max 80 mm 2 detector from Oxford Instruments to characterize the depletion profile and progressive consumption of elements involved in the oxidation reactions. Mass gain measurements together with XRD and micrograph observations enabled the characterization of the oxide scale, the oxidation kinetics as well as the microstructural evolutions up to potential breakaway phenomenon.

For the mechanical tests, three different metallurgical states were investigated: (1) as-received state, corresponding to pre-machining ring-rolled material, (2) "aged" state, corresponding to samples with ground surfaces after heat treatment to remove the surface/subsurface-affected layer (oxidized or depleted in Cr) and ( 3) "oxidized" state, corresponding to ground samples before heat treatment to investigate the surface/subsurface-affected layer effects. Mechanical tests were performed using specimens of 500 µm, 100 µm, 50 µm and 20 µm thicknesses for the three metallurgical states. Thicknesses of 250, 75 and 35 µm were added for as-received state to better characterize the effect of sample thickness onto the mechanical response. Tensile tests were conducted using an electromechanical INSTRON tensile machine equipped with a SCAIME 500 N load cell for samples thicknesses below 150 µm and 5 kN load cell for thicker samples with constant cross-head displacement rate. Elongation measurement of the gage area was assessed using contactless extensometry using digital image correlation techniques. A black speckle was sprayed onto a white background at the samples surface and was optically tracked synchronously to the room temperature tension tests. Displacement/deformation was computed using VIC digital image correlation (DIC) algorithm [START_REF] Sutton | Image Correlation for Shape, Motion and Deformation Measurements: Basic Concepts,Theory and Applications[END_REF].

Oxidation Behavior

The mass changes recorded for the 500-μm-thick samples for all oxidation durations are reported in Fig. 1a. A secondary y-axis is used to plot the mass gain in this large temperature window (left y-axis for 650 °C to 800 °C and secondary right y-axis for 900 °C and 1000 °C). The oxidation behavior reported in Fig. 1a follows a parabolic law in agreement with the Wagner's theory [START_REF] Wagner | Beitrag zur Theorie des Anlaufvorgangs[END_REF]. Experimental mass gain data are presented in Fig. 1a, alongside the associated oxidation parabolic rate constant k P value for each temperature on the upright corner. The oxidation kinetics are in agreement with those of similar alloys (see Fig. 11 in [START_REF] Malacarne | Long-term isothermal oxidation behavior of two industrial polycrystalline nickel base alloys at 700°C -Evaluation of intergranular oxidation distribution and kinetic[END_REF]). Figure 1b models the time to full Cr consumption expected for sample thicknesses in range 20-500 μm. The dots represent experimental points, and the lines model the couple thickness/ time for the studied temperatures according to Eq. (1). Experimental points located above the model lines are supposed to reach breakaway, with experimental confirmation of breakaway presence with full dots.

where t stands for time to full Cr consumption (hours), k P is the parabolic constant (g.cm -2 ), Mi the molar mass of the i element (g.mol -1 ), ρ the density of the alloy (g.cm -3 ), W i the weight percent of the i element (wt %) and e the thickness of the sample (cm).

With Eq. ( 1), it is assumed that chromium is the only chemical element being oxidized ( Cr 1) is based on a simple mass balance between the oxygen uptake to form chromia oxide layer with parabolic kinetics and the total amount of chromium available in the material. This equation is a simple form of Eq. 20 in ref. [START_REF] Young | A Simple Expression for Predicting the Oxidation Limited Life of Thin Components Manufactured from FCC High Temperature Alloys[END_REF], for a full depletion of Cr neglecting the diffusion of Cr in the alloy. Hence, it leads to an overestimation of the time to breakaway. It is then used as a first approximation.

+ 3 4 O 2 = 1 2 Cr 2 O 3 ). Equation (
Bulk specimens, i.e., non-limited materials, developed an outer and continuous Cr 2 O 3 layer topped with sparsely distributed TiO 2 rutile. Internal oxidation with formation of alumina Al 2 O 3 oxide for all the exposure times is also evidenced for every test performed at 800, 900 and 1000 °C (see Fig. 2). Chen et al. reported similar oxidation products in suchlike alloys [START_REF] Chen | Oxidation behavior of several chromia-forming commercial nickel-base superalloys[END_REF]. No spallation was observed after heat treatment up to 1000 °C. External surface oxidation evolves with the samples thickness, oxidation duration and temperature increase, leading to the formation of fast-growing oxides, i.e., spinels and NiO, also known as local breakaway phenomenon. The activation energy for the oxidation temperature range 650-1000 °C is Ea = 314kJ.mol -1 .

Some cross-sectional SEM analysis of oxidized 20-µm-thick samples is presented in Fig. 2, in the case of oxidation tests performed at 800 °C for 600 h (Fig. 2 a) and at 900 °C for 100 h (Fig. 2 b). XRD and EDS analyses confirmed the formation of oxides similar to the ones formed on thicker samples observed in Fig. 2 alongside NiO formation above the external chromia layer are also evidenced via XRD and in (Fig. 2b and Fig. 3e). Thébaut, Németh and Chateau drove the same conclusions studying the oxidation kinetics of AD730™ and Udimet 720Li, which are similar to the present material in terms of chemical composition, microstructure and processing method [START_REF] Chateau | Oxidation-assisted creep damage in a wrought nickel-based superalloy: Experiments and modelling[END_REF][START_REF] Thébaud | Etude des relations entre microstructure et propriétés mécaniques du nouveau superalliage base nickel AD730 TM[END_REF][START_REF] Németh | Environmentally-assisted grain boundary attack as a mechanism of embrittlement in a nickel-based superalloy[END_REF]. Ultrathin samples, i.e., volume-limited samples, demonstrate equivalent oxide growth kinetics for shorter times compared to thick samples until a threshold time for which the bulk (i.e., core) Cr concentration decreases (illustrated in Table 2). This results in the Cr concentration to drop at the oxide/metal interface. With regard to the depletion profile, the decrease of Cr content (i.e., Cr activity) enables other elements to oxidize forming faster growing oxides.

Microstructural Evolutions due to Aging and Oxidation

High-temperature exposure alters the microstructure of the studied material due to the effects of both aging and oxidation. First, topologically closed packed (TCP) µ phases alongside borides were found to form at 800 °C within the core of bulk samples for durations longer than 200 h. This microstructure evolution is illustrated in Fig. 3 b for a 500-µm-thick sample (white particles preferentially located at grain boundaries and primary precipitates). The formation of TCP phases is also evidenced at 700 °C for 4,000 h. The time-transformation-temperature (TTT) diagram built for Rene 65 by Wessman exhibits such metallurgical changes for such time/ temperature pairs [START_REF] Wessman | Thermal Stability of Cast and Wrought Alloy Rene 65[END_REF], also experimentally confirmed by Laurence [START_REF] Laurence | Impact of the Solution Cooling Rate and of Thermal Aging on the Creep Properties of the New Cast & Wrought René 65 Ni-Based Superalloy[END_REF][START_REF] Laurence | Impact du sur-vieillissement métallurgique sur le comportement et la durabilité du nouveau superalliage pour disque de turbine René 65[END_REF]. Aging also impairs the / ′ precipitation state in this temperature window, but this effect was not quantitatively investigated in the present study. Oxidation leads to subsurface microstructural modifications, as shown in Fig. 3. The depleted Cr layer grew with the exposure time and temperature due to the formation of the external Cr 2 O 3 layer. The oxide formation is accompanied by the extension of a subsurface primary-and secondary-precipitates-depleted layer, as reported in ref [START_REF] Amaro | High temperature oxidation and γ depletion in the single-crystal superalloy PWA 1484[END_REF]. Surface reactivity also led to an internal oxide at grain boundaries. In this affected region, the alloy is depleted from both the primary, secondary and tertiary γ' and of the TCP.

Depletion and oxide thicknesses were quantitatively analyzed using images of cross-sectional BSE and EDS observations. The edge detection method of elements and linear regression between elements in case of discontinuities were used to assess both the thickness of the depleted layers and oxide scales. The results are reported in Table 2 for oxidation conditions 800 °C -300 h, 900 °C -100 h and 300 h. Similar extension of the oxidation-affected subsurface region and oxide scales was found regardless of the sample thickness for 300 h at 800 °C, i.e., as long as breakaway condition is not met (Fig. 3). The disparities in external oxide thickness at 900 °C result from local breakaway phenomena associated with the formation of fast-growing spinels above the chromia layer. Intergranular oxide (Al 2 O 3 ) is microstructure dependent as it grows along grain boundaries, which results in important disparities due to grain boundaries discontinuous location. As-received alloy presents a γ′ volume fraction of 40%. Aging heat treatment induces microstructural evolutions with possible impact on the mechanical properties: for γ′ I primary precipitates (1 to 5 µm in initial size, evolving according to an LSW law r 3 -r 3 0 = kt [24]) and for γ′ II sec- ondary precipitates (40 nm in initial size, diffusion-controlled growth [START_REF] Wessman | Physical Metallurgy of Rene 65, a Next-Generation Cast and Wrought Nickel Superalloy for use in Aero Engine Components[END_REF]). The precipitates depletion zone was evaluated based on the primary precipitates distribution on 500-µm-thick samples. EDS line scans analyses demonstrate elements depletion thicknesses from metal/oxide interface to 99% nominal composition as summarized in Table 2. The bulk concentration of chromium in the 20-µm-thick oxidized samples is also presented since the Cr-depleted zone reaches the center of the smallest samples.

The chromium depletion ensues from supplying the chromia reaction. The precipitates provide aluminum and titanium to the external titania and internal alumina formations but also participate to the chromia reaction as a non-negligible chromium concentration can be found onto the primary precipitates [START_REF] Singh | Mechanisms related to different generations of γ' precipitation during continuous cooling of a nickel base superalloy[END_REF]. The TCP-depleted layer was unexpected to overtake the Cr depletion depth as entities depletions are expected to outcome from alloys starvation. Although chromium depletion of tens of micrometers is observed for bulk specimens, thin specimens with initial thickness of 20 µm manifest bulk concentration starvation. The γ matrix reveals a Cr concentration of 18 ± 1 weight percent. A bulk concentration reduction down to 50% can be observed in the 20-µm-thick sample exposed at 800 °C for 300 h. The Cr depletion does not yet affect the chromia layer thickness nor the internal intergranular oxidation depth and primary precipitates depletion width as corroborated by Fig. 3. No external spinel was observed above the chromia layer for this condition. However, spinel formation occurred for 900 h after 100 h with bulk chromium concentration of 1.5 weight percent. The breakaway mechanism is initiated with spinel formation yet controlled via diffusion of entities through the chromia layer leading to no apparent breakaway onto the mass gain measurements as observed by Fedorova et al. on AM1 alloy [START_REF] Fedorova | Quantification of growth kinetics and adherence of oxide scales formed on Ni-based superalloys at high temperature[END_REF]. Local fast-growing spikes were observed with a maximal oxide thickness of 45 µm for 900 °C -300 h on a 20-µm-thick sample (see Fig. 3e). As 1.5 wt% is very small in comparison with the initial content, and because there is almost no Cr concentration gradient when breakaway occurs, using the simplified approach of Eq. 1 to determine the time to breakaway is justified.

The microstructure gradient resulting from the surface reactions is expected to impact the mechanical behavior of the sample. The secondary and tertiary precipitates depletion layer, known as the hardening phase of γ/γ′ alloys, along with grain size evolution and a much wider size-reduced γ′ layer introduces an external ductile softer layer coherent with the external harder but brittle chromia layer. The impact of this microstructural gradient with regard to the thickness of the sample is to be addressed in the following mechanical results section.

Tensile Behavior of Pre-Oxidized Ultrathin Samples

Tensile tests were performed at room temperature on samples with thicknesses ranging from 20 to 500 µm with different metallurgical states, i.e., the "as-received" state, aged and oxidized states at 800 °C for 200 and 600 h. Stress-strain curves of pre-oxidized specimens during 200 h and 600 h at 800 °C are illustrated in Fig. 4. A significant thickness debit, i.e., a significant drop of the strength properties and ductility due to oxidation, is evidenced. Tensile properties are significantly affected with the increase of the pre-oxidation time and the decrease in sample thickness. While samples thicker than 50 µm demonstrate significant plasticity with strain hardening, 20-µm-thick samples failed in a nearly brittle manner due to the significant portion of the oxidation-affected region.

The evolution of the macroscopic tensile properties as a function of the sample thickness for all the sample variants has been estimated from the stress-strain curves. The values of yield strength (YS), the ultimate tensile strength (UTS) and the ductility have been normalized by same proprieties obtained for thick "asreceived" specimens and are compared Fig. 5. Each point in Fig. 5 represents an average of four to six mechanical tests. The normalization aims to directly evidence how aging and oxidation affect macroscopic tensile properties of the virgin material.

First, the effect of using thin specimen on the tensile test results can be evaluated on non-oxidized samples. While no evolution of the yield strength (YS) is observed for samples thicker than 100 µm, the yield strength of the as-received material dropped down for thinner samples, as illustrated in Fig. 5a. This size effect is typical of the polycrystal-to-multicrystal transition due to the presence of the free surface [START_REF] Keller | Microstructural size effects on mechanical properties of high purity nickel[END_REF]. Similar trends are evidenced for the evolution of the ductility and the UTS as a function of the thickness, with a transition thickness of by 67.5% for 20-µm-thick samples compared to the reference polycrystalline behavior. The free-surface grains are reported not to respond similarly to core grains due to change in grain confinements. The absence of grain boundaries to pin dislocations favors dislocation escapes and subsequently delays dislocation cell and network to form, participating in strain hardening [START_REF] Keller | TEM study of dislocation patterns in near-surface and core regions of deformed nickel polycrystals with few grains across the cross section[END_REF][START_REF] Mughrabi | Some consequences of surface and size effects in plastically deformed copper single crystals[END_REF][START_REF] Fourie | Sub-surface dislocation structure of deformed copper[END_REF].

Second, the effect of microstructural aging alone can be studied looking at the aged specimens behavior compared to as-received specimens. Indeed, it has been verified that aged material is deprived of any oxidation-affected layer. Furthermore, the microstructural evolutions are then present in the entire volume of the sample. Aged samples at thickness 20 µm and 50 µm exhibit similar mechanical properties in terms of YS, UTS and ductility compared to the as-received material for equivalent sample thicknesses. The aged 100-µm-and 500-µm-thick samples present minor variations around the as-received state for YS and UTS but noticeable decrease in ductility. A gap appears for 100 µm and 500 µm thicknesses among the 200 h and 600 h heat treatments, with superiority to the 200 h. The multicrystal-to-polycrystal transition is less explicit than as-received state with up to 8% variations in YS, UTS and ductility for both heat treatment times, even though a clear slope change is observed at 100 µm. The predominant differences between 200 and 600 h aged materials arise from the presence of TCP phases and γ′ II and γ′ III size evolutions. TCP appears along grain boundaries and primary precipitates in 600 h heat-treated samples. TCP particles are known to play a key role in the creep life of superalloys but reveal minor impact on their tension behavior, especially at room temperature [START_REF] Laurence | Impact du sur-vieillissement métallurgique sur le comportement et la durabilité du nouveau superalliage pour disque de turbine René 65[END_REF][START_REF] Wessman | Physical Metallurgy of Rene 65, a Next-Generation Cast and Wrought Nickel Superalloy for use in Aero Engine Components[END_REF]. Minor microstructural evolutions are expected for the primary precipitates, resulting in minor tension behavior changes. This quantitative evaluation for the modification of mechanical properties cannot be directly linked to the quantification of the depth of the subsurface-affected zone determined in Table 2 since the oxidized samples characterized do not correspond to same oxidation treatment as samples used for the tensile tests, not yet characterized with same details. However, a discussion on tendencies is still possible.

Oxidized samples demonstrate a drop of mechanical properties compared to both as-received and aged metallurgical states. 20-µm-thick samples broke at the onset of plasticity, around 2 to 3% of the maximal true strain, with a YS and UTS significantly below the as-received and aged states. Thin samples for 200 h and 600 h oxidation exhibit lower mechanical properties in YS and UTS, with up to 36.6 and 50.2% YS decrease, and up to 56.3% and 73.7% UTS decrease for 20-µm-thick sample for oxidation times of 200 h and 600 h, respectively. Local breakaway is expected to emerge for the condition 800 °C oxidized 600 h, even so the tendency does not reveal any major breakdown compared to the 200 h oxidized samples. Mechanical properties drop from oxidation due to the brittle external chromia layer, the presence of internal alumina spikes and a ductile primary precipitates-free layer. This observation is accentuated with an increase of the oxidation-affected thickness versus total thickness ratio. External and intergranular oxidation along with the Crand TCP-depleted layers (as evaluated previously in Table 2 and shown in Fig. 3) demonstrates the critical role of surface reactivity onto the mechanical properties, as revealed by the drop from aged to oxidized results. The low mechanical performance of samples oxidized during 600 h compared to the ones oxidized during 200 h arises from the extension of the oxidation-affected layer increasing with the oxidation duration due to premature damage within the oxidation-affected layer. Such low results on thin sample could emanate from the damaging of grain boundaries on the entire thickness from oxidation, leading to a major drop in ductility [START_REF] Németh | Environmentally-assisted grain boundary attack as a mechanism of embrittlement in a nickel-based superalloy[END_REF].

Conclusions

The study of oxidation kinetics and its influence on the mechanical behavior of a turbine disk γ/γ′ superalloy highlighted the following main conclusions: o Isothermal oxidations in air follow parabolic regime for all temperature studied between 650 °C and 1000 °C. The oxidation kinetics are in agreement with similar type of alloys. Oxidation leads to formation of several oxides: a dense external chromia layer, sparsely distributed titania islets and intergranular alumina regardless of the temperature and sample thickness. Spinel oxide phase is to occur for high temperature, long duration and low thickness samples, leading to local breakaway phenomenon. Size effect, i.e., volume-limited effects, has been here evidenced using ultrathin samples down to 15 µm. o Microstructural evolution inherent to oxidation at 800 °C leads to surface and subsurface alteration with chromium-, precipitates-and TCP-depleted layers, whose respective depth had been determined on sample cross-sectional observations in order to establish the influence subsurface changes on mechanical response. o The mechanical behavior of as-received metallurgical state using ultrathin samples evidenced a multicrystalline-to-polycrystalline transition for samples about 100 µm thick, affecting the YS, UTS and strain-to-failure. o Samples aged for 200 h and 600 h at 800 °C show moderate evolutions of tensile properties when compared to the as-received reference state. The impact of the metallurgical evolutions among the two heat treatment times is to be observed on YS, UTS and ductility for thickness even superior to 100 µm. o Oxidized specimens are highlighting the consequences of the microstructural gradient and subsurface embrittlement in the oxidation-affected layer. A significant drop is observed in YS, UTS and ductility for all thicknesses, especially compared to specimens without the surface/subsurface-affected layers. The thickness of the gradient of microstructure, larger for 600 h than for 200 h heat treatment, can be witnessed with the lower mechanical performances of samples oxidized during 600 h.

3 ⋅ 2 Fig. 1 a

 321 Fig. 1 a Mass grain (650 °C to 800 °C on the left y-axis, 900 °C to 1000 °C on the right y-axis) versus time and parabolic rate constant for 500-µm-thick samples, b time to full Cr consumption as function of the thicknesses for different temperatures ranging from 650 °C to 1000 °C

Fig. 2 Fig. 3 a 2

 232 Fig. 2 SEM BSE cross-sectional observation of initial 20-µm-thick samples oxidized at a 800 °C during 600 h and b 900 °C during 100 h

Fig. 4 Fig. 5

 45 Fig.[START_REF] Pineau | High temperature fatigue of nickel-base superalloys -A review with special emphasis on deformation modes and oxidation[END_REF] True strain vs true stress for oxidized condition at 800 °C for 200 h and 600 h

Table 1

 1

	Nominal composition of the studied nickel-based	Ni	Cr	Co	Al	Ti	Fe
	superalloy (wt %)	Bal	15 ± 2%	13.5 ± 2%	2 ± 0.5%

± 1% 1 ± 0.5%

Acknowledgements

The authors would like to acknowledge Karim Choquet (ICA) and Remi Roumiguier (MIDIVAL) for technical help. This study was supported by SAFRAN Tech, SAFRAN Aircraft Engine and the Agence Nationale de la Recherche (ANR) [ANR-18-CE08-0003; ANR-JCJC-COMPAACT project funded from the AAPG2018].

Authors and Affiliations

Charles Romain 1 • Damien Texier


Adélaïde Lerebours
email: adelaide.lerebours@univ-lr.fr

Svetlana Murzina

Knut You Song

Erik Tollefsen

Maura Benedetti

Francesco Regoli

Jeanette M Rotchell

Jasmine Nahrgang

Knut Erik Tollefsen

Susceptibility of polar cod (Boreogadus saida) to a model carcinogen

Keywords: Carcinogenesis, polar cod, benzo(a)pyrene, cancer genes, DNA repair genes

Introduction

Polycyclic aromatic hydrocarbons (PAHs) are a very large group of ubiquitous organic compounds that can originate from petrogenic, pyrogenic, biogenic and diagenic sources. A number of PAHs are identified as having carcinogenic properties and have been associated to an increased incidence of liver tumours in flatfish in highly polluted environments [START_REF] Malins | Toxic-Chemicals in Marine Sediment and Biota from Mukilteo, Washington -Relationships with Hepatic Neoplasms and Other Hepatic-Lesions in English Sole (Parophrys Vetulus)[END_REF][START_REF] Myers | Relationships between hepatic neoplasms and related lesions and exposure to toxic chemicals in marine fish from the U.S. west coast[END_REF][START_REF] Harshbarger | Epizootiology of neoplasms in bony fish of North America[END_REF][START_REF] Vogelbein | Hepatic neoplasms in the mummichog Fundulus heteroclitus from a creosote-contaminated site[END_REF][START_REF] Baumann | Long term trends in liver neoplasm epizootics of brown bullhead in the Black River, Ohio[END_REF]. This pathology has been used to monitor the effects of exposure to PAHs and the health of marine ecosystem since the 1980s [START_REF] Malins | Toxic-Chemicals in Marine Sediment and Biota from Mukilteo, Washington -Relationships with Hepatic Neoplasms and Other Hepatic-Lesions in English Sole (Parophrys Vetulus)[END_REF]Veethaak and Ap Rheinallt, 1992) and its assessment recommended by the International Council for Exploration of the Sea (ICES) and the Oslo and Paris Convention (OSPAR) Joint Assessments and Monitoring Programme (JAMP) [START_REF] Lyons | Using biological effects tools to define Good Environmental Status under the European Union Marine Strategy Framework Directive[END_REF].

Benzo(a)pyrene (BaP) is a well-known pyrogenic carcinogen in a plethora of animals such as marine mammals [START_REF] Acevedo-Whitehouse | Hepatic DNA Damage in Harbour Porpoises (Phocoena phocoena) Stranded along the English and Welsh Coastlines[END_REF][START_REF] Poirier | Intestinal Polycyclic Aromatic Hydrocarbon-DNA Adducts in a Population of Beluga Whales with High Levels of Gastrointestinal Cancers[END_REF], fish [START_REF] Wang | Expression of CYP1C1 and CYP1A in Fundulus heteroclitus during PAH-induced carcinogenesis[END_REF][START_REF] Wills | Comparative chronic liver toxicity of benzo[a]pyrene in two populations of the atlantic[END_REF] and mice [START_REF] Kasala | Benzo(a)pyrene induced lung cancer: Role of dietary phytochemicals in chemoprevention[END_REF][START_REF] Chen | Anti-Inflammatory and Apoptotic Signaling Effect of Fucoxanthin on Benzo(A)Pyrene-Induced Lung Cancer in Mice[END_REF]. The reference oral dose below which no effect is expected is 3.10 -4 µg BaP /g per day, based on animal and human studies (reviewed in EPA/635/R-17/003). The metabolites generated by endogenous metabolism (biotransformation) are highly genotoxic. Phase I biotransformation of BaP is mediated by cytochrome P450 (CYP) enzymes and produces highly reactive metabolic intermediates such as diol-epoxide, dihydrodiol and 3-hydroxybenzo(a)pyrene (3-OH-BaP) [START_REF] Karle | Crystal and molecular structure of a benzo[a]pyrene 7,8-diol 9,10-epoxide N2-deoxyguanosine adduct: Absolute configuration and conformation[END_REF]Zhu et al., 2008;Rey-Salgueiro et al., 2011). Those metabolites form DNA adducts that interfere with DNA repair and replication (Phillips and Arlt, 2007). This represents a critical event in the initiation of tumorigenesis, potentially leading to mutations within specific regions of DNA, such as proto-oncogenes and tumour suppressor genes [START_REF] Rotchell | Cloning of the Retinoblastoma cDNA from the Japanese medaka (Oryzias latipes) and preliminary evidence of mutational alterations in chemically-induced retinoblastomas[END_REF][START_REF] Corbier | Isolation of the retinoblastoma cDNA from the marine flatfish dab (Limanda limanda) and evidence of mutational alterations in liver tumors[END_REF][START_REF] Lerebours | Genetic alterations and cancer formation in a European flatfish at sites of different contaminant burdens[END_REF], 2016). The carcinogenicity of BaP has been well studied in several temperate fish species where specific DNA adducts are used as markers for exposure and potential genotoxic effects. Exposure to BaP specifically caused DNA adducts in fish such as pale chub (Zacco platypus) [START_REF] Lee | Cytochrome P450 System Expression and DNA Adduct Formation in the Liver of Zacco platypus Following Waterborne Benzo(a)pyrene Exposure: Implications for Biomarker Determination[END_REF] and killifish species (Fundulus grandis and F. similis) [START_REF] Willett | Exposure of killifish to benzo[a]pyrene: comparative metabolism, DNA adduct formation and aryl hydrocarbon (Ah) receptor agonist activities[END_REF][START_REF] Rose | DNA adducts in hematopoietic tissues and blood of the mummichog (Fundulus heteroclitus) from a creosote-contaminated site in the Elizabeth River, Virginia[END_REF][START_REF] Rose | Persistence of benzo[a]pyrene-DNA adducts in hematopoietic tissues and blood of the mummichog, Fundulus heteroclitus[END_REF]. Moreover, BaP exposure was associated with neoplastic lesions in brown bullhead (Ameiurus nebulosus) [START_REF] Ploch | Comparative in Vitro and in Vivo Benzo[a]pyrene-DNA Adduct Formation and Its Relationship to CYP1A Activity in Two Species of Ictalurid Catfish[END_REF], English sole (Parophrys vetulus) [START_REF] Reichert | Molecular epizootiology of genotoxic events in marine fish: Linking contaminant exposure, DNA damage, and tissue-level alterations[END_REF] and rainbow trout [START_REF] Hendricks | Hepatocarcinogenicity of benzo[a]pyrene to rainbow trout by dietary exposure and intraperitoneal injection[END_REF]. PAH-induced lesions have also recently been suggested in marine mammals such as harbour porpoises (Phocoena phocoena) [START_REF] Acevedo-Whitehouse | Hepatic DNA Damage in Harbour Porpoises (Phocoena phocoena) Stranded along the English and Welsh Coastlines[END_REF] and beluga whales [START_REF] Poirier | Intestinal Polycyclic Aromatic Hydrocarbon-DNA Adducts in a Population of Beluga Whales with High Levels of Gastrointestinal Cancers[END_REF].

Pollution induced cancer affects many aquatic species and represents a growing concern for aquatic wildlife (for a review see [START_REF] Baines | Linking pollution and cancer in aquatic environments: A review[END_REF].

Tumourigenesis is a progressive process characterised by different stages for which the underlying molecular steps and the role of environmental exposure are not always wellknown. Nonetheless, liver tumourigenesis has been well studied in flatfish [START_REF] Stentiford | Effect of age on liver pathology and other diseases in flatfish: Implications for assessment of marine ecological health status[END_REF][START_REF] Lerebours | Advanced diagnostics applied to fish liver tumours: relating pathology to underlying molecular aetiology[END_REF]2014;[START_REF] Elias | Conservation issues: Tundra ecosystems[END_REF] and in the model fish Japanese medaka (Oryzias latipes) [START_REF] Rotchell | Cloning of the Retinoblastoma cDNA from the Japanese medaka (Oryzias latipes) and preliminary evidence of mutational alterations in chemically-induced retinoblastomas[END_REF] and zebrafish (Danio rerio) [START_REF] Li | Males develop faster and more severe hepatocellular carcinoma than females in krasV12 transgenic zebrafish[END_REF][START_REF] Poirier | Intestinal Polycyclic Aromatic Hydrocarbon-DNA Adducts in a Population of Beluga Whales with High Levels of Gastrointestinal Cancers[END_REF]. DNA repair mechanisms have been associated with tumour formation perturbing several steps of the nucleotide excision repair (NER) pathway, which recognises and repairs DNA adducts induced by numerous environmental mutagens, including PAHs [START_REF] Gillet | Molecular mechanisms of mammalian global genome nucleotide excision repair[END_REF][START_REF] Rastogi | Molecular mechanisms of ultraviolet radiation-induced DNA damage and repair[END_REF]. While such mechanisms involved in the development of tumours in certain fish species are well characterised, a substantial knowledge gap exists for non-model and ecologically important species inhabiting remote regions in particular. The Arctic is currently experiencing a rapid decline in sea ice [START_REF] Kumar | Spatio-temporal change and variability of Barents-Kara sea ice, in the Arctic: Ocean and atmospheric implications[END_REF] that may lead to a significant increase in marine shipping [START_REF] Ho | The implications of Arctic sea ice decline on shipping[END_REF], oil and gas exploration and operation [START_REF] Elias | Conservation issues: Tundra ecosystems[END_REF], and tourism [START_REF] Meier | Arctic sea ice in transformation: A review of recent observed changes and impacts on biology and human activity[END_REF] and associated release of potential carcinogenic contaminants [START_REF] Elias | Conservation issues: Tundra ecosystems[END_REF]. The polar cod (Boreogadus saida) is a keystone fish species in the arctic marine ecosystem due to its abundance, distribution and central role in the food web [START_REF] Welch | Energy flow through the marine ecosystem of the Lancaster Sound region[END_REF]. Polar cod has been considered a model fish for arctic ecotoxicology studies [START_REF] Jonsson | The Arctic is no longer put on ice: evalua-tion of polar cod (Boreogadus saida) as a monitoring species of oil pollution in cold waters[END_REF][START_REF] Nahrgang | PAH biomarker responses in polar cod (Boreogadus saida) exposed to benzo(a)pyrene[END_REF]Nahrgang et al., , 2010a,b,c),b,c). The toxicity of petroleum compounds on the physiology of polar cod has been well studied [START_REF] Geraudie | In vivo effects of en vironmental concentrations of produced water on the reproductive function of polar cod (Boreogadus saida)[END_REF][START_REF] Bender | Effects of chronic dietary petroleum exposure on reproductive development in polar cod (Boreogadus saida)[END_REF][START_REF] Nahrgang | Early life stages of an arctic keystone species (Boreogadus saida) show high sensitivity to a water-soluble fraction of crude oil[END_REF][START_REF] Vieweg | Effects of dietary crude oil exposure on molecular and physiological parameters related to lipid homeostasis in polar cod (Boreogadus saida)[END_REF][START_REF] Nahrgang | Growth and metabolism of adult polar cod (Boreogadus saida) in response to dietary crude oil[END_REF] but the tumourigenic potential of a potent carcinogen remains unknown in that species. A few studies however have reported a potential susceptibility to carcinogenic contaminants including BaP. The hepatic metabolism of BaP is particularly efficient in polar cod and a significant increase of covalently bound reactive intermediates of BaP in the bile of fish has been found after dietary exposure to BaP [START_REF] Ingebrigtsen | Disposition and cellular binding of 3 H-benzo(a)pyrene at subzero temperatures: studies in an aglomerular arctic teleost fish -the polar cod (Boreogadus saida)[END_REF][START_REF] Bakke | Comparative absorption and tissue distribution of 14 C-benzo(a)pyrene and 14 C-phenanthrene in the polar cod (Boreogadus saida) following oral administration[END_REF].

These reactive intermediates were found to induce the formation of DNA adducts in the liver of that species [START_REF] Aas | DNA adduct levels in fish from pristine areas are not detectable or low when analysed using the nuclease P1 version of the 32 P-postlabelling technique[END_REF]. This genotoxic effect can in turn result in cellular abnormalities and cancer initiation. Finally, a recent study showed that expression of genes involved in DNA repair and cell cycle regulation processes was modified in liver of polar cod dietary exposed to BaP [START_REF] Song | Transcriptomic analysis reveals dose-dependent modes of action of benzo(a)pyrene in polar cod (Boreogadus saida)[END_REF].

In order to evaluate the susceptibility of polar cod to a carcinogenic compound, adult specimens were exposed under laboratory conditions for six months to control, 0.03 µg BaP/ g fish/ week and 0.3 µg BaP/ g fish/ week dietary BaP. Selected body condition indexes, bile metabolite concentrations and transcriptional responses of genes involved in DNA adduct recognition (xpc), helicase activity (xpd), DNA repair (xpf, rad51) and tumour suppression (tp53) were assessed after 0, 1, 3 and 6 months of exposure. Blood and spleen micronuclei, nuclear abnormalities and liver histopathological endpoints were assessed at the end of the experiment.

The sampling times were selected because carcinogenesis is a long-term process.

They were comparable to the exposure durations used in several studies interested in carcinogenesis in European eel [START_REF] Nogueira | Mutation analysis of ras gene in the liver of European eel (Anguilla anguilla L.) exposed to benzo[a]pyrene[END_REF], brown bullhead and channel catfish (Ploch et al., 1985) rainbow trout [START_REF] Hendricks | Hepatocarcinogenicity of benzo[a]pyrene to rainbow trout by dietary exposure and intraperitoneal injection[END_REF][START_REF] Black | A Reliable, Efficient, Microinjection Apparatus and Methodology for the In Vivo Exposure of Rainbow Trout and Salmon Embryos to Chemical Carcinogen[END_REF] and coho salmon [START_REF] Black | A Reliable, Efficient, Microinjection Apparatus and Methodology for the In Vivo Exposure of Rainbow Trout and Salmon Embryos to Chemical Carcinogen[END_REF] exposed to BaP. The BaP doses selected were lower than the concentrations frequently used in previous studies. They were 10 and 100 times lower than the concentration of 3 µg BaP/g of fish /week [START_REF] Colli-Dula | Transcriptome analysis reveals novel insights into the response of low-dose benzo(a)pyrene exposure in male tilapia[END_REF] that induced a decrease of body indexes in Nile tilapia after one month of exposure. In addition, our highest concentration was 4 times lower than the lowest concentration used in the study of [START_REF] Song | Transcriptomic analysis reveals dose-dependent modes of action of benzo(a)pyrene in polar cod (Boreogadus saida)[END_REF] that found gene expression changes in polar cod after two weeks of exposure, a twelve times shorter exposure duration.

Methods

Fish collection and exposure

Adult polar cod (4 years old) were collected along the west coast of the Svalbard archipelago (Norway) onboard RV Helmer Hanssen in January 2014 using a Campelen bottom trawl (at 200m depth) and a fish-lift [START_REF] Holst | Fish-lift: a device for sampling live fish with trawls[END_REF]. At the Tromsø aquaculture research station (Havbrukstasjon i Tromsø), fish were kept in 3000 L acclimation tank under a natural light and temperature (1.5 -3 °C) regime of 79°N (based on mooring data in [START_REF] Wallace | Comparison of zooplankton vertical migration in an ice-free and a seasonally ice-covered Arctic fjord: An insight into the influence of sea ice cover on zooplankton behavior[END_REF]. During this period, fish were fed until satiation with thawed Calanus sp. copepods (Calanus AS, Tromsø). Ninety fish were selected based on similar length (15 ± 1 cm) and weight (25 ± 7 g) for the experiment (June 2014).

Polar cod were dietarily exposed to 0, 0.03 and 0.3 µg BaP per gram fish per week, for 6 months (2 nd of July 2014 to 31 st January 2015). The experiment was conducted in compliance with the policies of the Norwegian animal welfare authorities (application ID 6571). Briefly, a BaP (Sigma Aldrich, St. Louis, USA) solution in acetone was mixed with Calanus spp (Calanus AS) to yield 0.5 or 5 µg BaP per g feed or acetone alone (acetone control). The acetone was volatilized by constant stirring on a magnetic stirrer for 2.5 hours at 30 °C. Small pellets were then created with the addition of 0.5 mL gelatin per g feed. Fish were fed pellets corresponding to 4% of their body wet weight (bw) 5 days a week. On the first, third and fifth day of a week, fish were exposed to dietary BaP or a solvent control by receiving the 2% bw exposed feed (or solvent control) and 2% bw of unexposed feed (no BaP, no acetone). Feeding was done by distributing the pellets to the surface of the tank. Thus, feeding hierarchies may have occurred resulting in some intra-tank individual exposure variations. On the remaining 2 days of a week, all fish were fed 4% bw of unexposed feed.

The amount of food given to each tank was adjusted at each sampling point to account for both growth and sampling of specimens. With this feeding regime, the fish nominally received an average of 0, 0.03 and 0.3 µg BaP per gram of fish per week. After 1 (2 nd August), 3 (3 rd October) and 6 (31 th of January) months, 10 fish per condition were anaesthetized and killed by a sharp blow to the head. Total body weight (g) and fork length (cm) were measured and the presence of parasites recorded. Liver and gonads were removed and weighed. Bile was snap frozen in liquid nitrogen and stored at -80°C until 3-OH BaP metabolite determination. A liver section was snap frozen in liquid nitrogen and stored at -80°C for molecular analyses. During the final sampling (6 months of exposure), a standardized liver cross-section was fixed for 24 hrs in neutral buffered formaldehyde (4%) before being transferred to 70% ethanol for subsequent histological assessment. Blood and spleen samples were preserved in Carnoy solution (3 methanol: 1 acetic acid) and stored at +4°C for subsequent identification of nuclear abnormalities and micronuclei. Finally, somatic weight (g) was determined as weight of eviscerated fish. Gonadosomatic index (GSI) and hepatosomatic index (HSI) were calculated as follows: GSI = (gonad weight/somatic weight) x 100 HSI = (liver weight/ somatic weight) x 100.

3-OH-benzo[a]pyrene measurement

Biliary 3-OH-benzo[a]pyrene metabolite concentration was determined after 1, 3 and 6 months of exposure following the procedure detailed in Song et al., (2018). Preparation of hydrolysed bile samples was performed as described in [START_REF] Krahn | Mass-spectrometric analysis for aromatic-compounds in bile of fish sampled after the Exxon-Valdez oil-spill[END_REF]. Briefly, bile (1-20 μL) was mixed with an internal standard (triphenylamine) and diluted with demineralised water (10-50 μL) and hydrolysed with β-glucuronidasearylsulphatase (20 μL, 1 h at 37 °C). Methanol (75-200 μL) was added and the sample was mixed thoroughly before centrifugation. The supernatant was then transferred to vials and analysed. High pressure liquid chromatography (Waters 2695 Separations Module) was used to separate 3-OH-BaP in a Waters PAH C18 column (4.6 × 250 mm, 5 μm particle size). The mobile phase consisted of a gradient from 40:60 acetonitrile:ammonium acetate (0.05 M, pH 4.1) to 100% acetonitrile at a flow rate of 1 mL/min, and the column was heated to 35 °C. A 2475 fluorescence detector measured fluorescence at the optimum for each analyte (excitation/emissions: 380/430). A total of 25 μL extract was injected for each analysis. The results were calculated by use of the internal standard method (Grung et al., 2009). The calibration standards utilized were obtained from Chiron AS, Trondheim, Norway, and were in the range 0.2-200 ng/g. Values below the limit of detection were considered as equal to 0 ng/g in the analyses.

Histopathological analyses

Livers were processed in a vacuum infiltration processor (Shandon Citadel 1000) using standard histological protocols [START_REF] Feist | Biological effects of contaminants: use of liver pathology of the European flatfish dab (Limanda limanda L.) and flounder (Platichthys flesus L.) for monitoring[END_REF]. The tissues were embedded in paraffin using an STP-120 spin tissue processor (Thermo Fisher Scientific, USA). Sections of 4 μm thickness were cut using a microtome HM 450 (Thermo Fisher Scientific, USA) and subsequently stained with haematoxylin and eosin (H&E). The liver sections were examined for microscopic pre-tumour and tumour lesions according to BEQUALM and ICES criteria [START_REF] Feist | Biological effects of contaminants: use of liver pathology of the European flatfish dab (Limanda limanda L.) and flounder (Platichthys flesus L.) for monitoring[END_REF]. The pre-tumour lesions sought were the vacuolated, basophilic and eosinophilic foci of cellular alteration (FCA). Tumour lesions were the benign hepatocellular adenoma and the malignant hepatocellular carcinoma (HCC). Lesions associated to nuclear and cellular polymorphism, cell death, inflammation and regeneration were also examined. A total of 5, 6 and 4 fish were assessed from control, low and high exposure condition, respectively.

Micronucleus test and nuclear abnormalities

The micronuclei and nuclear abnormalities frequencies were measured in blood and spleen of polar cod tissues fixed in Carnoy's solution; subsequently separated cells were dispersed on glass slides, and stained with the fluorescent dye 4′,6-diamidino-2-phenylindole at 100 ng/ mL. For each experimental condition, a range of 6 to 8 fish were investigated, and for each specimen 2000 cells with preserved cytoplasm were scored to assess the presence of micronuclei and nuclear abnormalities. Micronuclei are defined as round structures, smaller than 1/3 of the main nucleus diameter, on the same optical plan and clearly separated from nucleus; Nuclear abnormalities include (i) binucleated: cell with two nuclei, (ii) notch nuclei: looks like nucleus but do not have nuclear materials, (iii) nuclear bud: evagination of bud-like structure from the nucleus, and (iv) blebbed nuclei: small euchromatin evagination of the nuclear membrane [START_REF] Gorbi | Biological effects of diethylene glycol (DEG) and produced waters (PWs) released from offshore activities: A multi-biomarker approach with the sea bass Dicentrarchus labrax[END_REF][START_REF] Islam | Influence of extreme ambient cold stress on growth, hematological, antioxidants, and immune responses in European seabass, Dicentrarchus labrax acclimatized at different salinities[END_REF].

Gene expression analyses

Following 1, 3 and 6 months of exposure, a cross section of each liver, next to the one dedicated to histological analyses at 6 months was used for gene transcriptional response analyses. Total RNAs were extracted using the High Pure RNA Tissue kit (Roche Diagnostics Ltd, West Sussex, U.K.) according to the supplier's instructions which included a DNase treatment. RNA quality (integrity of 18S and 28S ribosomal bands) was evaluated by electrophoresis on a 1% agarose-formaldehyde gel. RNA purity was assessed by measuring the ratios of absorbance: A 260 /A 280 and A 260 /A 230 using a spectrophotometer (NanoDrop, ThermoFisher). All samples were of high purity (ratios' values > 2.1).

First strand cDNAs were synthesized from 1 µg of total RNA using the AffinityScript Multiple Temperature cDNA Synthesis Kit (Agilent Technologies, Stockport, U. K.) using random hexamer primers and according to the supplier's instructions. Putative coding sequences (Figure S1) were identified by nucleotide and protein BLAST searches on the NCBI database (http://blast.ncbi.nlm.nih.gov/Blast.cgi) and sequence homologies across fish species on the EMBL-EBI platform (https://www.ebi.ac.uk/Tools/msa/clustalo/). The contigs produced in the study of [START_REF] Song | Transcriptomic analysis reveals dose-dependent modes of action of benzo(a)pyrene in polar cod (Boreogadus saida)[END_REF] were also used. Primer pairs and FAM™-TAMRA™ dye probes used to amplify the target sequences were designed using the Prime Express software (Applied Biosystem) (Table 1). Ten ng of the reverse transcribed product measured by a qubit fluorometer (Thermo Fisher Scientific) was used as a template for subsequent polymerase chain reaction (PCR) in a 20 µL final volume using 1x of TaqMan® Fast Advanced Master Mix (Life technologies, Paisley, U.K.), 900 nM primers and 250 nM probe (final concentrations) according to the supplier's protocol. PCR reactions were performed in the Applied Biosystems™ ViiA™ 7 Real-Time PCR System using the following programme: one cycle at 95°C for 20 s and 40 amplification cycles at 95°C for 3 s and 60°C for 30 s. Primer efficiencies were determined by 10 times dilution series of the cDNA template and were about 100%. The optimal normalization gene was selected by testing the expressions of 3 reference genes (β tubulin, hprt1 and 28S) on all the samples using the NormFinder algorithm. The expression of the β tubulin gene displayed the highest stability. The melting curves were carefully checked after each qPCR run. The gene expression was calculated according to the delta delta Ct method.

Statistical analyses

Statistical analyses were performed using R (version 3.1.2). The effect of the BaP exposure concentration and time of exposure were assessed on all the biological parameters measured using 1-way ANOVA. When the normality of the residuals was not verified by the Shapiro-Wilk test, the non-parametric Kruskal-Wallis tests were used. Post-hoc comparisons were performed using the least-square mean test for parametric test and the Wilcoxon rank test for non-parametric test. The  error was adjusted using the Bonferroni correction for each posthoc test.

Results

Over the course of the six-month exposure, all specimens grew significantly in weight and underwent gonadal maturation with mean GSI ranging from 1.5 ± 0.7 to 20.8 ± 3.9 % (Table 2). Endoparasites were commonly found across all treatments and sampling times.

Nematodes on the liver surface were the most common parasites with a frequency of occurrence of 26%. Parasites of the phylum Platyhelminthes were less common (7%). No mortality was observed.

The dietary BaP exposure of polar cod led to a dose-dependent production of biliary 3-OH BaP metabolites for the low (0.03 µg BaP/g of fish/week) and high (0.3 µg BaP/g of fish/week) BaP exposure conditions after 1 and 6 months of exposure (Figure 1). The concentrations of bile 3-OH BaP ranged from 20 to 40 ng/g of bile for the low exposure condition and were approximately 10 times higher, from 132 to 390 ng/g of bile, for the highest exposure condition.

The transcriptional responses related to DNA adduct recognition (xpc), helicase activity (xpd), DNA repair (xpf, rad51) and tumour suppression (tp53) were not significantly changed by any BaP dietary exposures as compared to controls (p > 0.05) (Figure 2).

The number of micronuclei recorded in the blood and spleen of polar cod (p > 0.05) exposed to BaP did not significantly vary as compared to control (Figure 3 A,B). Nuclear abnormalities in polar cod spleen were significantly increased in the high dose group (p = 0.03), while close to significant in the low dose group (p = 0.057) (Figure 3 C,D). No significant nuclear abnormalities were observed after BaP exposure in blood cells (p > 0.05).

Histopathological analyses revealed one basophilic focus of cellular alteration in liver of two individuals exposed to the low exposure condition after six months of exposure (Figure S2). No tumour-related lesions were observed in livers of control and highly exposed individuals.

Discussion

The present study showed that polar cod grew steadily through the entire experiments for all exposure conditions (0, 0.03 and 0.3 µg BaP/g of fish/ week). These results were expected under chronic low dose exposure scenarios and are consistent with similar results obtained in polar cod exposed to higher dietary BaP concentrations (1.2 and 60.9 µg BaP/ g of fish/ week) but for a shorter period of two weeks [START_REF] Song | Transcriptomic analysis reveals dose-dependent modes of action of benzo(a)pyrene in polar cod (Boreogadus saida)[END_REF]. In Nile tilapia exposed to intraperitoneal injections of 3 µg BaP/g of fish/ week for 4 weeks, K, GSI and GSI were slightly decreased [START_REF] Colli-Dula | Transcriptome analysis reveals novel insights into the response of low-dose benzo(a)pyrene exposure in male tilapia[END_REF]. This suggests that there is a dose and time dependent threshold above which exposure to BaP induce significant body condition indexes changes. The fish species and the mode of BaP administration (injected intraperitoneally versus dietary) may also be important factors to consider.

The bile concentration of 3-OH-BaP has been used as an indicator of BaP exposure and biotransformation in many fish species including polar cod (Baake et al., 2016;[START_REF] Baali | Bile metabolites of polycyclic aromatic hydrocarbons (PAHs) in three species of fish from Morocco[END_REF][START_REF] Kammann | PAH metabolites in fish bile: from the seine estuary to Iceland[END_REF][START_REF] Song | Transcriptomic analysis reveals dose-dependent modes of action of benzo(a)pyrene in polar cod (Boreogadus saida)[END_REF]. Indeed, previous studies led on polar cod exposed to either PAHs or crude oil have shown a very high correlation between bile metabolites of PAHs and both cyp1a mRNA expression and EROD activity [START_REF] Bakke | Comparative absorption and tissue distribution of 14 C-benzo(a)pyrene and 14 C-phenanthrene in the polar cod (Boreogadus saida) following oral administration[END_REF][START_REF] Bender | Effects of chronic dietary petroleum exposure on reproductive development in polar cod (Boreogadus saida)[END_REF][START_REF] Vieweg | Effects of dietary crude oil exposure on molecular and physiological parameters related to lipid homeostasis in polar cod (Boreogadus saida)[END_REF], Nahrgang et al., 2019[START_REF] Song | Transcriptomic analysis reveals dose-dependent modes of action of benzo(a)pyrene in polar cod (Boreogadus saida)[END_REF]. The increase in biliary 3-OH-BaP metabolite concentrations after 1 and 6 months exposure supported that of a similar, albeit shorter, exposure study [START_REF] Song | Transcriptomic analysis reveals dose-dependent modes of action of benzo(a)pyrene in polar cod (Boreogadus saida)[END_REF]. When exposed to a four times higher exposure dose (1.2 µg BaP/ g of fish/ week) than the highest dose used in the present study, a 3-OH-BaP concentration of 800 ng/ g of bile was found, which was two to six times higher than the metabolite concentration range identified in our study. The reactive BaP intermediates have been found to accumulate and covalently bind DNA in the biliary system of polar cod one month after exposure to a single dietary concentration (Baake et al., 2016) equivalent to the cumulative dose received in the high BaP exposure condition during the first month of our study. Those reactive BaP metabolites covalently bind to biological molecules such nucleic acids and form DNA adducts that can lead to tumour formation. For instance, higher levels of BaP-7,8-diol metabolites and DNA binding activity were found in bile of English sole (Parophrys vetulus) a fish species more sensitive to carcinogenesis than the more resistant starry flounder (Platichthys stellatus) (Varanasi et al., 1986). In polar cod dietarily exposed to higher BaP concentrations (from 5 µg BaP/ g of fish in a single injection) DNA adducts were found [START_REF] Aas | DNA adduct levels in fish from pristine areas are not detectable or low when analysed using the nuclease P1 version of the 32 P-postlabelling technique[END_REF], revealing an increased risk of liver tumour formation later on. Indeed, 50% of rainbow trout displayed pre-tumour (basophilic FCA) and tumour (HCC) liver lesions after six months of exposure to a similar dose injected intraperitoneally [START_REF] Hendricks | Hepatocarcinogenicity of benzo[a]pyrene to rainbow trout by dietary exposure and intraperitoneal injection[END_REF]. In their study, 25 % of the trouts displayed similar liver lesions after twelve months of dietary exposure to a high dose of BaP (estimated to 1-2 mg BaP/ g fish/ week) [START_REF] Hendricks | Hepatocarcinogenicity of benzo[a]pyrene to rainbow trout by dietary exposure and intraperitoneal injection[END_REF]. In the present study, the potential genotoxic damage generated by the BaP metabolites produced did not cause significant tumour lesions. This could be the result of several factors potentially in combination, including low dose, low exposure duration and effective DNA repair mechanisms.

The DNA repair system and cell cycle regulators can prevent DNA adducts and the onset of tumorous events. In the present study, the transcriptional response of genes involved in the nucleotide excision repair (NER) process (xpc, xpd, xpf), DNA double strand breaks repair (rad51) and cell division regulation (tp53) did not vary significantly after 1, 3, and 6 months of exposure to both BaP dietary concentrations. The exposure levels of BaP may have been too low to cause significant accumulation of cell damage and trigger a significant gene transcriptional response. Interestingly, a dose-specific transcriptional response of some genes has been observed in liver of polar cod dietary exposed to BaP [START_REF] Song | Transcriptomic analysis reveals dose-dependent modes of action of benzo(a)pyrene in polar cod (Boreogadus saida)[END_REF]. For instance, some genes involved in apoptosis (bax and casp9), a process that eliminates damaged cells and prevent the proliferation of abnormal cells in tumour formation, were upregulated in polar cod exposed to the high exposure level (60.9 µg BaP/ g of fish/ week).

The expression levels of those genes were not modified in fish exposed to the low exposure dose (1.2 µg BaP/ g of fish/week) suggesting a threshold above which gene transcription is modified [START_REF] Song | Transcriptomic analysis reveals dose-dependent modes of action of benzo(a)pyrene in polar cod (Boreogadus saida)[END_REF]. The basal gene expression level may also be sufficient to repair DNA and/or delay the cell cycle to maintain the genetic integrity. Moreover, the DNA repair gene measured in our study, rad51, may not be involved in the repair of specific DNA damage induced. Similarly to the results herein, this gene was not differentially expressed in liver of polar cod dietary exposed to BaP [START_REF] Song | Transcriptomic analysis reveals dose-dependent modes of action of benzo(a)pyrene in polar cod (Boreogadus saida)[END_REF]. Rad51 is involved in the repair of DNA double strand breaks, which belong to a different pathway than the NER. The mechanism of DNA damage induced by BaP exposure is more likely to involve DNA adducts than double strand breaks. Other genes involved in DNA repair processes and control of cell cycle have been found induced at higher exposure regimes. For example, the gene encoding for the growth arrest and DNA damage inducible beta gene (gadd45b) was induced in the liver of the tropical fish, Nile Tilapia (Oreochromis niloticus) after one month of exposure to 3 µg BaP/ g of fish/week [START_REF] Colli-Dula | Transcriptome analysis reveals novel insights into the response of low-dose benzo(a)pyrene exposure in male tilapia[END_REF]. In polar cod exposed to 1.2 and 60.9 µg BaP/g of fish/ week, genes involved in the excision DNA repair process (such as hmgb2b and rad23a) were differentially expressed [START_REF] Song | Transcriptomic analysis reveals dose-dependent modes of action of benzo(a)pyrene in polar cod (Boreogadus saida)[END_REF]. Shorter timepoints may have also been necessary to observe a gene expression modulation as an early response to stressors. In the liver of polar cod, transcriptional responses of genes involved in DNA damage repair were changed after two weeks of dietary exposure to BaP [START_REF] Song | Transcriptomic analysis reveals dose-dependent modes of action of benzo(a)pyrene in polar cod (Boreogadus saida)[END_REF]. Some studies using a reference genotoxic compound showed that DNA damage was rapidly repaired with increased transcription of DNA repair genes such as rad51 in zebrafish larvae, as early as 6 hours [START_REF] Reinardy | Changes in expression profiles of genes associated with DNA repair following induction of DNA damage in larval zebrafish Danio rerio[END_REF]). The addition of early sampling times seems relevant to include in future studies.

Other biological processes such as detoxification mechanisms could have prevented polar cod from the genotoxic effects of BaP exposure. Activation of detoxification events could explain the resistance of polar cod to BaP exposure and the absence of liver tumours in the present study. Variation in the expression of genes and proteins belonging to the cytochrome P450 family involved in phase I of BaP detoxification process has been well described in liver of fish [START_REF] Nahrgang | PAH biomarker responses in polar cod (Boreogadus saida) exposed to benzo(a)pyrene[END_REF][START_REF] Lee | Cytochrome P450 System Expression and DNA Adduct Formation in the Liver of Zacco platypus Following Waterborne Benzo(a)pyrene Exposure: Implications for Biomarker Determination[END_REF][START_REF] Colli-Dula | Transcriptome analysis reveals novel insights into the response of low-dose benzo(a)pyrene exposure in male tilapia[END_REF]. Interestingly, cyp1a1 and cyp1b1 genes were upregulated in liver of polar cod following dietary exposure to 60.9 µg BaP/g of fish/ week but were not differentially expressed after exposure to a lower dose of BaP (1.2 µg BaP/g of fish/ week) [START_REF] Song | Transcriptomic analysis reveals dose-dependent modes of action of benzo(a)pyrene in polar cod (Boreogadus saida)[END_REF]. This suggests a dose threshold for activating the detoxification mechanisms during a chronic exposure. Activation of genes and proteins involved in phase II detoxification process has been also described in liver of fish exposed to BaP (Nahgang et al., 2009). For instance, gstA1 gene expression was modified in the liver of Nile tilapia exposed to 3 µg BaP/ g of fish/ week [START_REF] Colli-Dula | Transcriptome analysis reveals novel insights into the response of low-dose benzo(a)pyrene exposure in male tilapia[END_REF]. Interestingly, GST activity was higher in starry flounder, a tumour resistant species, than in English sole, a tumour sensitive species, after exposure to a BaP dose that induced carcinogenesis (Varanasi et al., 1987). Finally, phase III detoxification process based on active efflux of chemicals by ATP-binding cassette (ABC) transporters could be involved in BaP elimination. For example, a rainbow trout ABCG2 transporter was found to interact with BaP (Zaja et al., 2016).

In accordance with relatively low biliary BaP metabolite concentrations observed throughout the exposure and limited responses of genes involved in DNA damage identification and repair, no effect of dietary BaP on the micronuclei and nuclear abnormalities was recorded in the present study except in spleen where nuclear abnormalities increased in the high dose group. Micronuclei are formed during the anaphase stage of the cell division. They are considered as a reliable index of chromosomal breakage, chromosomal loss and cellular spindle malfunction [START_REF] Bolognesi | Micronucleus assay in aquatic animals[END_REF].

Additionally, micronuclei constitute an irreversible form of genotoxic damage compared to DNA strand breaks and their induction are regulated by a large number of experimental carcinogens, including chlorinated hydrocarbons, benzidine, aflatoxins, methylcholanthrene, and common carcinogenic pollutants, such as PAHs, heavy metals, and pesticides [START_REF] Bolognesi | Micronucleus assay in aquatic animals[END_REF]. Many research studies reported the increased in micronuclei frequency in erythrocytes of different fish species exposed to PAHs [START_REF] Shirmohammadi | Using cell apoptosis, micronuclei and immune alternations as biomarkers of phenanthrene exposure in yellowfin seabream (Acanthopagrus latus)[END_REF].

Contrary to micronuclei, nuclear abnormalities origin has not been clearly explained; some suggest that nuclear abnormalities can be a primary response, prior to the micronuclei formation, highlighting their relevance in the evaluation of genotoxic damage [START_REF] Bolognesi | Micronucleus assay in aquatic animals[END_REF][START_REF] Seriani | Hematology, micronuclei and nuclear abnormalities in fishes from São Francisco River, Minas Gerais state, Brazil[END_REF]. An increase of erythrocytic nuclear abnormalities and strand breaks was observed in eels (Anguilla anguilla L.) and juvenile sea bass (Dicentrarchus labrax) exposed to a range of 0.3 to 2.7 µM of BaP and naphthalene [START_REF] Maria | Anguilla anguilla L. biochemical and genotoxic responses to benzo[a]pyrene[END_REF][START_REF] Teles | Anguilla anguilla L. liver ethoxyresorufin Odeethylation, Glutathione S-transferase, erythrocytic nuclear abnormalities, and endocrine responses to naphthalene and β-naphthoflavone[END_REF][START_REF] Gravato | Juvenile sea bass liver P450, EROD induction, and erythrocytic genotoxic responses to PAH and PAH-like compounds[END_REF], while on the contrary, lower concentrations of BaP (0.1 µM) did not affect DNA integrity [START_REF] Nogueira | Mutation analysis of ras gene in the liver of European eel (Anguilla anguilla L.) exposed to benzo[a]pyrene[END_REF]. The induction of micronuclei and other nuclear abnormalities were also caused by crude oil exposure in turbot (Scophthalmus maximus) and Atlantic cod (Gadus morua) [START_REF] Baršienė | Analysis of micronuclei in blue mussels and fish from the Baltic and North Seas[END_REF]2006). The exposure duration and levels are extremely important in determining micronuclei and nuclear abnormalities formation; long-term chemical exposures can cause genetic changes and consequently physiological alterations or pathologies including cancer development [START_REF] Depledge | Methods to assess effects on brackish, estuarine, and near-coastal water organisms[END_REF]. In a study led on the European flounder, Platichthy flesus, [START_REF] Köhler | Nuclear changes in blood, early liver anomalies and hepatocellular cancers in flounder (Platichthys flesus L.) as prognostic indicator for a higher cancer risk?[END_REF], first suggested that nuclear anomalies inside liver lesions of hepatocellular cancers were correlated with micronuclei frequencies in fish blood and that the histopathological grading of cancers from preneoplastic, benign to malignant types was clearly associated with micronuclei increase.

The present study showed that polar cod were consistently exposed to dietary BaP through the entire experiment and biotransformed the mother compound to intermediate metabolites. However, this exposure did not lead to significant changes in the transcription of selected genes, nor in chromosomal alterations and significant tissue lesions. Some early responses to stress may have occurred prior to the first sampling time point at one month of exposure, and basal expression of genes or potentially activated compensatory mechanisms may have been sufficient to control the damage caused by the reactive metabolites.

Moreover, protective mechanisms such as detoxification and apoptosis could have prevented the cells from the accumulation of cell damage caused by the reactive metabolites. Therefore, we deduce that the BaP exposure concentrations were below the threshold of observable effects. As a whole, our results showed that polar cod exposed to 0.03 and 0.3 µg BaP/ g fish/ Table 1. Sequences of primer pairs and FAM/TAMRA probes used in RT-qPCR reactions for each of the target genes studied. β tubulin was used as the reference gene.

Table 2. Fulton condition (K), hepatosomatic index (HSI), gonadosomatic index (GSI), liver, gonad and body weight (g), and fork length (cm) (mean ± SD, n = 10) and sex ratio determined after 0, 1, 3, and 6 months of exposure to different BaP treatments (acetone control, low and high exposures).

  week was not sensitive to the model carcinogen and liver carcinogenesis. The present work encourages the addition of earlier sampling points and indicators of detoxification mechanisms in future studies. killifish (Fundulus heteroclitus) with different exposure histories. Environmental Health Perspectives, 118: 1376-1381. Zaja R, Popovic M, Loncar J, Smital T. 2016. Functional characterization of rainbow trout (Oncorhynchus mykiss) Abcg2a (Bcrp) transporter. Comparative Biochemistry and Physiology, Part C, 190: 15-2. Zhu S, Li L, Thornton C, Carvalho P, Avery BA, Willett KL. 2008. Simultaneous determination of benzo[a]pyrene and eight of its metabolites in Fundulus heteroclitus bile using ultra-performance liquid chromatography with mass spectrometry. Journal of Chromatography B, 863: 141-149.

Figure

  Figure and TableLegend. Figure1. Concentrations of biliary 3-OH-benzo[a]pyrene (ng/g bile, n = 4 to 9) in fish

Figure 2 .

 2 Figure 2. Relative expression of genes (mean ± SD, arbitrary units) in liver of polar cods (n =

Figure 3 .

 3 Figure 3. DNA damage in the form of micronuclei per thousand in the blood (A) and spleen

  Figure1

Figure S2 .

 S2 Figure S2. Basophilic foci of cellular alteration diagnosed in liver of a polar cod exposed to

Table 1 Gene name Primers and Probe Sequences 5'-3'

 1

	β tubulin	F: GCCCGGCACCATGGA
		R: TGGCCGAAAACGAAGTTGTC
		P: TCCGGTGCTTTCGGTCAGATCTTCA
	XPC	F: GCTTCGACTTCCATGGAGGAT
		R: CTTCGTGCTCCTCACACACAA
		P: CGCATGCTGTGACCGACGGCTAC
	XPD	F: TCATGTTCGGAGTCCCTTATGTT
		R: GGAACTGGTCCCGGAGGTA
		P: ACACACAGAGCCGCATTCTGAAGGC
	XPF	F: ATCTGGACCTGGCGAGGAA
		R: TCCTGCTTTGCGGGTGTT
		P: CTGGAGCCCGCCAACGCTACC
	Rad51	F: AAGAAGCCGATTGGAGGAAAC
		R: CGCCCCTTCCTCAGGTACA
		P: TCATGGCCCACGCCTCCACC
	tp53	F: CCTCTGAGGGGCATGTTCTC
		R: GGGGCTCTTTCTTTTTTTTGG
		P: TCCTGGGCGCGACCGCA

Table 2 819

 2 Putative coding sequences used to design the primers and probe for the 6 genes studied. The pink marks indicate the location of the introns in the DNA sequence. Primers were designed to overlap the introns whenever possible to check the specificity of the qPCR reactions.

		0 month	1 month Control Low	High	Control	3 months Low	High	6 months Control Low	High
	Fork l. (cm) 15.9 ± 1.6	16.4 ± 1	16.7 ± 1.6	16.9 ± 1.7	17.8 ± 1.6	16.8 ± 1.9	17.3 ± 1.5 16.4 ± 1.5 17.2 ± 0.7 17.3 ± 1.2
	Total w. (g) 25.6 ± 7.1	29.3 ± 5.6 32.4 ± 8.8	33.2 ± 9.1	38.3 ± 11.6	32.1 ± 8.5	35.6 ± 10.4 32.7 ± 10	37.3 ± 4.5 36.7 ± 8
	K	5.2 ± 0.5	5.2 ± 0.4	5.5 ± 0.5	5.4 ± 0.3	5.3 ± 0.7	5.4 ± 0.5	5.3 ± 0.4	5.2 ± 0.7	5.3 ± 0.4 5 ± 0.4
	Liver w. (g) 2 ± 0.9	2.2 ± 0.7	2.5 ± 0.9	2.6 ± 0.7	2.7 ± 1.2	2.3 ± 0.6	2.8 ± 1	2.7 ± 0.9	2.6 ± 0.5 3 ± 0.9
	HSI	9.6 ± 3.3	9.4 ± 2.1	9.4 ± 2.1	10.4 ± 2.8	8.6 ± 2.6	9.3 ± 2.3	9.8 ± 2.1	12 ± 3.9	9.5 ± 2	11.3 ± 2.3
	Gonad w. (g) 0.3 ± 0.2	0.5 ± 0.2	0.5 ± 0.3	0.5 ± 0.3	1.4 ± 0.5	1.1 ± 0.6	1.6 ± 0.9	4.2 ± 2.3	5.6 ± 1.3 5 ± 1.8
	GSI	1.5 ± 0.7	2 ± 0.5	2.2 ± 1	1.8 ± 0.6	4.6 ± 1.2	4.1 ± 1.8	5.6 ± 2.3	16.9 ± 6.8 20.8 ± 3.9 19.3 ± 6.4
	Sex ratio	40	60	30	50	50	40	30	50	22	50
	820
	821


Maxime Amblard

Karën Fort

Bruno Guillaume

Philippe De Groote

Chuyuan Li

Pierre Ludmann

Michel Musiol

Siyana Pavlova

Guy Perrier

Sylvain Pogodalla

The Annotators Did Not Agree on Some of the Guidelines Examples

We propose the annotation of 7 sentences out of the 31 provided in the ISA-17 shared task, according to our understanding of the guidelines. We include here several remarks to improve the annotation and provide some tools to make the task easier.

Introduction

We annotated a fifth of the proposed sentences in the ISA-17 shared task. We tried and covered the interactions, within the QuantML annotation scheme, between quantification and various phenomena: negation, relative clauses, cardinal determiners, etc. Our annotations of the following sentences are provided as supplementary material:

(1) Some of the students failed the exam.

(5) The editors didn't see a misprint. (7) A man who walks in the park whistles. (8) Mary visits a museum every day. (11) Not all the students passed the exam. (12) Most of the students passed the exam. (31) More than four hundred ships are waiting to pass through the Suez Canal. In Section 2, we describe the methodology of our participation to the ISA-17 shared task; in Section 3, we comment on the task per se. After these practicalities, Sections 4 and 5 provide feedback on the QuantML annotation scheme and its guidelines. Section 6 draws a parallel between QuantML and the Parallel Meaning Bank (PMB) scheme. In the final Section, we detail theoretical suggestions as well as the tools we deployed for the shared task.

Methodology

A Collaborative Effort We organized eight different collective meetings, of approximately two hours each, in addition to individual work. The results we provide were therefore obtained through collaborative discussions. It has to be noted that, although most of us have a very good command of the language, there is no native speaker of English in the team.

We based our annotations on the documentation resources directly mentioned in the Call for Papers of the ISA-17 Shared Task: (1) the provided working document for the ISO at hand (ISO WD 24617-12:2019(E)); (2) Semantic Annotation of Quantification in Natural Language [START_REF] Bunt | Semantic annotation of quantification in natural language[END_REF];

(3) A Semantic Annotation Scheme for Quantification [START_REF] Bunt | A semantic annotation scheme for quantification[END_REF]; and, (4) Towards an ISO Standard for the Annotation of Quantification [START_REF] Bunt | Towards an ISO standard for the annotation of quantification[END_REF]. Whenever discrepancies showed up in the documentation, we tried and used the resources in that preference order.

We noted an important learning curve effect, as we spent quite a long time on our second annotation (Sentence 5), as compared to the following ones.

Tooling Up In order to be as consistent as possible in our annotation and easily share them in the group, we used several tools (see Section 7):

(1) XML processing tools (well-formedness and validation); (2) visualization tools to provide a graphical structure of the annotated elements; (3) a framework dedicated to automatic building of semantic representations. Applying this set of tools to a subset of 10 annotation examples provided in the guidelines also helped identify inconsistencies.

3 Remarks on the Shared Task Some issues in the shared task organization hindered our participation, without fully concerning the annotation scheme nor its guidelines.

The first issue is the lack of a unique and explicit reference document (guidelines), as is the case for other resources such as the Penn Treebank [START_REF] Santorini | Part-of-speech tagging guidelines for the Penn Treebank Project[END_REF]. The call for papers pointed to some relevant ones, but their selection and ranking is left to the participants, even though the standard is not published yet and there are discrepancies in the pointed documents. Section 5 develops this issue further.

Markables The proposed markables biased our annotation. For instance, in Sentence 7, we needed to mark in the park, but we were reluctant to use a markable that is not provided. Yet, Example (66) in the ISO draft features such a markable.

Since deciding on the markables is the very first annotation step, it determines the finesse of the final result: a more careful examination and explanation is necessary for the scheme and its guidelines. One suggestion is to use markable variables including offsets, as m<n>-<p> for a markable spanning from the (n + 1)-th token to the p-th, so that annotators are not biased by the pre-identified markables.

Unavailability of a Resource

We had a doubt when annotating the semantic roles (namely, a @semRole in Sentence 31), as the proposed semantic roles are defined in ISO 24617-4, a document which is not freely available. We thus referred to VerbNet for a suitable role. But we are not sure if it is presented in the ISO list.

Adequacy of the Scheme

The QuantML annotation scheme tackles important and intricate phenomena about quantification. It relies on a thorough and helpful introduction and state of the art, allowing non-specialists of the domain to better understand the logic underlying the annotation [START_REF] Fort | Collaborative Annotation for Reliable Natural Language Processing[END_REF] and therefore to evolve from a teacher-like to a peer relation [START_REF] Akrich | Le mode d'emploi, genèse, forme et usage[END_REF]. Some phenomena we encountered during the annotation phase could, however, be more developed. For instance, more space could be devoted to the interaction between quantification and negation, in particular when the polarity induced by a negation on an event differs between participants in that event.

Tricky Events We also encountered difficulties with the @clause attribute possible values of the relClause element (see for instance Sentence 7). In particular, if event elements are the expected concrete values, the guidelines should be explicit about it. Besides, we wondered whether a participation element could be sufficient, as both elements share most attributes.

Having events as attribute values also occur for instance in Sentence 31, with the attitude verb to wait, where the theme or goal participant should be annotated with the clause or the event the agent is waiting for. It is not clear whether attributing an event is possible, as events and participants seem clearly disjoint in the QuantML metamodel.

Genericity Finally, QuantML declares genericity as a loose end, which is unfortunate since they induce major variations in quantification. The scheme could also account for generics (with dedicated involvement values, like "generic a", or an attribute @genericity), even though it could not interpret them to their full extent. Doing so would boost the understanding of generics and their semantics. Otherwise, users will have to retrace all their annotations when if the scheme introduces genericity at a later stage. Worse, users may tinker and tamper with their annotations, e.g., with @involvement, but in non-standard ways then.

About the Annotation Guidelines

We will use the term guidelines here to refer to the four documents listed in Section 2. As a matter of fact, there is an appendix holding that title of guidelines in both the technical report and the ISO working document, but it was insufficient to annotate the provided sentences.

The biggest issue with this, apart from the difficulty of searching into several documents, is the inconsistency between and among them.

Scattered Examples

In terms of the assignments of different attributes, explanations and examples are so scattered that we spent a lot of time comparing the sentences and the semantic differences. A comprehensive list of all the possible values for each attribute would help to ease the work. A precise definition of the different attributes should also be provided. Some of them are only glossed (e.g., the @rep attribute for events, while the @repetition attribute for participation links are described in different places), some of them are only defined in the QuantML semantics (e.g., the @semRole attribute of the relClause element), some of them are never mentioned but in the list of attributes of a given element (e.g., the @clause attribute of the relClause element).

Terminology Issues The terminology used in the annotation scheme did not appear very intuitive to us. For example, the value unspecific for the @distr attribute does not tell us much about what it is supposed to mean. Our understanding of the guidelines leads us to believe that this should be interpreted as what we consider as a cumulative distribution and propose that the attribute value be changed accordingly. Also, the guidelines raise a distinction between definiteness and determinacy, but mix them up in both the abstract and the concrete syntax. Since the only values are det and indet, a boolean attribute @determinacy seems a better choice of terminology.

A Parallel with a Meaning Bank

With the interpretation to DRT, the QuantML annotation scheme allows developing resources coupling raw text and corresponding DRS. The Parallel Meaning Bank (PMB, [START_REF] Abzianidze | The Parallel Meaning Bank: Towards a Multilingual Corpus of Translations Annotated with Compositional Meaning Representations[END_REF] is such a resource, yet built on an automated annotation pipeline. In a nutshell, the pipeline builds syntactic analysis in a Combinatory Categorial Grammar and associates λ-DRS to tokens [START_REF] Bos | Towards a large-scale formal semantic lexicon for text processing[END_REF]. This yields DRS from a compositional syntax-semantics interface.

However, syntactically similar sentences lead to similar relative scopes of quantifiers, which can be erroneous as Examples (71/1608) and (56/3237) from the PMB show: (71/1608) She has a bottle of milk every morning (56/3237) I am keeping a diary every day They both are annotated with the universal quantification (over morning or day) outscoping the existential one (over bottle or diary). It should, however, be the opposite in Example (71/1608), i.e., the diary is the same every day.

On the other hand, QuantML is not bound to a specific syntactic framework, and formulates the semantic difference between these two examples with the dedicated structure scoping (see Sentence 31).

There also are divergences between QuantML and PMB. For instance, looking at prepositional modifications of events, specifically locations with in and under for instance, the PMB features spatial operators to discriminate them (00/2412 and 06/1633) whereas QuantML mixes them up.

Apart from the differences mentioned above, QuantML and PMB also differ on other aspects, for instance on dealing with determinacy and predicate distribution over plurals. In the QuantML scheme, determinacy is interpreted as reference domains over source domains while it triggers presuppositions through pointers in the PMB [START_REF] Venhuizen | Parsimonious semantic representations with projection pointers[END_REF]. Furthermore, tense is annotated in PMB whereas QuantML does not offer this option. Finally, the PMB works at the moment with the simplifying assumption that any predicate is distributive.

Going Further

Theoretical Suggestions In the annotations we provide, we propose two different solutions for Sentence 5 where the difference is at the level of the scope of the negation. We could not find in the guidelines how this kind of ambiguity should be handled.

Similarly, we did not find whether specifying forbidden scoping and reading is possible and/or desirable. Finally, the annotation process led us into interesting theoretical questions about the effect of collective/distributive readings under the scope of a negation (see the @distr attribute in both annotations we provided for Sentence 5).

Providing Tools In addition to the sentences we annotated, we provide a Document Type Definition (DTD) to check the produced XML, and an implementation of the scheme's interpretation. This helped us identify inconsistencies among the XML attributes, e.g., @individuation or @indiv (ISO WD 24617-12:2019(E)). The DTD regulates the XML format as it requires a single and coherent way to represent these attributes. Further, it regulates values for closed classes: e.g., it would reject def and indef, as typos for det and indet respectively ((C62) and (C21) in ISO WD 24617-12:2019(E)).

We have noted that the same attribute @involvement is currently used both with a reference to an XML identifier or with a string value (both are used in Sentence (B59) of the guidelines). This prevents from a precise definition of @involvement the DTD.

In order to investigate the semantics, we also used the framework of Abstract Categorial Grammars (ACG) to define the compositional syntaxsemantic interface I Q , and implemented it in ACGtk. We use Continuation-Based Dynamic Logic instead of Discourse Representation Theory, for it features no free variables. At the moment, our implementation ignores individuation, and treats all merges as dynamic conjunctions.

While unrefined, this implementation allows us to investigate the semantic interpretations of annotation choices.

For instance, the guidelines lead us to annotate some of the students in Sentence 1 with <entity ... involvement="some" definiteness ="det"/> since it features a definite plural. Yet, it yields an unexpected logical formula stating all the students in the reference domain failed their exam. A similar situation arises for Sentence 12.

We also used a graph-based concrete syntax representation annotation with an automatic conversion from the XML format into a graph (see Figure 1). By converting examples 1 from the TiCC report [START_REF] Bunt | Semantic annotation of quantification in natural language[END_REF], we noticed some annotation errors (for instance, participation links are missing in Examples (A5) and (A7) and some errors in attributes in Example (A6), see Figure 2). 1 The first 11 examples, provided in the file annotation.pdf in the supplementary material

Conclusion and Perspectives

Our participation in this shared task allowed us to spend time working on difficult issues in a collaborative way. This was also an opportunity to deploy a range of tools that assisted our understanding of the QuantML and our participation to the ISA-17 shared task.

Figure 1 :

 1 Figure 1: Graph representation of Sentence 11 (Not all the students passed the exam).

Figure 2 :

 2 Figure 2: Wrongly annotated sentence A6 (Three men moved both pianos) in the TiCC report.