T Riberi -Béridot

M G Tsoutsouva

G Regula

G Reinhart

F Guittonneau

L Barrallier

N Mangelinck-Noël

STRAIN BUILDING AND CORRELATION WITH GRAIN NUCLEATION DURING SILICON GROWTH

Keywords: Strain, growth competition, twins, nucleation, in situ X-ray imaging

This work is dedicated to the grain structure formation in silicon ingots with a particular focus on the crystal structure strain building and its implication in new grain nucleation process. The implied mechanisms are investigated by advanced in situ X-ray imaging techniques during silicon directional solidification. It is shown that the grain structure formation is mainly driven by Σ3 <111> twin nucleation. Grain competition phenomena occurring during the growth process lead to the creation of higher order twin boundaries, localised strained areas and associated crystal structure deformation. On the one hand, it is demonstrated that local strain building can be directly related to the characteristics of the twin boundaries created during silicon growth due to grain competition. On the other hand, space restriction due to competition during growth can be at the origin of local strain building as well. Finally, the accumulation of all these factors generating strain is responsible for spontaneous new grain nucleation. When occurring, both grain nucleation and subsequent grain structure reorganisation contribute to lower the strain in the growing ingot.

It is demonstrated as well that the local distribution of the strained areas created during silicon growth is retrieved after cooling down, from melting temperature to room temperature, on top of an additional larger scale deformation of the sample due to the cooling down only.

Introduction

Aiming to the production of low cost and high efficiency silicon based solar cells for photovoltaic (PV) applications, current research focuses on several key targets from silicon purification to cell manufacturing including the silicon ingot fabrication process. When using the conventional multi-crystalline silicon (mc-Si) growth technique, the final silicon grain structure cannot be controlled at all. In addition, the distribution of dislocations is highly inhomogeneous from one grain to another as shown for example by Chen et al. [START_REF] Chen | An optical microscopy study of dislocations in multicrystalline silicon grown by directional solidification method[END_REF]. The presence of dislocations and/or deformed regions can be directly linked to low PV efficiency highlighting their importance for the final properties. Indeed, dislocations play a major role on the efficiency of solar cells, because they can act as preferential segregation sites for impurities, ultimately reducing the carrier lifetime [START_REF] Ekstrøm | Structure and dislocation development in mono-like silicon[END_REF][START_REF] Trempa | Defect formation induced by seed-joints during directional solidification of quasi-monocrystalline silicon ingots[END_REF][START_REF] Oriwol | Small-angle subgrain boundaries emanating from dislocation pile-ups in multicrystalline silicon studied with synchrotron white-beam X-ray topography[END_REF]. As such, dislocations remain one of the most important efficiency limiting defects in Si for PV applications [START_REF] Kivambe | On the role of stacking faults on dislocation generation and dislocation cluster formation in multicrystalline silicon[END_REF][START_REF] Binetti | Study of defects and impurities in multicrystalline silicon grown from metallurgical silicon feedstock[END_REF] for which defect engineering is needed [START_REF] Woo | An insight into dislocation density reduction in multicrystalline silicon[END_REF]. At a higher scale, subgrain boundaries and grain boundaries of high planar mismatch can be more detrimental than high symmetry grain boundaries such as symmetric coincidence site lattice (CSL) twin boundaries, also due to decoration by impurities [START_REF] Adamczyk | Recombination activity of grain boundaries in high-performance multicrystalline Si during solar cell processing[END_REF].

Within this context, three alternative technologies have been proposed to optimize the Si growth process and to improve the crystalline quality of the final ingot. These methods aim at mastering the initial grain nucleation and defect generation from the first stage of solidification:

the dendritic casting method [START_REF] Usami | Implementation of faceted dendrite growth on floating cast method to realize high-quality multicrsytalline Si ingot for solar cells[END_REF][START_REF] Fujiwara | Growth of structure-controlled polycrystalline silicon ingots for solar cells by casting[END_REF], the mono-like solidification (ML-Si) [START_REF] Jouini | Improved multicrystalline silicon ingot crystal quality through seed growth for high efficiency solar cells[END_REF][START_REF] Oliveira | Formation mechanism and properties of twinned structures in (111) seeded directionally solidified solar grade silicon[END_REF][START_REF] Stoddard | Casting Single Crystal Silicon: Novel Defect Profiles from BP Solar's Mono2 TM Wafers[END_REF] and the high performance multi-crystalline silicon (HP mc-Si) [START_REF] Lan | The emergence of high-performance multi-crystalline silicon in photovoltaics[END_REF]. Both ML-Si and HP mc-Si techniques, which are used in the industry, produce ingots with a lower dislocation density compared to the conventional mc-Si while allowing the use of low-cost casting solidification methods. However, huge and inherent challenges still exist and are related to: the control of the initial grain nucleation, twinning frequency occurrence, grain competition, defect generation, the interaction of the later with grain boundaries and their evolution during growth so that further research is needed. A major issue shared by all techniques without exception is to control and lower the density of strained regions of the crystal structure that are at the origin of dislocation emission. Few works are available in the literature discussing those phenomena and studying grain formation, grain boundary interaction [START_REF] Fujiwara | In situ observation of Si faceted dendrite growth from low-degree-ofundercooling melts[END_REF][START_REF] Fujiwara | Grain growth behaviors of polycrystalline silicon during melt growth processes[END_REF][START_REF] Riberi-Béridot | On the impact of twinning on the formation of the grain structure of multi-crystalline silicon for photovoltaic applications during directional solidification[END_REF][START_REF] Tsoutsouva | In situ investigation of the structural defect generation and evolution during the directional solidification of <110> seeded growth Si[END_REF][START_REF] Chuang | In situ observation of interaction between grain boundaries during directional solidification of Si[END_REF] and dislocation generation mechanisms [START_REF] Kutsukake | Generation mechanism of dislocations and their clusters in multicrystalline silicon during two-dimensional growth[END_REF][START_REF] Stokkan | Relationship between dislocation density and nucleation of multicrystalline silicon[END_REF][START_REF] Stokkan | Study of evolution of dislocation clusters in high performance multicrystalline silicon[END_REF][START_REF] Ryningen | Growth of dislocation clusters during directional solidification of multicrystalline silicon ingots[END_REF].

Dislocations can originate from plastic relaxation in deformed crystalline zones. However, it is still not clarified if deformed regions and dislocations develop during both crystal growth and cooling down or only during cooling down. Indeed, the origin of the dislocation or strained regions distribution observed in multi-crystalline silicon is still under debate [START_REF] Ryningen | Growth of dislocation clusters during directional solidification of multicrystalline silicon ingots[END_REF]. The first explanation relies on the formation of dislocations by cooling down due to the Alexander-Haasen model [START_REF] Alexander | Dislocations and plastic flow in the diamond structure[END_REF]. In this model, during cooling down, inhomogeneity in the stress pattern between different grains due to the solidification step and to the crystallographic orientation creates an inhomogeneous distribution of dislocations linked to the elastic anisotropy of Si single crystal inducing stresses between grains. The second model is based on the local nucleation of dislocation clusters during crystal growth [START_REF] Takahashi | Generation mechanism of dislocations during directional solidification of multicrystalline silicon using artificially designed seed[END_REF] near the solid-liquid interface. These two models to explain dislocation clusters in Si ingots are related to two different steps of the Si ingot fabrication: solidification and cooling down so that it is not possible to propose process improvement methods to reduce dislocation clusters. In the work of Ryningen et al. [START_REF] Ryningen | Growth of dislocation clusters during directional solidification of multicrystalline silicon ingots[END_REF], dislocation clusters were followed as a function of height in a multi-crystalline silicon ingot. From these observations, it was inferred that dislocations should have nucleated during growth oppositely to the cooling down origin mechanism.

Apart from the above controversy, the mechanisms are most likely interlinked as dislocations may originate during growth close to the solid-liquid interface but they can be generated and multiplied due to thermal stress during cooling down as well.

Moreover, recent molecular dynamics simulations of silicon growth highlighted the interrelation between, strain field, dislocation generation relatively to the growth direction and twin nucleation [START_REF] Zhou | Formation of Dislocations in the Growth of Silicon along Different Crystallographic Directions-A Molecular Dynamics Study[END_REF].

As a consequence, the formation mechanisms of dislocations and of crystal structure deformed regions during solidification must be understood beyond their fundamental interest. There is a need to confirm the strain building dynamics during crystal growth and near the solid-liquid interface at high temperature.

Fundamental understanding of the mechanisms is limited by the difficulty of accessing, from the ex situ study of the solidified ingots, to the origin of deformed and defective areas. The present paper is devoted to the mechanisms involved during the solidification process by advanced in situ characterisation methods. A particular focus is done on the crystal structure strain building during growth and grain competition and to its spreading and its possible role in grain nucleation. This work aims at deepening the fundamental understanding of the strain-related phenomena that occur during the Si crystal growth. Ultimately, this knowledge could be used to optimize the directional solidification processes to reduce the dislocation density and inhomogeneity.

Experimental

Solidification furnace and sample

The directional solidification experiment is carried out in a dedicated installation of IM2NP named GaTSBI (Growth at high Temperature observed by Synchrotron Beam Imaging), which is presently installed at beamline ID19 (previously at BM05) at the European Synchrotron Radiation Facility (ESRF). GaTSBI is a unique device that allows following in real time the solidification process during growth. It is a specially designed instrument composed of a high temperature directional solidification (DS) furnace employed in conjunction with synchrotron radiation X-ray imaging techniques (radiography and Bragg diffraction imaging). GaTSBI not only provides information on the solid-liquid interface dynamics, but also gives access to the single grain dynamic evolution during growth, the crystalline orientation / misorientation, the qualitative level of crystal structure local deformation as well as the nature and interaction of structural defects in general. A detailed description of the equipment, of the imaging techniques and of the previously obtained results can be found elsewhere [START_REF] Tandjaoui | Real time observation of the directional solidification of multicrystalline silicon: X-ray imaging characterization[END_REF][START_REF] Tandjaoui | Twinning occurrence and grain competition in multicrystalline silicon during solidification[END_REF].

In the present work, the initial monocrystalline Si sample was cut (38 mm × 5.8 mm × 0. The sample is heated by applying the same temperature at the bottom and top resistances of the furnace up to 1373 K. Then, a temperature gradient of 30 K/cm is imposed between the two resistive heaters until melting. A local temperature gradient of 12 K/cm was measured in our previous work for this applied temperature gradient [START_REF] Stamelou | {111} facet growth laws and grain competition during silicon crystallization[END_REF]. The sample is partially melted and thus a seed crystal, preserving the initial orientation of the sample, is kept within the field of view of the X-ray imaging as can be seen on the diffraction pattern just after partial melting (Figure 1.a).

After the partial melting and the stabilization of the solid-liquid interface, a cooling rate of -1 K/min is applied on both heaters until the region of the silicon sample observed within the field of view is fully solidified. Because of the imposed temperature gradient, the solidification is directional in the upward direction. Then, the sample is cooled down until 923 K by applying cooling rates of -13 K/min and -10.4 K/min on the top and bottom heaters, respectively. Below 923 K, the natural cooling down to room temperature of the furnace and sample is not anymore monitored.

In situ X-ray imaging during crystal growth

The sample is constantly illuminated by the X-ray synchrotron white beam during the whole melting, solidification and cooling down sequence. The solid-liquid interface progresses and its morphology is recorded by X-ray radiography using a specific CCD camera developed at the ESRF named FReLoN (Fast Readout Low-Noise) with 2048×2048 image pixel size. An optics giving 5.8 μm pixel size and 11.9 × 11.9 mm 2 field of view is used. Radiography images are recorded every 3 s with an exposure time of 1 s. The obtained X-ray radiography images are direct images of the beam transmitted through the sample. The image contrast is based on the material absorption. The absorption coefficient of the solid and liquid Si depends on their mass densities which are not much different (2.31 g/cm 3 and 2.56 g/cm 3 for solid and the liquid, respectively) meaning that the solid-liquid interface in the raw images is hardly distinguishable. As a consequence, an image processing method consisting of successive image division is applied to reveal the shape of the solid-liquid interface more accurately as explained in our previous work [START_REF] Tandjaoui | Twinning occurrence and grain competition in multicrystalline silicon during solidification[END_REF].

White beam X-ray Bragg diffraction patterns with a large beam (also named topography) show diffraction spots corresponding to different {hkl} diffracting planes. These diffraction patterns are regularly recorded on X-ray sensitive films (AGFA Structurix D3-SC, 17.6×12.5 cm 2 ) positioned at a distance of about 300 mm from the sample (see for example Figures 1.a and 1.b).

In the experiment described in the following, the exposure time used to record the diffraction patterns is 0.5 s. Due to the small beam divergence and to its large size, the whole width of the sample (5.8 mm) can be illuminated; the diffracting volume corresponds to the width of the sample (5.8 mm) × the height of the field of view (10 mm) × the thickness of the sample (0.3 mm). Each diffraction spot contains information on the level of crystal perfection and/or crystal structure strain field. Bragg diffraction imaging consists in studying the detailed structure of diffractions spots. This is a powerful technique that can be used for the visualization of defects (dislocations, twins, domain walls, inclusions, impurity precipitates) present in the crystal volume. Indeed, it allows characterizing the long range distortion fields and / or the strain fields associated with a macroscopic crystal deformation. This is made possible because these distortion fields in the crystal structure diffract at different positions, according to the Bragg law and depending on the different wavelengths of the white beam, compared to the perfect crystal. As a consequence, these distortion fields are at the origin of superimpositions of the diffracted rays, so that a contrast (non -homogeneous intensity distribution) is created in the recorded image and corresponds to crystal structure strain fields [START_REF] Burns | HPHT growth and x-ray characterization of high-quality type IIa diamond[END_REF]. Bragg diffraction imaging technique was used previously by Oriwol et al. [START_REF] Oriwol | Small-angle subgrain boundaries emanating from dislocation pile-ups in multicrystalline silicon studied with synchrotron white-beam X-ray topography[END_REF] to study dislocations and the formation of sub-grain boundaries ex situ in silicon ingots and by IM2NP in situ during crystal growth (e.g. [START_REF] Tsoutsouva | In situ investigation of the structural defect generation and evolution during the directional solidification of <110> seeded growth Si[END_REF]). The originality of our work is to combine both radiography and diffraction imaging in situ during growth. Indeed, the combination of both imaging techniques provides complementary dynamic information about crystal growth and competition and about the crystal structure deformation.

Ex situ complementary investigations 2.3.1 Electron Backscatter Diffraction (EBSD)

Ex situ electron backscatter diffraction (EBSD) measurement has been performed after solidification and cooling down to room temperature using a FEG-SEM JEOL JSM 7001F equipped with a HKL Nordlys camera with either a 7 μm or a 0.7 μm step size depending on the studied area. In order to image the three -dimensional orientation of the crystals in the sample, inverse pole figure (IPF) orientation maps are generated with respect to the three space directions:

normal to the sample surface (z), transverse direction (y) and in the growth direction (x).

Moreover, the coincidence site lattice map (CSL) is reconstructed to evidence the orientation relationship between adjacent grains and the boundaries with a special character. In this paper, Σ3 <111> (red color in the maps), Σ9 <110> (blue) and Σ27a <110> (yellow) twin boundaries labeling refer to rotations around <h k l> axis that satisfy the misorientation ranges given by the Brandon criterion, which are (60 ± 8.66)°, (38.94 ± 5)° and (31.58 ± 2.89)°, respectively.

Additionally, the grain orientation spread (GOS) and local misorientation (LM) maps are extracted as well from the EBSD results. The GOS map is constructed by calculating the difference between the orientation of each pixel in the grain and of the grain average orientation to evidence the more distorted grains within a grain structure. A color code is used to map the grains without deformation (perfect Si crystal appears in blue) and having an average crystal structure deformation (red color for the highest deformation). The LM map provides the local misorientation of each pixel (kernels of 5 × 5 pixels) to locate deformed regions.

Experimental results

Diffraction spots corresponding to the orientation of the maintained Si seed can be seen in the highlight the fact that the projection of the solid-liquid interface can be seen in these images.

There is a {111} facet at the solid-liquid interface whose inclination relatively to the sample is directly related to the seed crystal orientation (Figure 1.c) as also seen in our previous work [START_REF] Tsoutsouva | In situ investigation of the structural defect generation and evolution during the directional solidification of <110> seeded growth Si[END_REF]. After a few millimeters of growth, grains nucleate at the edges of the sample and enter in competition with the initial seed and finally block its progress as can be seen in the sequence of diffraction pattern images recorded during growth in Figure 2. The new grains have a particular orientation relationship with the seed and with each other, they are in twin relationship. This fact is evidenced by the presence of a common diffraction plane, (1 � 11 � ), for the left twin crystal and the seed as can be seen by comparing Figures 2.a i) to v) and 2.b i) to v) [START_REF] Tandjaoui | Twinning occurrence and grain competition in multicrystalline silicon during solidification[END_REF]. Additionally, the zebra aspect and the spots in Figure 2 show that the twins are nucleating regularly during growth and that the crystallographic orientation is alternately repeated. The right zebra pattern is also related to successive twinning. This cannot be evidenced with the diffraction spots showed in Figure 2 but is confirmed by ex situ EBSD characterization (Figure 3). Competition among twinned grains takes place as they meet in the middle of the sample. At this level, grain boundaries are formed including Σ3 and in some cases higher order twin CSL boundaries. At last, the right twins take over all other grains as growth proceeds. As can be noticed in Figure 4, the solid-liquid interface is inclined which indicates the existence of a radial local temperature gradient. The full lines on The grain boundary formation at the encounter of twins coming from both sides continues regularly until the dynamics of the competition disturbs the arrangement and a new grain nucleates (grain VI in Figure 7). The nucleation of this grain is of particular interest because it does not take place at the level of the edge facets as for all earlier nucleation during growth.

Moreover, it has a different crystallographic orientation from the other previously existing grains.

The dynamics of this particular grain nucleation are recorded by X-ray radiography during the solidification (Figure 7). The grain boundary groove created by the encounter of grains IV and V can be seen in the As observed on the radiography images (Figure 4.a), the solid-liquid interface is flat except from the side region where it is inclined. It is higher on the right hand side indicating that there is a transverse temperature gradient which can be estimated from the radiography images to about 0.8 K/cm. The origin of this transverse temperature gradient can be due to the positioning of the sample in the crucible or to its shape after the melting segment creating slight thermal field asymmetries. It is worth noting that it is smaller than the gradient in the vertical solidification direction measured at 12 K/cm which ensures a directional growth in the upward direction as can be seen during the experiment. However, this is sufficient for the right hand side of the interface to be in advance compared to the left hand side. As a consequence, the first twin nucleates on the right hand side {111} facet (Figure 2) and all the subsequent twins nucleating on the right hand side are in advance compared to the twins coming from the left side of the sample. The twin nucleation from the ingot edge has been reported in medium scale [START_REF] Trempa | Mono-crystalline growth in directional solidification of silicon with different orientation and splitting of seed crystals[END_REF] as well as in industrial scale mono-like ingots [START_REF] Jay | Advanced process for n-type mono-like silicon a-Si:H/c-Si heterojunction solar cells with 21.5% efficiency[END_REF]. As a consequence, this phenomenon and the particular final grain structure observed in Figure 3 can be generalised although our samples are small size model ingots.

In our experiments, all twins observed ex situ after growth and after subsequent cooling down to room temperature are issued from the growth segment without exception as can be seen from the comparison between diffraction images during growth (Figure 2) and the grain structure after cooling down (Figure 3). As a consequence, from this experiment as well as from our previous work [START_REF] Tsoutsouva | In situ investigation of the structural defect generation and evolution during the directional solidification of <110> seeded growth Si[END_REF], it is demonstrated that all twins and new grains nucleate during the solidification/growth segment disregarding of shear band phenomena described in the literature [START_REF] Commin | Identification of shear bands in wrought magnesium alloy friction stir welds and laser beam welds[END_REF][START_REF] Mirzadeh | EBSD study of a hot deformed austenitic stainless steel[END_REF] as e.g. in wrought magnesium alloys submitted to laser or friction stir welding. In the case of shear band phenomena, a highly twinned microstructure and localized strain is observed.

Commin et al. [START_REF] Commin | Identification of shear bands in wrought magnesium alloy friction stir welds and laser beam welds[END_REF] state that during welding, the stored energy is relieved plastically through the shear bands. In our experiments, although twin nucleation is not observed during cooling down, plastic deformation can take place following the Alexander-Haasen [START_REF] Alexander | Dislocations and plastic flow in the diamond structure[END_REF] model creating dislocations. The initial strain is built at the encounter of grains during growth competition phenomena as will be further discussed in the following.

Twin growth

Once the twin grains have nucleated at the left and right sides of the sample, they grow towards the central part along their respective {111} facets. Since the growth of the right twins is in advance, compared to the left, each time they meet, the growth of the twin grains coming from the left side is blocked. The progress of the pristine grain issued from the seed is also stopped by the left/right twin grain competition (Figures 2 and3). Due to the higher undercooling at the level of the edge facets compared to the solid-liquid interface, the nucleation on the {111} edge facets is favored and subsequently, edge nucleated twin grains grow at a faster velocity than the general interface [START_REF] Stamelou | {111} facet growth laws and grain competition during silicon crystallization[END_REF].

Grain competition and associated strain building

The grain competition between the left and right twins leads to the formation of a groove at the solid-liquid interface and to the generation of a grain boundary.

The grain boundary crystallographic characteristic is directly linked to the relative crystallographic orientations of the two meeting grains. Due to the alternated twin crystallographic orientations, the same grain boundary types are retrieved alternately. In this experiment, Σ3 and Σ9 twin boundaries are formed and are observed in sequence. As mentioned in section 3, strain is not observed at the level of the Σ3 twin grain boundaries whereas local strain is clearly observed each time a Σ9 twin boundary is formed (Figures 5 and6).

During the heating segment of the experimental procedure, dislocations appear in the sample as seen before [START_REF] Tsoutsouva | In Situ Imaging of Dislocation Expansion in FZ-Si Seeds During Temperature Ramp Heating Process[END_REF] and remain in the seed kept after partial melting. During solidification, these dislocations develop, usually along the growth direction and can encounter grain boundaries.

Dislocations can be stopped and accumulate, unless they are able to cross-slip at the level of a grain boundary which is most likely to occur at Σ3 type twin boundaries because of the presence of a {111} plane [START_REF] Tsoutsouva | In situ investigation of the structural defect generation and evolution during the directional solidification of <110> seeded growth Si[END_REF]. This situation was also discussed by Lim et al. [START_REF] Lim | Interaction between lattice and grain boundary dislocations and their role in mechanical properties of interfaces[END_REF]. They conclude that in nickel, coherent Σ3 twin boundaries act as effective barriers to slip except in the case of screw dislocations which can direct or cross slip across the boundary using the boundary plane itself as a glide plane because it is a {111} plane. Then, the screw dislocations do not leave behind any residual dislocation in the boundary. Ultimately, they can cross slip and propagate along the {111} planes until they reach a free surface or meet another interface. The strained areas observed on the projection of the solid-liquid interface (see Figure 2.a.iii)) and corresponding to the positions of the twin planes can be associated to the gliding of dislocations along the {111} facets that exit at the solid-liquid interface.

Oppositely, local deformed crystal structure zones are created each time a Σ9 twin boundary is formed due to grain competition. This is expected as in the case of Σ9 twin boundaries, cross slip is unlikely. For instance, only the dislocations having the Burgers vector direction of a common rotation axis <110> to build a symmetric grain boundary could cross slip; for other rotation axes, no cross slip is possible. As a consequence, dislocations and strain can accumulate at the level of the Σ9 twin boundary. On top of the local strain, it is observed that the crystal structure strain field can propagate at longer ranges when due to competition by comparison of Figure 6.a and c.

Clearly in our experiments, strain and dislocation emission build during growth as inferred by

Ryningen et al. [START_REF] Ryningen | Growth of dislocation clusters during directional solidification of multicrystalline silicon ingots[END_REF]. With these results, it is showed that dislocations and strain can be generated by growth features during solidification only, even though dislocation generation and/or multiplication can occur during heating up and cooling down steps.

Spontaneous grain nucleation inside the grain boundary groove and subsequent strain level decrease

The sequence of the grain boundary groove evolution can be generalized according to our previous work [START_REF] Tsoutsouva | In situ investigation of the structural defect generation and evolution during the directional solidification of <110> seeded growth Si[END_REF][START_REF] Riberi-Béridot | On the impact of twinning on the formation of the grain structure of multi-crystalline silicon for photovoltaic applications during directional solidification[END_REF] and to the results here. First, when a facetted / facetted grain boundary groove is formed, both facets generally grow at the same growth velocity. If there is no twin relationship between both grains, the direction of the generated grain boundary follows the bisector of the angle between the two facets. This is in agreement with the model proposed by

Duffar and Nadri [START_REF] Duffar | The grain-grain-liquid triple phase line during solidification of multi-crystalline silicon[END_REF][START_REF] Nadri | Two-dimensional numerical modeling of grain structure in multi-crystalline silicon ingot[END_REF]. Ultimately, grain VI nucleation is observed inside the grain boundary groove (Figure 7.e) when it is deeper at this instant as can be seen in Figure 7.d compared to Consequently, a Σ3 twin boundary is formed between grain V and the nucleated grain VI (Figure

5.c).

Several phenomena can concur to the nucleation of the grain VI inside the grain boundary groove. The first reason is that the undercooling inside a grain boundary groove is higher than at the level of the solid-liquid interface as shown in our previous work [START_REF] Stamelou | {111} facet growth laws and grain competition during silicon crystallization[END_REF] which facilitates grain nucleation in this area. However, higher undercooling values (ranging 0.2 to 0.8 K) compared to the undercooling inside grain boundary grooves (ranging from 0.1 to 0.4 K) are measured at the edges. It means that the nucleation probability is higher at the edges than inside grain boundary grooves although grain nucleation inside grain boundary grooves remains possible. As a consequence, the main contribution to the grain structure formation is due to nucleation on {111} facets at the edges and the inevitable upcoming grain competition as seen in this experiment.

As seen above, the nucleation of the purple grain VI occurs on the left {111} facet of the grain boundary groove. The attachment energy and the contact area are the key factors for the heterogeneous nucleation of twins as modelled by Jhang et al. [START_REF] Jhang | Heterogeneous twinning during directional solidification of multi-crystalline silicon[END_REF]. This model was specifically applied to the particular experiment described here and to the grain boundary groove in Figure 7 (Case 1, model 1.1 in [START_REF] Jhang | Heterogeneous twinning during directional solidification of multi-crystalline silicon[END_REF]). For both facets, the attaching energy of a twin at the level of the crucible wall/Grain IV/Grain V is negative so that twinning is thermodynamically favourable for the experimentally measured undercooling of about 0.4 K [START_REF] Stamelou | {111} facet growth laws and grain competition during silicon crystallization[END_REF]. Additionally, from the experimental EBSD data, the 3D configuration of the {111} facets at the level of the grain boundary groove can be quantitatively described. Larger facet angles relatively to the wall are calculated for the left {111} facet in Figure 7 so that it has a lower contact area with the crucible wall. Finally, both low attachment energy and lower contact area concur to a higher twining probability of 8 x 10 -7 on the left facet (facet 1 in reference [START_REF] Jhang | Heterogeneous twinning during directional solidification of multi-crystalline silicon[END_REF]) compared to the a twinning probability of 8 x 10 -8 on the right facet (facet 2 in reference [START_REF] Jhang | Heterogeneous twinning during directional solidification of multi-crystalline silicon[END_REF]). Thus, according to this model, the left {111} facet (Grain V in Figure 7) is the most thermodynamically favourable for the nucleation of a new grain. Experimentally, nucleation is indeed observed at the level of this particular {111} facet. Additionally, the origin of the nucleation of this grain is due to the competition between twins growing from the left and from the right sides. Indeed, grains are not free to grow due to neighbour grains during competition.

The most stable situation is obtained when the Σ3 twin boundaries formed by competition can follow a common plane {111} or {112}, corresponding to a symmetric coherent and most densely packed and to a symmetric/asymmetric incoherent and second most densely packed Σ3 twin boundary, respectively [START_REF] Ervik | Dislocation formation at Σ=27a boundries in multicrystalline silicon for solar cells[END_REF][START_REF] Zhao | Carbon segregation at Σ3 {1 1 2} grain boundaries in silicon[END_REF]. In the case of Σ9 twin boundaries, three cases have been reported

by Ervik et al. [START_REF] Ervik | Dislocation formation at Σ=27a boundries in multicrystalline silicon for solar cells[END_REF]: two twin boundaries with {122} or {114} common planes, the first situation, corresponding to the best crystalline case and to the most densely packed CSL, and an asymmetric Σ9 twin boundary with {111}/{115} planes with a low density of coincidence sites.

At the first encounter of twin grains coming from the left and right sides of the sample, a small vertical Σ9 twin boundary is formed (α location in Figure 5.b). This Σ9 twin boundary corresponds to a boundary with a {122} common plane, lying along the macroscopic grain boundary direction as can be seen on the pole figure (orange circles highlight the common poles in Figure 8.a). This is the most stable configuration as the Σ9 {122}1,2 is associated with the lowest energy. Moreover, this boundary is aligned with the temperature gradient which would ensure that both neighbor grains would grow without competition if no further nucleation would occur.

However, in the present experiment, subsequent twin nucleation take place on both sample edges as discussed above. Because of competition and of the fact that the twin grains on the right nucleate and grow in advance, the next Σ9 twin boundary cannot develop vertically (β in Figure 5.b). In this case, the Σ9 twin boundary adopts the asymmetric {111}/{115} configuration (common poles are highlighted with the green circles in Figure 8.b) which corresponds to a higher grain boundary energy [START_REF] Brokman | Atomistic faceting of asymmetric tilt boundaries[END_REF]. This structure has been revealed by TEM (Transmission Electron Microscopy) and its energy has been calculated by DFT (Density Functional Theory) [START_REF] Stoffers | Complex Nanotwin Substructure of an Asymmetric Σ9 Tilt Grain Boundary in a Silicon Polycrystal[END_REF]. It was reported that on the contrary to the Σ9 {122}1,2 grain boundary, the atomic structure of the asymmetric Σ9 {111}/{115} one shows strong distortions. Its energy was found to be in the range of the highly symmetric low-Σ GBs but about twice the one of the symmetric Σ9 {122}1,2. As a consequence, this situation is unstable from the energetic point of view and not favorable. During growth, rearrangements tend to create a more stable configuration. Indeed, later during growth, twin grains from the left grow laterally towards the center at the expense of the twin grain from In general, non-symmetrical grain boundaries are deformed at the atomic scale [START_REF] Stoffers | Complex Nanotwin Substructure of an Asymmetric Σ9 Tilt Grain Boundary in a Silicon Polycrystal[END_REF] and offer greater resistance for dislocation crossing, thereby creating higher strain [START_REF] Lim | Interaction between lattice and grain boundary dislocations and their role in mechanical properties of interfaces[END_REF] and structure deformation, promoting dislocation emission. Concomitantly, an increasing strain is observed. Indeed, the competition goes along with an increasing accumulation of strain when Σ9 twin boundaries are present that can be seen on both diffraction images (Figure 6.d) and grain orientation spread (GOS) in Figure 9.b as well as on the local misorientation (LM) in Figure 9.c EBSD maps.

The local misorientation map (Figure 9.c) gives local information about the strain probably related to the presence of higher density of dislocation clusters. What can be added from the analysis of this map, is that strain accumulates at the twin boundary from the left just before the grain nucleation. Moreover, strain is generated at the encounter of the twins and can propagate in neighbor grains up to several millimeters. As a following step, the grain VI nucleates inside the grain boundary groove on the left {111} facet that belongs to grain V.

The nucleated grain VI is in average less strained than the incoming twins from the right (Figure 9.b). Figure 9.a is a diffraction image of the grain VI on which it can be seen that the highest strain level (darker contrast) is localized at the position of its nucleation and beginning of its growth. Later, the strain level decreases as evidenced by the lighter contrast. On the one hand, after the nucleation, grains above this nucleation event are generally less deformed at the scale of the grain structure (Figure 9.b) and more locally inside the grain that nucleated (Figure 9.a). Indeed, it can be noted that the GOS decreases in average for the last twin (orange color in Figure 9.b) as the nucleation of the grain VI leads to a lower level of distortion in the above growing grains. On the other hand, in the region on the right, after nucleation of the grain inside the grain boundary groove, local strain and dislocation emission (Figure 9.a) are observed. It is due to the competition with the newly nucleated grain that tends to extend in the solidification direction and to the fact that grain VI is incidentally found in a Σ27 twin relationship with the right grain. This type of twin boundary is prone to crystal structure deformation and associated dislocation emission [START_REF] Tsoutsouva | In situ investigation of the structural defect generation and evolution during the directional solidification of <110> seeded growth Si[END_REF][START_REF] Stokkan | Study of evolution of dislocation clusters in high performance multicrystalline silicon[END_REF].

Strain redistribution cannot be invoked in our experimental case as the existing strain field built during growth remains. However, the new grain nucleation contributes to have a better crystalline quality in the upper part of the ingot. Indeed, after this grain nucleation, the upper growing grains recover a lower strain density.

So, the nucleation can be triggered by energetic reasons discussed above, by grain competition space constraints as well as by the existence of a high density of dislocations that can favor nucleation by decreasing the nucleation undercooling value. It is worth mentioning that this result can be generalized as for the same crystallographic orientation in other samples processed with similar conditions, comparable grain structure can be retrieved and nucleation events similar to the one of the grain VI are observed.

Strain evolution during cooling down

In Figure 10, the evolution of the (2 � 20) diffraction spot is followed during the cooling down after solidification. A global deformation of the sample is revealed by the evolution of the shape of the diffraction images as clearly seen in Figure 10.e. It corresponds to a bending of the crystal planes at the sample scale. However, although this global strain can be at the origin of dislocations, seeing the very dark contrast of this diffraction image, it is not at the origin of new grains and twin nucleation as can be confirmed by comparing the diffraction images during growth (Figure 2) and the EBSD maps after cooling down (Figure 3). Moreover, another major result is that although there is a clear evolution of the global deformation at the scale of the sample, local strains created during growth still exist after cooling down and are not redistributed as can be seen by comparing the X-ray diffraction images during growth ( As a consequence, one of the main result of this work is also to show that strain that is created during solidification and locally distributed can be retrieved in the ingot after cooling down even though additional sample scale plastic deformation able to generate dislocations is added during cooling down.

Conclusion

Strain building during growth has a high impact on the generation of dislocations but it is also associated to the spontaneous new grain nucleation. It is shown that localized strain building is the result of the twin growth dynamics and competition during growth while the associated dislocation emission mainly depends on the character and the type of the generated twin boundary.

One of the main results of this work is that a new grain nucleation can be triggered by the strain accumulation during growth induced by grain competition. Subsequently, this nucleation event contributes to the recovery of a lower strain level in the upper growing grains. The nucleation in presence of strain accumulation can be triggered by energetic reasons as well as by the existence of the associated dislocations. Indeed, dislocations can favor nucleation by decreasing the nucleation undercooling. It is shown that all new grains and twins are only due to nucleation and competition during solidification and are not at all associated to release of plastic deformation during cooling down in the experiment presented here. It should be noted that this also is in agreement with our previous observations.

Another main result is that strain, which is created during solidification and nonhomogeneously locally distributed, can be retrieved in the ingot after cooling down even though additional strain is created during cooling down. Moreover, no detectable additional twin nucleation is observed during cooling down.

Aknowlegments

The work has been supported by the ANR Project CrySaLID (Nº ANR-14-CE05-0046-01).

The ESRF (European Synchrotron Radiation Facility) BM05 team is greatly acknowledged for support during the X-ray imaging experiments.

  3 mm) from a double side mechanochemically polished intrinsic (resistivity beyond 5000 Ω.cm) FZ wafer 50.8 mm, {110} in surface orientation, provided by SIL'TRONIX Silicon Technologies. Inside GaTSBI, solidification experiments are performed under dynamic vacuum (~10 -6 mbar) while the sample is housed inside a pyrolytic BN crucible with dimensions: length 40 mm, width 6 mm and depth 0.3 mm. The sample is oriented <100>, <110> and < 11 � 0 > (Figure 1.c) in the solidification direction, in the direction normal to the wafer surface and in the transverse direction, respectively.

Figure 1 :

 1 Figure 1: White beam diffraction patterns recorded during the experiment: a) initial state after partial melting of the sample to keep an oriented seed: TU (upper resistor) = 1717 K and TL (lower resistor) = 1564 K, b) at the end of the solidification in the field of view: TU (upper resistor) = 1704 K, TL (lower resistor) = 1551 K, c) macroscopic orientation of the initial sample. Applied temperature gradient: 30 K/cm and cooling rate of -1 K/min applied on both heaters. 𝐠𝐠 �⃗ is the projection of the diffraction vector on the film.

Figure 1 .

 1 Figure 1.a. The diffraction pattern recorded after the end of the solidification process at a cooling

Figures 2 .

 2 Figures 2.a and b show zoomed images of the diffraction spots (1 � 11 � ) and (2 � 20) recorded from

Figure 2 :

 2 Figure 2: Zoomed images of two diffraction spots extracted from the diffraction patterns during growth (applied temperature gradient: 30 K/cm and cooling rate of -1 K/min applied on both heaters at t0). a) (𝟏𝟏 � 𝟏𝟏𝟏𝟏 � ) and b) (𝟐𝟐 � 𝟐𝟐𝟐𝟐) diffraction spots. t0 corresponds to the beginning of solidification and 𝐠𝐠 �⃗ is the projection of the diffraction vector on the film.

Figure 3 :

 3 Figure 3: Inverse Pole Figure (IPF) maps obtained by EBSD after growth and cooling down. a) x: along the growth direction, b) z: direction normal to the sample wafer surface, c) y: transverse direction, d) CSL (Coincidence Site Lattice) map.

Figure 4 :

 4 Figure 4: X-ray radiography image of a) the solid-liquid interface during growth with facets at both edges, b) zoom at the level of the grain boundary groove formed due to competition between twins from the left (II) and right (III). The blue lines on Figures 4.a and b correspond to the {111} facet traces. c) Pole figure of the {111} planes issued from the EBSD measurements. The poles of the seed (I), left (II) and right (III) grains are highlighted respectively in red, light blue and dark blue on the pole figure.

Figure 4 .

 4 a corresponds to the inclination of the facets at the edges. These lines are reported on the {111} pole figure (Figure4.c) and correspond directly to the traces of the {111} planes confirming that the facets observed in Figure4.a are {111} facets. A first twin nucleation can be observed on the {111} facet situated on the right of the sample and at a later stage on the {111} facet situated on the left. At the level of the edge facets, the measured maximum undercooling is lower than 1 K ranging from 2 × 10 -1 to 8 × 10 -1 K as previously evaluated[START_REF] Stamelou | {111} facet growth laws and grain competition during silicon crystallization[END_REF].The twins nucleate first at the edges of the sample and grow along the {111} plane towards the center[START_REF] Riberi-Béridot | On the impact of twinning on the formation of the grain structure of multi-crystalline silicon for photovoltaic applications during directional solidification[END_REF]. This sequence is repeated several times during growth on both edges. In this experimental configuration, twins growing from the right and left hand sides reach about the middle of the sample where they meet forming a grain boundary. The corresponding grain boundary groove is evidenced at the solid-liquid interface in Figures 4.a and b. The pole figure of the {111} planes, extracted from the ex situ EBSD measurements, confirms that both grain boundary groove facets are {111} facets (dotted lines on Figure 4) similarly to the edge facets.

Figure 5 :

 5 Figure 5: a) Inverse Pole Figure (IPF) map obtained by EBSD after growth along the growth direction (equivalent to Figure 3.a), b) CSL map of the competition region, c) CSL map of the region of the purple grain (VI) nucleation.

Figure 6 :

 6 Figure 6: a) �𝟏𝟏𝟏𝟏𝟏𝟏� diffraction spot of the seed and twins at t0 + 13 min during growth (equivalent to Figure 2.a.v), b) complementary spot showing the left twins, c) complementary spot showing the right twins, d) zoom at the level of the growth competition region.

  Figures 7.a to d. It is a facetted/facetted grain boundary groove constituted of two {111} facets existing in both grains IV and V as can be seen in the pole figure (Figure 7.f).

Figure 7 :

 7 Figure 7: Radiography images of the solid-liquid interface at the level of grain IV/V competition leading to the nucleation of grain VI during growth (purple in the IPFX in Figure 3) : a) ti, b) ti + 3 s, c) ti + 6 s, d) ti + 9 s, e) ti + 12 s. f) Pole figure of the {111} planes corresponding to a twin grain on the right (IV, dark blue) and left (V, light blue) sides and to the grain inside the grain boundary groove (VI, purple) from EBSD measurement after crystal growth.

Figure 7 .

 7 Figure 7.a. A deeper grain boundary groove corresponds to a higher undercooling inside the grain

Figure 8 :

 8 Figure 8: Two possible configurations of the Σ9 twin boundaries between grains of type IV and V in Figure 7, dark and light blue in Figure 5.a, respectively. a) Vertical grain boundary trace (orange lines) corresponding to a Σ9 {122}1,2 twin boundary. Common poles are highlighted by orange circles, b) inclined grain boundary trace (green lines) corresponding to a Σ9 {111}1/{115}2 twin boundary. Common poles are highlighted by green circles.

  the right. Then, vertical Σ9 {122}1,2 twin boundaries are formed again (Figure 5.c). The important effect of the growth dynamic competition between twin grains coming from the left and right is also evidenced by the presence of a distorted Σ3 twin boundary at first encounter (χ in Figure 5.c) that evolve to the ideal orientation and straightness of the Σ3 twin boundary. At a later time, at the level of the encounter with a new twin grain from the right (Figures 2.a and b.iv)), a tiny asymmetric Σ9 {111}/{115} twin boundary (δ in Figure 5.c) is created just before the nucleation of the grain VI (purple grain in Figure 5.a).

Figure 9 :

 9 Figure 9: a) Diffraction image of the grain VI during solidification at the same instant as Figures 2.a and b.v). b) Grain Orientation Spread (GOS) map, colours range from blue (perfect Si crystal) to red (highest level of the average deformation of the crystal structure) and c) Local Misorientation (LM) map, the highest misorientation corresponding to the red colour. Both GOS and LM maps are extracted from EBSD measurement after growth and cooling down.

  Figures 6.a & d) and the LM and GOS maps after cooling down (Figures 9.b and c).

Figure 10 :

 10 Figure 10: a) to d) Images of the (𝟐𝟐 � 𝟐𝟐𝟐𝟐) diffraction spot during cooling down to 923 K (Applied cooling rate are -13 K/min and -10.4 K/min on the top and bottom heaters, respectively). e) Image of the (𝟐𝟐 � 𝟐𝟐𝟐𝟐) diffraction spot at room temperature. TU and TL are the temperatures of the upper and lower heaters, respectively. The same scale is used for all images.


Mirlande Présumé

Angélique Simon-Deckers

Céline Tomkiewicz-Raulet

Béatrice Le Grand

Jeanne Tran Van Nhieu

Gregory Beaune

Olivier Duruphty

Jean Doucet

Xavier Coumoul

Jean-Claude Pairon

Jorge Boczkowski

Sophie Lanone

Pascal Andujar
email: pascal.andujar@inserm.fr

Exposure to metal oxide nanoparticles administrated at occupationally-relevant doses induces pulmonary effects in mice

Keywords:

In spite of the great promises that the development of nanotechnologies can offer, concerns regarding potential adverse health effects of occupational exposure to nanoparticle (NP) is raised. We recently identified metal oxide NP in lung tissue sections of welders, located inside macrophages infiltrated in fibrous regions. This suggests a role for these NP in the lung alterations observed in welders.

We therefore designed a study aimed to investigate the pulmonary effects, in mice, of repeated exposure to NP administrated at occupationally-relevant doses. We therefore chose 4 metal oxide NP representative of those found in the welder's lungs: Fe 2 O 3 , Fe 3 O 4 , MnFe 2 O 4 and CrOOH. These NP were administrated weekly, for up to 3 months, at 2 different doses: 5 µg, chosen as occupationally-relevant to welding activity, and 50 µg, chosen as occupationally-relevant to the context of a NP-manufacturing facility. Our results show that 3 month-repeated exposures to 5 µg NP induced limited pulmonary effects, characterized by the development of a mild peribronchiolar fibrosis observed for MnFe 2 O 4 and CrOOH NP only. This fibrotic event was further extended in terms of intensity and localization after the repeated administration of 50 µg NP: all but Fe 2 O 3 NP induced the development of peribronchiolar, perivascular, and alveolar fibrosis, together with an interstitial inflammation.

Our data demonstrate for the first time a potential risk for respiratory health posed by repeated exposure to NP at occupationally-relevant doses. Given these results, the development of occupational exposure limits specifically dedicated to NP exposure might therefore be an important issue to address.

INTRODUCTION

Atmospheric pollution has long been known to potentially induce adverse health effects in the general population [START_REF] Logan | Mortality in the London fog incident[END_REF]. Particulate pollution represents one component of the atmospheric pollution that is associated with an increased morbidity and mortality, in particular at the cardiovascular and respiratory levels [START_REF] Atkinson | Epidemiological time series studies of PM2.5 and daily mortality and hospital admissions: a systematic review and meta-analysis[END_REF][START_REF] Qiu | Coarse particulate matter associated with increased risk of emergency hospital admissions for pneumonia in Hong Kong[END_REF][START_REF] Lepeule | Long-term effects of traffic particles on lung function decline in the elderly[END_REF][START_REF] Delfino | Asthma morbidity and ambient air pollution: effect modification by residential traffic-related air pollution[END_REF]. Nanoparticles (NP) constitute an important and still growing part of particulate pollution not only in the environmental context, but also in occupational settings.

At workplace, NP pollution can be emitted secondary to various industrial processes, such as arc-welding. During this process, welding fumes contain NP that represent up to 10% in mass and 80% in number of the total particles emitted [START_REF] Antonini | Persistence of deposited metals in the lungs after stainless steel and mild steel welding fume inhalation in rats[END_REF][START_REF] Gomes | Determination of airborne nanoparticles from welding operations[END_REF][START_REF] Dasch | Physical and chemical characterization of airborne particles from welding operations in automotive plants[END_REF][START_REF] Stephenson | Workplace exposure to submicron particle mass and number concentrations from manual arc welding of carbon steel[END_REF]. NP can also be emitted intentionally at workplace, thereby generating manufactured NP. Exposure of workers to NP can occur during the initial production of NP, as well as during their further processing, dispersion and/or integration in secondary products. NP concentrations up to several mg/m 3 have been detected in workplaces manufacturing nanosized iron oxides, titanium dioxide, silver or nanostructured materials used for the electric industry for example [START_REF] Plitzko | Workplace exposure to engineered nanoparticles[END_REF][START_REF] Lee | Exposure assessment of workplaces manufacturing nanosized TiO2 and silver[END_REF][START_REF] Xing | Exposure characteristics of ferric oxide nanoparticles released during activities for manufacturing ferric oxide nanomaterials[END_REF][START_REF] Pietroiusti | Health implications of engineered nanomaterials[END_REF][START_REF] Curwin | Exposure characterization of metal oxide nanoparticles in the workplace[END_REF].

A growing number of workers is estimated to be involved in work processes linked to the intentional or unintentional production of NP; up to 6 millions of workers will potentially be exposed to NP in 2020 [START_REF] Roco | Nanotechnology Research Directions for Societal Needs in[END_REF]. In spite of the great promises that the development of nanotechnologies can offer, concerns regarding potential adverse health effects of such occupational exposure to NP is raised. However, very few data are currently available, probably because of the relatively recent development of nanotechnologies and production of manufactured NP. On the other hand, NP unintentionally emitted during an occupational activity such as welding is however not new, and we can, as such, take advantage of the literature available on this population of workers. Various adverse respiratory outcomes have been described in welders, among which inflammation and lung remodeling are largely

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reported [START_REF] Antonini | Comparative Microscopic Study of Human and Rat Lungs After Overexposure to Welding Fume[END_REF][START_REF] Antonini | Evaluation of the Pulmonary Toxicity of a Fume Generated from a Nickel-, Copper-Based Electrode to be Used as a Substitute in Stainless Steel Welding[END_REF]. We recently identified metal oxide NP (Fe, Mn, Cr oxides essentially) in lung tissue sections of welders [START_REF] Andujar | Role of metal oxide nanoparticles in histopathological changes observed in the lung of welders[END_REF]. These metal oxide NP were located inside macrophages present in the alveolar lumen and infiltrated in fibrous regions, which suggests a role for these NP in the lung alterations observed in welders.

To deepen our findings and extend them to occupational settings where NP are intentionally emitted, we designed an in vivo study aimed to investigate the pulmonary effects, in mice, of repeated exposure to NP administrated at occupationally-relevant doses. Our underlying hypothesis was that repeated exposure to NP at occupational levels could lead to lung remodeling similar to that observe in lung tissue from welders. Therefore, 4 metal oxide NP in relationship with the occupational exposure of welding, and that we previously identified in lung tissue sections from welders [START_REF] Andujar | Role of metal oxide nanoparticles in histopathological changes observed in the lung of welders[END_REF] were selected: Fe 2 O 3 , Fe 3 O 4 , MnFe 2 O 4 and CrOOH.

These NP were weekly administrated to mice up to 3 consecutive months, at 2 doses each one being relevant to occupational settings: 5 µg, chosen as occupationally-relevant to the welding activity, and 50 µg, chosen as occupationally-relevant to the context of a NP-manufacturing facility. Our results demonstrate that, while repeated exposures to 5 µg NP induced only limited pulmonary, administration of 50 µg NP induced the development of peribronchiolar, perivascular, and alveolar fibrosis, together with an interstitial inflammation in response to all but Fe 2 O 3 NP. Overall, these data provide new evidence for a potential risk for health of repeated exposure to NP at occupationally-relevant doses. were chemically synthetized and characterized as powder and as suspension after sonication as previously described [START_REF] Andujar | Role of metal oxide nanoparticles in histopathological changes observed in the lung of welders[END_REF]. Stock solutions of NP suspensions (2 mg/mL) were prepared in ultrapure water and stored at -80°C. Just before use, the suspensions were sonicated for 10 min in ultrasonic bath (Elmasonic S30H). NP suspensions were observed using a JEOL1400-TEM at 120 kV. The observation of at least 200 NP per suspension showed that all NP are spherical and present an average diameter of 20-25 nm (Fe 2 O 3 , Fe 3 O 4 and MnFe 2 O 4 ) or 15 nm (CrOOH) (Supplemental Figure S1). Dynamic light scattering experiments demonstrate that all NP formed aggregates in solution, their size largely depending on the chemical nature of the NP considered (Supplemental Figure S2).

Animal housing and handling

Animal housing

Male C57BL/6 mice 6-7 weeks of age were purchased from Janvier (Le Genest-St-Isle, France) and acclimated during 1 week. All mice were supplied with food (SAFE, Auguy, France) and tap sterilized water ad libitum in standard wire-topped cages in a controlled environment, with a 12 h light/dark cycle. All experiments were approved by our local Institutional Animal Care and Use Committee (Comité d'éthique -ComEth, ANSES/ENVA/UPEC, #C2EA-16).

Animal exposure to NP

To estimate the amount of NP to administrate in our study in order to be relevant for realistic occupational exposure, we utilized the following equation [START_REF] Antonini | Evaluation of the Pulmonary Toxicity of a Fume Generated from a Nickel-, Copper-Based Electrode to be Used as a Substitute in Stainless Steel Welding[END_REF]:

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Daily deposited dose of NP = occupational exposure limit (OEL) x NP mass fraction in occupational emission x minute ventilation x duration x NP deposition efficiency with 1/ OEL for welding fumes at 5 mg/m 3 , 2/ a mass concentration of NP in welding fumes at 10% of the total particle emitted 3/ a mean minute ventilation at 22.500 mL/min (tidal volume of 1500 mL and respiratory frequency of 15/min for a worker with moderate activity), 4/ a daily occupational exposure of 8 hours, and 5/ a mean deposition efficiency of NP in the alveolar region of 50% for an average NP aerodynamic diameter of 20 nm [START_REF] Andujar | Role of metal oxide nanoparticles in histopathological changes observed in the lung of welders[END_REF][19][START_REF]NIOSH: Appendix G: 1989 Air contaminants update project -exposure limits NOT in effect[END_REF][START_REF] Paek | Breathing patterns during varied activities[END_REF].

Importantly, these calculations do not account for particle clearance, but provide an estimate of the plausible worker exposure concentrations. Overall, the daily NP lung burden was estimated at 2.7 mg per worker per day. Given the average worker weight (70 kg) and the average weight of the mice (25 g), the daily deposited dose of NP for a mouse was estimated at 1 µg. Therefore, the dose of 5 µg weekly was considered as representative of occupational exposure of workers in the context of an unintentional process such as welding [START_REF] Dasch | Physical and chemical characterization of airborne particles from welding operations in automotive plants[END_REF][START_REF] Stephenson | Workplace exposure to submicron particle mass and number concentrations from manual arc welding of carbon steel[END_REF]. The dose of 50 µg weekly was subsequently calculated for a NP only-containing aerosol, within the 5mg/m 3 OEL.

Once a week, mice received Saline (Control group), 5 or 50 µg NP (Fe 2 O 3 , Fe 3 O 4 , MnFe 2 O 4 , CrOOH groups) administrated by pharyngeal aspiration performed under anesthesia (1.6 mg ketamine (Virbac, Carros, France) plus 300 mg xylazine (Bayer®, Puteaux, France)). Mice were sacrificed as previously described 24h, 1 week, 1 or 3 months after the first exposure [START_REF] Présumé | Design and Characterization of an Inhalation System of Iron and Manganese Oxide Nanoparticles for Rodent Exposure[END_REF]. A schematic representation of the experimental protocol is given in Supplemental Figure S3.

Lung tissue collection

At the time of the sacrifice, mice were anesthetized and their right lung was inflated, fixed with 10% of OCT (Optimal Cutting Temperature) at a constant pressure of 20 cm H 2 O and paraffin-embedded for further histological and immunohistochemical analysis. Before being

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frozen in liquid nitrogen and stored at -80°C, left lung was lavaged with twice with 1 mL of physiological saline, removed from the chest cavity, and immediately frozen at -80 °C for use in further experiments.

Histological and immunohistochemical analysis

Paraffin-embedded lung tissue sections (5 μm) were stained with hematoxylin-eosin-saffron (HES) for histological examination or with Sirius Red for collagen deposition. The occurrence, localization and severity of histological lesions were assessed using a semiquantitative score adapted from Ashcroft [START_REF] Ashcroft | Simple method of estimating severity of pulmonary fibrosis on a numerical scale[END_REF]. At least 10 fields per lung tissue section were analyzed (magnification x200), and a scale of 4 semi-quantitative fibrosis scores was utilized: [0: normal; 1: minimal fibrous thickening; 2: moderate thickening of walls without obvious damage to lung architecture; 3: increased fibrosis thickening of walls with definite damage to lung structure and formation of fibrous bands or small fibrous masses] for peribronchial, perivascular or alveolar wall thickness extension.

A similar scale of 4 semi-quantitative scores was used for inflammatory infiltrates in the alveolar, bronchiolar or vessel lumen, peribronchiolar or perivascular walls, as well as in the interstitium [0: < 20 inflammatory cells/field; 1: 21 to 50; 2: 51 to 100; 3: > 100]. Finally, the number of NP agglomerate/aggregate per field was also quantified (magnification x200).

Additionally, immunohistochemical (IHCh) analysis was performed using specific antibodies directed against Heme oxygenase (HO-1) protein as previously described [START_REF] Andujar | Role of metal oxide nanoparticles in histopathological changes observed in the lung of welders[END_REF].

BALF analysis

Cellularity

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Free alveolar cells were recovered from the lavage fluid by centrifugation at 400g for 15 min at 4 °C. The cellular pellet was suspended in 150 μL of physiological saline. An aliquot of the cell suspension was then examined using a hemocytometer to evaluate the total white cell number. For differential counts, the cell suspension was cytospun (cytospin-2, Shandon Products Ltd.), fixed in methanol, and stained with Diff Quick solution (Medion Diagnostics, Plaisir, France).

Alveolar epithelial barrier integrity and cytotoxicity evaluations

Total protein content and LDH concentrations in BALF were determined by the Bio-Rad protein assay (Bio-Rad, Marne-La-Coquette, France) and by the Cytotoxicity Detection Kit (Roche®), respectively.

Lung inflammation

In order to evaluate lung inflammation, BALF content in Transforming growth factor beta (TGFß), Tumor Necrosis Factor alpha (TNFα) and Interleukin 1beta (IL1β) proteins was quantified by DuoSet® ELISA as per the manufacturer's instructions (R&D Systems, Lille, France).

Quantification of mRNA expression in lung homogenates

mRNA were isolated from frozen lung tissue samples using the RNeasy Mini Kit (Qiagen, Courtaboeuf, France). mRNA expression of genes related to inflammatory (TNFα, Monocyte Chemoattractant Protein 1 -MCP-1, Interleukin 6 -IL-6, Prostaglandin-endoperoxidase 1S.

Statistical analysis

Eight animals per experimental group were used for each time-point and each dose, except for one week exposure at 50 µg where 10 mice per experimental group were used. Taking into account the possibility of non-normal distribution in the hypothetic mice population, and the number of mice (lower than 30 independent mice), non-parametric tests (Kruskal-Wallis statistical test followed by Dunn's multiple comparison test) were used (GraphPad Prism software, version 5.01, USA). For all statistical tests, p values smaller than 0.05 were considered as significant. Values are expressed as the mean or median and interquartile range for histological semi-quantitative scores. At the dose of 50 µg administrated weekly during 3 months, all NP induced a pronounced lung fibrotic remodeling characterized by the thickening of alveolar, peribronchiolar as well as perivascular walls, together with the presence of interstitial lung inflammation (Figure 2). These modifications were not present at shorter exposure durations (data not shown).

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RESULTS

Histological analysis of lung tissue sections

Whatever the experimental condition, neither granuloma formation nor the development of emphysema could be observed. Moreover, no bronchiolar hyperplasia could be detected.

The presence of microscopically observable NP aggregates/agglomerates was not detected in 5 µg-exposed animals, but was detectable in all animals exposed to 50 µg weekly, essentially in alveolar and bronchiolar lumens, with a significantly higher number of aggregates/agglomerates present in animals weekly exposed to 50 µg MnFe 2 O 4 or CrOOH NP as compared to those exposed to Fe 2 O 3 or Fe 3 O 4 NP (Figure 3).

Alveolar epithelial barrier integrity

We next addressed the integrity of the epithelial barrier by assessing the total protein content and LDH release in the BALF of NP-exposed mice. As shown in Figures 4 and5, there was a significant increase of BALF total protein content only in the MnFe 2 O 4 -exposed mice after 1

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month of weekly exposure to 5 µg NP (Figure 4, Panel A). No significant modification of BALF total protein content was observed for the other experimental conditions. Moreover, no significant modification of LDH release was observed, whatever the exposure duration, the dose or the chemical nature of the NP administered (Figures 4,5, Supplemental Figure S4).

Pulmonary inflammatory and oxidative responses

As the presence of NP in the lungs has been related to the induction of inflammatory and oxidative responses [START_REF] Boczkowski | Respiratory toxicities of nanomaterials -A focus on carbon nanotubes[END_REF][START_REF] Sarkar | Nanotoxicity: oxidative stress mediated toxicity of metal and metal oxide nanoparticles[END_REF], we next explored the total cellularity of BALF. As shown in Figures 4,5, and Supplemental Figure S4, BALF total cellularity was significantly increased only in CrOOH-exposed mice as compared to unexposed animals, whatever the dose received (5 or 50 µg weekly), after a 3 months-exposure. The cell differential was kept unchanged, whatever the experimental condition; only macrophages and rare neutrophils were detected in BALF samples (data not shown).

We next evaluated the release in the BALF of NP-exposed mice, of 3 inflammatory cytokines known to represent important markers of the pulmonary response to NP exposure: TNFα, TGFß and IL1ß [START_REF] Boczkowski | Respiratory toxicities of nanomaterials -A focus on carbon nanotubes[END_REF][START_REF] Sarkar | Nanotoxicity: oxidative stress mediated toxicity of metal and metal oxide nanoparticles[END_REF][START_REF] Park | Inflammatory responses may be induced by a single intratracheal instillation of iron nanoparticles in mice[END_REF]. For the dose of NP relevant to welding activity (5 µg), only CrOOHexposed animals present a transient but significant increase in TNFα secretion 24 hours after the first exposure as compared to unexposed mice (Supplemental Figure S4 exposure to 50 µg NP (Figure 5). IL1ß BALF content was not modified by NP exposure, whatever the chemical nature of the NP, the dose or the exposure duration studied.

We further evaluated inflammation at the level of the whole lung, by qPCR analysis. As shown in Figures 6,7 and Supplemental Figure S5, a transient increase in MCP-1 mRNA expression was observable after 1 day of exposure to 5 µg Fe-based NP (Fe 2 O 3 , Fe 3 O 4 and MnFe 2 O 4 ). This increase was also observed after 1 day and 1 month weekly repeated exposure to CrOOH NP. Moreover, TNFα mRNA expression was increased in response to all NP after 3-months weekly repeated exposure to the occupationally-relevant dose of 5 µg (Figure 7). This increase in TNFα mRNA expression was also detectable in the 50 µgexposed groups, either transiently (Fe 2 O 3 and MnFe 2 O 4 ) or steadily (Fe 3 O 4 , starting after 1 month, and CrOOH, at all exposure durations). Moreover, in Fe 3 O 4 -and CrOOH-exposed mice, MCP-1 and PTGS mRNA expressions were increased as compared to that measured in unexposed mice after 1 and 3 months. The same was true for IL-6 mRNA expression after 1 month (Figure 6, Panel B).

As for the oxidative response, no major modification of NQO1 or CYP1A1 expression could be detected in the 5 µg-exposed animals, except for a significant increase in CYP1A1 expression after 1 day of exposure to Fe 3 O 4 NP (Supplemental Figure S5). However, in 50 µg-exposed animals, the antioxidant NQO1 mRNA expression was significantly increased in Fe 3 O 4 -, MnFe 2 O 4 -and CrOOH-exposed mice after 1 month repeated exposure as compared to unexposed animals, and was still significantly increased after 3 months exposure duration in CrOOH-exposed mice (Figures 6 and7). Neither modification of CYP1A1 mRNA expression nor that of HO-1 protein was detected, whatever the experimental condition (Figures 6,7,8 and Supplemental Figure S5).

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DISCUSSION

The general aim of our study was to investigate the pulmonary effects, in mice, of repeated exposure to NP administrated at occupationally-relevant doses. To achieve this aim, we developed an experimental protocol designed to mimic at best an occupational exposure in 2 working contexts: welding (unintentional NP production) and NP-manufacturing facility (intentional NP production).

Our results demonstrate that repeated exposure of mice to metal oxide NP at doses relevant to welding activity induce no (after Fe 2 O 3 and Fe 3 O 4 NP exposure) or only limited (after MnFe 2 O 4 and CrOOH NP exposure) lung remodeling; in these latter animals, only a slight although significant peribronchiolar fibrosis could be observed after 3 months, without any major sign of inflammation. To the best of our knowledge, these results are the first to address the specific issue of the pulmonary effects of repeated exposures to metal oxide NP administrated at doses relevant to the occupational activity of welding. Indeed, although the relevance of the exposure dose is a long-time ongoing issue in the literature dedicated to NP exposure [START_REF] Oberdörster | Lung dosimetry -Considerations for noninhalation studies[END_REF], the majority of the studies published so far have been conducted using doses that are largely over the calculated doses relevant to the welding activity: at least 1 mg/kg (corresponding to 25 µg per animal for a 25 µg mouse), and up to 500 µg per animal.

Moreover, these doses were most often administrated as a single bolus, which dampens the overall relevance of the results obtained in these studies [28,[START_REF] Zhu | Comparative study of pulmonary responses to nano-and submicron-sized ferric oxide in rats[END_REF]. Our results are therefore difficult to compare with data from the literature. However, considering the results obtained with the lowest doses used in previously published studies, the effects of weekly pulmonary exposures to Fe or Cr oxide NP can be described as limited; absence of acute inflammatory cell recruitment in the BALF in most cases, low levels of inflammatory cytokines secretion, and no histological modification after 1 month (longest time point analyzed in these studies) [26, 30-32, 33 6264]. Our results are in accordance with these data, and further extend them,

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not only to doses relevant to the occupational activity of welding, but also to longer exposure durations (up to 3 months) and to a more realistic scenario of repeated exposures instead of administration of NP as a single bolus. Interestingly, lung TNFα mRNA expression was increased in response to all NP after 3 months weekly exposure to 5 µg. Given the implication of this cytokine in the early events of inflammation, one could infer that longer exposure duration to doses of NP relevant to welding activity could lead to more pronounced lung alterations. We are fully aware that welding fumes cannot be summarized to only metal oxide NP, as a complex mixture of particles (micrometric and nanometric) together with a number of potentially toxic gases (e.g. ozone, nitrogen oxide, nitrogen dioxide) are generated during the welding process [START_REF] Antonini | Health effects of welding[END_REF]. However, the relevance of our findings is supported by the fact that we were recently able to link the presence of NP aggregates/agglomerates in lung tissues samples of arc-welders to the presence of similar fibrotic lesions [START_REF] Andujar | Role of metal oxide nanoparticles in histopathological changes observed in the lung of welders[END_REF]. Various adverse outcomes have been described in welders, and are not limited to pulmonary endpoints [START_REF] Antonini | Health effects of welding[END_REF].

Therefore, although we found only limited pulmonary effects of repeated exposure to occupationally-relevant doses of metal oxide NP, extra-pulmonary adverse effects could have been detected. This could be particularly true for the brain for example, as exposure to Mn is associated with neurotoxicity in welders [START_REF] Antonini | Health effects of welding[END_REF]. Moreover, an increase in the incidence of lung cancer has been described particularly in those exposed to Cr [START_REF] Antonini | Health effects of welding[END_REF]. However, as the maximum exposure duration was 3 months in our study, this was probably not enough to evaluate the carcinogenicity of the exposure, even for a mouse presenting a 2.5 years life expectancy. As Mn-and Cr-based NP were the most potent to induce lung remodeling when utilized at doses relevant to welding, their extra-pulmonary effects could have been particularly interesting to evaluate. These outcomes should deserve further studies.

The mild pulmonary alterations detected at doses relevant to the occupational activity of welding were further extended both in terms of intensity and spatial distribution after repeated

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exposure of mice to doses relevant to the context of a NP-manufacturing facility; not only a more pronounced thickening of the peribronchial wall could be observed, but also that of perivascular and alveolar walls, together with the presence of an interstitial inflammation. These findings were observed in response to all but Fe 2 O 3 NP, given at the same mass concentration. In an attempt to decipher the underlying molecular mechanisms of these fibrotic lesions, the expression pattern of MCP-1 mRNA in lung homogenates could be of interest, as MCP-1 expression was increased after 1 and/or 3 months of repeated exposure to 50 µg of all but Fe 2 O 3 NP. Indeed, MCP-1 is a chemokine synthetized by a large variety of cells including macrophages and fibroblasts, and which exerts chemotactic and activating effects on macrophages [START_REF] Liu | Role of human pulmonary fibroblast-derived MCP-1 in cell activation and migration in experimental silicosis[END_REF]. Moreover, it has been recently described that pulmonary fibroblast-derived MCP-1 drives fibroblast proliferation and migration in response to silicon dioxide [START_REF] Liu | Role of human pulmonary fibroblast-derived MCP-1 in cell activation and migration in experimental silicosis[END_REF]. It is also important to note that the secretion of TGFß was increased only in the BALF obtained from MnFe 2 O 4 -exposed animals during 3 months. Although we did not evaluate longer exposure durations, and given the essential role of this cytokine in the development of lung fibrosis, this result suggests that MnFe 2 O 4 -exposed animals could be particularly prone to lung remodeling, thanks to the implication of molecular mechanisms specific to this speciation of NP. This is also suggested by the fact that MnFe 2 O 4 -exposed animals develop peribronchiolar wall thickening after 3 months weekly exposure at doses relevant for welding activity. However, such remodeling was also present in animals repeatedly exposed to 5 µg CrOOH NP, which indicates the occurrence of other, yet unidentified, molecular mechanism(s) that could be responsible for lung remodeling in response to these NP.

Our data showed that Fe 2 O 3 NP were overall less reactive than Fe 3 O 4 NP. This difference could be due to different degrees of solubility between NP. However, previous data from our lab using the same metal NP as those used in the present study indicate a very small if no J U S T A C

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solubility at all whatever the NP tested [START_REF] Andujar | Role of metal oxide nanoparticles in histopathological changes observed in the lung of welders[END_REF]. Another explanation for this difference could be the different accumulation profiles between Fe 2 O 3 and Fe 3 O 4 NP. However, the number and localization of NP aggregates/agglomerates were similar for these NP, thus underlying the importance of chemical speciation in NP's effects. Interestingly, previous data from our team demonstrated that Fe 2 O 3 NP were more reactive than Fe 3 O 4 NP in terms of pro-inflammatory cytokines secretion by macrophages [START_REF] Andujar | Role of metal oxide nanoparticles in histopathological changes observed in the lung of welders[END_REF]. This suggests that macrophages might not be the essential or only target cell type to drive lung remodeling in response to iron oxide NP.

Interestingly, although aggregates/agglomerates of metal oxide NP could be detected in all groups of animals in the present study (at the 50 µg dose), yet all but Fe 2 O 3 NP were able to induce fibrotic lesions. However, the number of aggregates/agglomerates observed 3 months after repeated exposure to Fe 3 O 4 NP, which was lower than after exposure to MnFe 2 O 4 or CrOOH NP, didn't affect the intensity of the remodeling, which was similar for the 3 NP. It must be noted that, as our study was dedicated to investigate the pulmonary effects of repeated exposure to NP administrated at occupationally-relevant doses, the critical endpoint we focused on was therefore the initial mass concentration of the NP, not their size or agglomeration state immediately after sonication, i.e., just after administration to mice.

Finally, although we cannot rule out the technical difficulty of observing and characterizing such aggregates/agglomerates with a regular optical microscope, this result confirms the multifactorial origins potentially linking the presence of NP to any biological effect [START_REF] Lanone | Determinants of carbon nanotube toxicity[END_REF].

Our results overall demonstrate that repeated exposure to NP at doses relevant to occupational exposure represents a potential risk for respiratory health. There is currently no international consensus regarding which OEL can be applied to NP, although they could represent a major component of the occupational pollution [START_REF] Pietroiusti | Engineered nanoparticles at the workplace: current knowledge about workers' risk[END_REF][START_REF] Shaughnessy | Occupational health risk to nanoparticulate exposure[END_REF]; the proposed OEL for NP is 10% of the exposure limits established for the respective micro-scale industrial aerosols [START_REF] Katsnelson | Some inferences from in vivo experiments with metal and metal oxide nanoparticles: the pulmonary phagocytosis response, subchronic systemic toxicity and genotoxicity, regulatory proposals, searching for bioprotectors (a self-overview)[END_REF]. Indeed, with the exception of nanometric TiO 2 for which a recommended exposure limit (REL) at 0.3

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mg/m 3 has been proposed by the US, OEL have been defined for respirable particles only i.e. most of them in the micrometric range [START_REF] Pietroiusti | Engineered nanoparticles at the workplace: current knowledge about workers' risk[END_REF]40,41]. Some NP-specific values have been proposed, but they differ according to the different proposing countries or agencies, in terms of the metric used to quantify the relative exposure to NP or the categorization of NP in various risk groups, background upon which these OEL have been developed [START_REF] Pietroiusti | Engineered nanoparticles at the workplace: current knowledge about workers' risk[END_REF], which makes these proposed values highly provisional. Moreover, the current OELs don't consider the speciation of NP, although it represents an important determinant of their biological effects.

CONCLUSION

Overall, our data demonstrate for the first time a potential risk for respiratory health posed by repeated exposure to NP at occupationally-relevant doses. Given these results, the development of OEL specifically dedicated to NP exposure might therefore be an important issue to address in the near future.

  Four NP representative of welding occupational exposure (Fe 2 O 3 , Fe 3 O 4 , MnFe 2 O 4 , CrOOH)

synthase 2 -

 2 PTGS, Regulated upon activation normal T-cell express, and presumably secreted -RANTES) and oxidative responses (NAD(P)H/quinone oxidoreductase -NQO-1, Cytochrome P450-family 1 subfamily A-polypeptide 1 -CYP1A1) was measured by relative quantification method using GAPDH (glyceraldehyde-3-phosphate dehydrogenase) as the E D housekeeping gene and performed with a PCR ABI 7700 apparatus (Applied Biosystems, Courtaboeuf, France) with SybrGreen technologies (Applied Biosystems). Primer sets are shown in Table

Figure 1

 1 Figure 1 shows representative HES (Panel A) and Sirius Red (Panel B) images of lung tissue

  , Panel C). For the highest dose of NP (50 µg weekly), all but Fe 2 O 3 NP induced significant increases in BALF TNFα content after 1 day (Supplemental Figure S4, Panel D). This upregulated secretion could still be observed after 1 month (for Fe 3 O 4 -and CrOOH-exposed animals) or 3 months (for MnFe 2 O 4 -and CrOOH-exposed mice, Figures 4 and 5) repeated exposure. Interestingly, TGFß secretion was significantly increased in MnFe 2 O 4 -exposed mice, after 3 month repeated

Figure 1 :

 1 Figure 1: Histological analysis of lung tissue sections after repeated exposure to 5 µg NP.

Figure 2 :

 2 Figure 2: Histological analysis of lung tissue sections after repeated exposure to 50 µg

Figure 3 :

 3 Figure 3: Analysis of agglomerates/aggregates accumulation in lung tissue sections.

Figure 4 :

 4 Figure 4: BALF analysis of mice exposed to NP for 1 month. BALF total protein content,

Figure 5 :

 5 Figure 5: BALF analysis of mice exposed to NP for 3 months. BALF total protein content,

Figure 6 :

 6 Figure 6: Total lung mRNA analysis of mice exposed to NP for 1 month. Quantification of

Figure 7 :

 7 Figure 7: Total lung mRNA analysis of mice exposed to NP for 3 months. Quantification

Figure 8 :

 8 Figure 8: Heme-Oxygenase-1 (HO-1) expression in lung tissue sections. Representative

ACKNOWLEDGMENTS

This work was supported by funds from INSERM, Agence Nationale de la Recherche (grant ANR-09-CESA-017), Université Paris Est-Créteil, Chancellerie des Universités de Paris (Legs POIX), Fonds de Dotation-Recherche en Santé Respiratoire (SPLF), and C'Nano (grant AAP09-NanoSoud). Mirlande Présumé was a fellow from Agence nationale de sécurité sanitaire de l'alimentation, de l'environnement et du travail (ANSES) and Agence de l'Environnement et de la Maîtrise de l'Énergie (ADEME). Angélique Simon-Deckers and Grégory Beaune were both fellows from ANR. Sophie Lanone and Jorge Boczkowski were both recipients of a Contrat de Recherche Translationnelle, between Inserm and CHI Créteil (SL), or CHU Mondor (JB). This work also received the support of Labex SERENADE 11-LABX-0064 and DHU A-TVB (Département Hospitalo-Universitaire Ageing-Thorax-Vessel-Blood).

DECLARATION OF INTEREST

The authors declare that they have no competing interests.

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