H Popescu

J Perron

B Pilette

R Vacheresse

V Pinty

M Sacchi

R Delaunay

F Fortuna

K Medjubi

R Gaudemer

J Luning

N Jaouen

COMET a new end-station at SOLEIL for coherent magnetic scattering in transmission

We have developed and built a new instrument, named COMET for COherent Magnetic scattering Experiments in Transmission, using polarized soft X-rays. This high-vacuumset-up is placed at the intermediate focal point of the elastic branch of the SEXTANTS beamline at Soleil. The main application is in solid state physics, the instrument being optimized for studying material propertie using coherent scattering of soft X-rays with an emphasis on imaging, with chemical selectivity, the magnetic domains of artificially nano-structured materials. The instrument principal features are presented and illustrated through recently performed experiments.

Introduction:

The magnetic materials have a very important role in key technologies like information handling (storage, logic and sensor devices), high power gigahertz nano-oscillators or high performance magnets for motors. Nano-magnetism is therefore a very promising research topic. In order to understand the fundamental magnetic properties of the matter, powerful characterization techniques and tools are needed. Macroscopic information, like the average magnetization over a large volume, is often not sufficient to understand the local microscopic details. For this, one needs high resolution imaging of the magnetic nano structures with the possibility to record the evolution of the system at different timescales ranging from ns down to fs. Thus many magnetic imaging techniques have been developed, like scanning probe (MFM), electron probe (TEM) or photon probe. In the photon probe category we can distinguish techniques based on the optical microscopy (Kerr and Faraday) and on the use of polarized X-rays. The new instrument presented in this paper is part of this last category. x-rays or X-ray, always the same. Soft x-rays cover the binding energies of the 1s electrons in carbon, oxygen and nitrogen and the 2p electrons of transition metals like Fe, Co and Ni and 3d electrons in rare earth metals. The wavelength ranges between ~0.5 and ~10 nm, making soft x-rays a proper tool for imaging nanoscale structures. The polarization properties of soft x-rays play a crucial role for the study of the magnetic materials. The specific interaction of polarized x-rays with magnetic matter gives rise to various dichroism effects. X-rays magnetic circular dichroism (XMCD) is the difference in absorption of left and right circularly polarized light when the photon energy is tuned to an absorption edge of a given element. This effect is proportional to the projection of the magnetic moment of the element onto the photon propagation direction. XMCD is adapted to the study of ferromagnetic and ferrimagnetic materials. The x-rays magnetic linear dichroism (XMLD) is the difference in absorption of linearly polarized photons between the polarization parallel and perpendicular to the magnetization axis. XMLD, related to the spatial anisotropy of the squared magnetization, can be observed in both ferromagnetic and anti-ferromagnetic materials, but such measurements are more often applied to the study of the latter.

In the following we will give a brief overview of different techniques and existing instruments dedicated to magnetic imaging with polarized soft x-rays, which we classify in two main categories: using xray optics and lens-less imaging.

For the soft x-ray regime the refractive index is close to unity, thus imaging with conventional lenses is impossible. This limitation was overcome in the 80s with the development of the nanolithography where a new type of lens was developed: the Fresnel zone plate (FZP). This is a diffractive optics, typically circular gratings, where the line density is increasing from the center in the radial direction, while keeping constant the wavelength difference between neighboring lines. The performance of a FZP, i.e. the spatial resolution that can be obtained and the focal point, is determined by the number of zones and the width of the outermost zone [D. Attwood, Soft X-Rays and extreme Ultraviolet Radiation. Cambridge, U.K.: Cambridge Univ. Press, 1999]. Two different imaging techniques can be done using FZP optics: full-field transmission soft x-ray microscope (TXM) and scanning transmission soft x-ray microscope (STXM). In a TXM full-field microscope the FZP forms a complete image of the sample at the 2D detector, typically a CCD camera. One example is the XM-1 x-ray microscope at the Advanced Light Source (ALS) in Berkeley. The spatial resolution that can be attained is around 25 nm for around 15 microns of field of view. Obtaining a complete real space image can take few seconds only.

Differently, a STXM uses the FZP to focus monochromatic radiation to a small spot on the sample. An image is obtained by scanning the sample across the focus spot of the FZP. The transmitted photons can be recorded with fast point detectors. One example is the MAXYMUS(M?) microscope in BESSY II (Berlin) [http://www.helmholtz-berlin.de], reporting resolutions below 20 nm.

Both TXM and STXM are routinely used for imaging the magnetic structures in transmission, where the detected signal contains thickness averaged information. The STXM microscope makes it possible to form an image using detection modes other than transmission, for instance photoelectron and fluorescence emission, widening the range of application to non-transparent samples.

A complementary approach to provide only surface sensitivity is the X-PEEM [A. Scholl, "Applications of photoemission electron microscopy (PEEM) in magnetism research", Current Opinion Solid State Mater. Sci., vol. 7, no. 1, pp. 59-66, 2003.], which is a hybrid between an x-ray source and a high resolution electron microscope. The emission of secondary electrons from the sample is proportional to the x-ray absorption. Only the electrons from the first few nm can escape the sample, making XPEEM a surface sensitive magnetic imaging technique. As an example, a leading instrument is PEEM3 at ALS with a spatial resolution down to 20 nm [https://als.lbl.gov/beamlines/11-0-1].

The lensless imaging approach is using the coherent scattering of soft x-rays in resonant conditions and requires a high degree of longitudinal and transverse coherence. These imaging techniques can be classified in two main parts: x-ray holography techniques, based on the interference between the main beam and a reference beam, and imaging techniques based on retrieval algorithms (ex. ptychography). For the holography technique a simple Fourier inversion of the diffraction diagram is reconstructing the real space image. The new COMET instrument, described in this paper, is based on these lensless techniques for imaging magnetic structures.

The SEXTANTS beamline [START_REF] Sacchi | The SEXTANTS beamline at SOLEIL: a new facility for elastic, inelastic and coherent scattering of soft X-rays[END_REF] at Soleil is dedicated to the use of resonant scattering of polarized soft xrays, implementing several techniques as Resonant inelastic X-ray scattering, diffraction, reflectivity and coherent scattering, with four specialized experimental end-stations open to users: AERHA [START_REF] Chiuzbăian | Design and performance of AERHA, a high acceptance high resolution soft x-ray spectrometer[END_REF], RESOXS [START_REF] Jaouen | An apparatus for temperature-dependent soft X-ray resonant magnetic scattering[END_REF], IRMA [START_REF] Sacchi | IRMA-2 at SOLEIL: a set-up for magnetic and coherent scattering of polarized soft x-rays[END_REF] and the new one here presented, COMET. The photon energy range covered by the beamline is from 50 eV to 1700 eV, with full control of polarization (linear horizontal, vertical or custom, and circular left or right). The flux at the sample position for the different stations exceeds 10 12 photons/sec over the optimized 100-1000 eV energy range.

The COMET instrument is placed at the intermediate focal point of the elastic branch of the SEXTANTS beamline (figure 1). COMET has been designed to have an easy beam switch between this instrument and the KB end-station placed downstream on the same elastic branch of the beamline. Two differential pumping blocks, placed upstream and downstream from the COMET instrument, ensure fast beam availability, downstream and upstream, after venting. Typically, after one hour of pumping, the pressure in the main chamber enters the 10-6 mbar range, which is enough in order to open towards the rest of the beamline.

Figure 1: Top view schematics of the SEXTANTS beamline, with the four experimental end-stations: AERHA on the inelastic branch, and COMET (intermediate focal point), RESOXS and IRMA2 (KB focal point) end-stations on the elastic branch. In the standard Soleil coordinate system, the S direction in along the beam and the X direction perpendicular to the beam, in the horizontal plane. Z (not shown here) is the vertical direction.

Instrument design

Vacuum chambers

This station has been designed to have a very high mechanical stability(?). Two main vessels compose this instrument: the sample environment and the 2D detector ones, placed on the same marble table. The sample chamber (cubic) has removable flanges with double viton gaskets (figure 2), primary pumped in between. The periphery flanges are standard CF40 and CF63, for regular electric and fluid feed-throughs and for view-ports. The lateral rectangular flange is a two point quick access door, for easy sample exchange and set-up pre-alignment while vented. The modular removable walls permit later upgrades of the chamber. The typical pressure available in user operation is between 10 -6 to 10 -7 mbars. Part of this instrument was developed in collaboration between SOLEIL and the LCPMR team [Sorbonne University, Paris, France].

The 2D detector chamber (cylindrical) has only CF flanges and is isolated from the main chamber by a CF150 gate valve. It remains under vacuum for very long periods allowing a base pressure of 10 -7 mbar despite the presence of the CCD detector and cabling. These chambers have turbo pumps equipped with rubber covered bellows for vibration damping.

Beam conditioning

The spatial (transverse) coherence at the sample is directly related to the angular acceptance of the beamline. The coherence of the SEXTANTS beamline was measured [START_REF] Sacchi | IRMA-2 at SOLEIL: a set-up for magnetic and coherent scattering of polarized soft x-rays[END_REF] using Non Redundant Apertures (NRA). While the vertical coherence distance is naturally ensured by the vertical small size of the beam even in full acceptance mode, for the horizontal coherence the beamline front-end slits have to be reduced. At 700 eV the transverse coherence distance obtained is better than 25 x 25 µm 2 , with a remaining current of monochromatic and coherent beam of around 1 µA measured using an AXUV-100 diode (from Opto Diode Corp, USA), corresponding to 10 10 coherent and monochromatic (R≈10 4 )? 700 eV photons per second arriving at the sample of the COMET instrument.

A piezoelectric shutter is placed just behind the exit slits of the monochromator, where the beam has a vertical size that depends on the slit opening, typically between 30 and 50 microns. The shutter is composed by a 4 cm long piezo blade, driven by high voltage, which can bend vertically by several hundred microns, ensuring a complete blanking of the beam. The open/close states are trigged directly by the CCD controller, with a minimum response time around 10 ms. During acquisitions, this device blocks the x-ray beam while reading out the detector.

Sample positioner

The optical table inside the sample chamber is directly mounted on a synthetic marble, through stable columns. The flexible bellows around these columns permit vibration damping from the pumping system.

Several under vacuum motorized blocks (figure 3) compose the sample environment, in the beam arrival order:

1) Guard-hole: An XZ piezo stage [SmarAct, Germany], holding different pinholes for cleaning the incoming beam and choosing the precise illuminated region on the sample (typically few tens of microns). The travel in each direction is 21 mm, with encoded positioning to 500 nm resolution. The high quality pinholes are fabricated by focused ion beam (FIB) using the facility operated by the CSNSM team.

2) Mask: An SXZ piezo stage (SmarAct) with the same parameters as the ones previously described. These masks are used for FTH (Fourier Transform Holography) imaging technique [START_REF] Eisebitt | Lensless imaging of magnetic nanostructures by x-ray spectro-holography[END_REF], in the separated mask and sample approach [START_REF] Tieg | Overcoming the field-of-view restrictions in soft x-ray holographic imaging[END_REF][START_REF] Spezzani | X-ray holographic imaging of magnetic order in patterned Co/Pd multilayers[END_REF][START_REF] Popescu | Four-state magnetic configuration in a tri-layer asymmetric ring[END_REF], or for ptychography experiments [START_REF] Fienup | Phase retrieval algorithms: a comparison[END_REF]. Both techniques require a very small distance between the mask and the sample, ranging from contact to 1 mm, depending on the experiment. In the case of contact mode, for the separated mask and sample approach in holography, the parallelism between the mask and the sample is done by autocollimation using the synchrotron white beam. The mask and the sample are parallel to each other when the visible light reflected by either one is sent back through the same path of the incoming light. For this aligning procedure the mask holder is equipped with two supplementary manual rotations (shown in figure 3 under the mask piezo block).

3) Sample a): a high load stepper motor SXZ bloc [PI-MiCos, Germany], with 35 mm travel in each direction and 150 nm encoded resolution is holding the sample. The high load makes it possible to install a variety of sample environments, like electromagnetic coils, cooling stripes, RF feed-throughs, etc. Sample b): An additional piezo SXZ block (SmarAct), with 31 mm travel and 50 nm encoded resolution can be added for high resolution scans, dedicated to the ptychography experiments. Two miniaturized piezo rotation encoded stages (SmarAct) are available for custom experiments (ex. Tomography) to be developed on User's demand. 4) diode: the 1x1 cm 2 Al capped AXUV-100 diode is placed 15 cm behind the sample for the fine alignment of all the previous elements. This retractable diode is mounted on a horizontal 100 mm travel stage (SmarAct), together with a protection screen for the CCD detector.

5) The mask and sample systems are placed on an intermediate table which can rotate around the Z axis (typically +/-45°). The table rotation is ensured by a heavy load motorized rotation stage. Figure 3 shows this intermediate table in two positions: normal incidence (left) and 45° incidence (right). This 45° incidence is useful for getting the projection, for example, of the in-plane magnetic components [START_REF] Tieg | Imaging the in-plane magnetization in a Co microstructure by Fourier transform holography[END_REF]. In this case we can use for holography either a reference slit [START_REF] Duckworth | Magnetic imaging by x-ray holography using extended references[END_REF] or specific angle drilled reference holes [START_REF] Popescu | Four-state magnetic configuration in a tri-layer asymmetric ring[END_REF] for the masks.

Sample environment

We developed an environment capable of delivering static magnetic fields of up to 1 Tesla in all the directions in the horizontal plane. The system is based on four rotatable, transversely-magnetized permanent magnet rods [O. Cuget, P. Hansson and J. M. D. Coey, IEEE Transcactions on Magnetics, Vol. 30, No. 6 (1994)].

The four cylindrical permanent magnets (NdFeB rods with 26 mm diameter) have independent under vacuum motorized and encoded rotations (figure 4 a). The diagonal distance between the magnets and the sample (located at the central axis of the four magnetic rods) can be varied by using a mechanical Archimedean spiral system (green part on the 3D representation in figure 4 a) driven by a single stepper motor in order to simultaneously change and keep symmetrically the same gap on the four magnets. All the motors have gear mechanisms with great reduction ratio in order to maintain the considerable torque forces. The combination of the gap and the individual angles permits the tuning of the resulting static field at the sample position to a precise direction and magnitude in the horizontal plane.

Figure 4. Four permanent magnet system delivering at the sample position (i.e. on the central axis) a total static field of up to 1 Tesla. a): cut over the 3D engineer design. b): real setup, here shown in the commissioning phase. At present this device is installed and available at the COMET end-station (the upper part shown in figure 3). c): magnetic field measured in the s direction (along the beam) for one single magnet. d): magnetic field measured in the x direction (perpendicular to the beam) for one single magnet. e): magnetic field measured in the vertical direction for one single magnet. f): resultant field for the 4 magnets together, matched in order to maximize the field in the s direction while cancelling it in the perpendicular direction.

For the commissioning phase we used a 3D Hall sensor, placed at the sample position, in order to calibrate the magnetic field as a function of the angles and gap for each magnet. We recorded the Bs, Bx and Bz magnetic field intensities as angle/gap maps for each magnet, separately. Figure 4 (c, d ande) shows the measured magnetic field created by a single magnet in the s, x and z directions, respectively.

The resultant field created by the four magnets together is then the vector addition of the individual components. Figure 4f shows the total field resulting from the combination of the four magnets, here with the angles matched in order to maximize the field in the s direction (along the beam), while cancelling the resulting field in the x, perpendicular to the beam, direction. In a similar way, other preferential directions for the resultant field can be obtained in the horizontal plan.

For this commissioning phase the minimum gap measured was limited to 10 mm because of the size of the Hall sensor holder. In normal conditions, without the Hall sensor, the gap can go to 7 mm, corresponding to a maximum field of 1T (the four magnets phased together) at the sample level. In the results chapter we present the example of a real experiment showing the evolution of magnetic domains versus the applied external static field using a test Co/Pd multilayer sample.

The homogeneity of the resultant field is better than 3% within a radius of 2 mm around the center of the four magnets, in the closest gap configuration (the highest gradients). The beam was aligned in the center of the four magnets with a precision better than 0.5 mm by using a mechanical adapter mounted on the magnets and specially designed for this alignment procedure. The standard beam size on the sample is few tens of microns and the imaged fields of view are typically few microns, so we can consider that the field strength is homogenous and well known along the whole imaged area.

The material of the permanent magnets is NdFeB with a remanent field Br = 1.37 Tesla. The field strength changes with the temperature within 0.1% per degree. Since the permanent magnets are not in direct mechanical contact with the sample, they remain close to room temperature and the low/high temperature of the sample is compatible with the high magnetic static field, allowing combined sample environments. This magnetic environment is also compatible with the separate mask and sample approach and with the normal or tilted transmission geometry, as shown in figure 3.

The temperature environment can be controlled via a commercial He cryostat/heater [Janis, USA] ending with a local flexible stripe, the actual temperature on the sample being from 20K to 800K (magnet heating by radiation. The temperature regulation is done via a Lakeshore 350 controller with stability better than 1 mdeg. The temperature environment might not be compatible with the extendable field of view techniques, as the thermic drifts can affect the alignment between the mask and the sample.

RF pumping is also possible via an external RF generator and SMA feedthrough (as shown in results paragraph [START_REF] Bukin | Time-resolved imaging of magnetic vortex dynamics using holography with extended reference autocorrelation by linear differential operator[END_REF]).

Detection

The main detector is a 16 bit PI-MTE CCD camera, with 2048 x 2048 pixels. The chip and pixel size are 27.? mm and 13.5 µm respectively. The real dynamic range of the detector goes from 100 counts (background level) to around 20000 counts, this being the maximum value before producing saturation effects.

A movable frame is integrated to the CCD head using a motorized XZ piezo system. The frame travel is 31 mm in both transversal directions, with position encoded to 50 nm resolution (encoded ???). This frame can hold two different spherical beam-stops with typical sizes of few hundred microns, placed at 2 cm in front of the chip. An Al filter (100 nm thick) protects the CCD against the visible light coming mainly from the encoders.

The CCD is mounted on a stable optical table using three columns coupled to the marble (figure 5). The under vacuum CCD carriage is motorized and can slide the CCD along the beam propagation direction (s direction). The travel of the carriage covers a sample-to-CCD distance from = 15 to = 45 cm (z is used elsewhere, no?, and is chosen in order to have the best tradeoff between the pixel size and the field of view of the reconstructed image, both depending on the CCD pixel size and on the number of pixels.

The reconstructed pixel size is directly proportional to the wavelength and to the maximum angular momentum collected by the CCD: λd/2048x13.5µm, where d is the sample to CCD distance. As an example, for the Co edge (778 eV), the theoretical reconstructed pixel size is 9 nm when the CCD is at a distance of 15 cm from the sample, and 26 nm when the CCD is placed at a distance of 45 cm. In reality the size of the reconstructed pixel is defined by the actual spreading of the diffraction diagram within the CCD chip size.

The CCD can also move in x direction (perpendicular to the beam propagation direction), but only in retracted position, with 5 cm travel, in order to let the beam pass through and reach the downstream KB experimental station.

Figure 5: Internal mechanics holding the retractable CCD. The XZ beam-stop and a 100 nm Al filter are integrated to the CCD head.

Control Command:

All the motors and detectors are integrated in the SOLEIL standard Tango control system and fully interfaced with the SEXTANTS beamline acquisition software. Automatic scans are possible using the Python environment, depending on the complexity of the experiment.

Multi-dimensional scans, using the CCD as detector, can be used in an automatic mode for ptychography or tomography measurements, where diffraction diagrams are recorded for different (x, z) sample positions, scanned with custom trajectories (spiral, mesh, etc.). Fast 2D maps (figure 6) can also be done using a scalar detector (the rear diode) coupled to a high sensitivity Keithley 6517 electrometer in order to localize and identify the sample patterns or mask coordinates.

Figure 6: sample fast mapping in order to localize the nano-patterns of interest. a): SEM image of CoPd nano-patterns on Si3N4 membrane, taken while FIB milling. The white scale-bar is 20 µm. b): 2D fast mapping of the same sample using the piezo motors and the transmission diode into the Comet chamber. c) and d): zoom on the identified nano-objects.

Photon energy scans can be performed in transmission mode using either the diode (for the integrated signal through the sample) or the 2D detector, in order to get the XAS or the energy-dependent evolution of speckles.

Different communication ports (serial, GPIB, analog voltage in/out, power, etc.) are available and interfaced with the Tango control, for driving and triggering the eventual user custom material, like RF generators, coils, power supplies, etc.

First experimental results:

This instrument was commissioned and has already served for a few user experiments. Here the performance of the instrument will be illustrated by a brief description of early results.

Time resolved holographic imaging in integrated mask and sample approach:

Time resolved experiments were done at Sextants beam-line using the COMET instrument. User custom samples with integrated RF antenna and holography mask permitted to excite a magnetic vortex and image the core displacement as a function of time [START_REF] Bukin | Time-resolved imaging of magnetic vortex dynamics using holography with extended reference autocorrelation by linear differential operator[END_REF]. The diameter of the holography object aperture was 3.7 microns, and the Co square pattern inside was 2 microns. The sample to CCD distance was 27 cm. The delay between the x-ray pulse and the local magnetic excitation was controlled via a RF generator triggered by the synchrotron clock. These experiments were done either in 45° transmission geometry in order to get the in plane magnetic domains, or at 0° to see the out of plane magnetic components of the domain walls. The holography mask had a slit for encoding the reference beam, and the reconstruction was done using the Heraldo technique [START_REF] Duckworth | Magnetic imaging by x-ray holography using extended references[END_REF]. In figure 7 up-left we display a typical diffraction diagram where we can observe the Airy rings from the object hole and the vertical diffraction of the slit. The observed core oscillations have a periodicity of 3.5 ns [START_REF] Bukin | Time-resolved imaging of magnetic vortex dynamics using holography with extended reference autocorrelation by linear differential operator[END_REF].

Figure 7. a): diffraction diagram from a sample with integrated slit reference. Axis coordinates are in pixels and the intensity scale-bar in counts. The maximum scattering vector corresponding to the shown diffraction diagram (i.e. at 300 pixels from the direct beam) is 4π sin(θ)/λ = 0.112 nm -1 . b): reconstructed magnetic vortex (in plane magnetic component, done at 45° geometry). c): vortex core position versus time. d): charge (non-magnetic?) image of the square. e): intensity profile along the red line in d) for testing the imaging resolution. The intensity change over the topological border takes place within one reconstructed pixel, where the pixel size is 21.7 nm.

In the figure 7 d we show the reconstructed image of the 2 μm Co square (topological part only, obtained by adding the signal from the two helicities) and the intensity profile (figure 7 e) corresponding to the red line crossing the gold mask to the left and the Co square to the right. The measured pixel size is 21.7 nm, calculated from the known size of the square pattern. We observe that the intensity changes when crossing the edges of the FIB defined structures takes place within one pixel, thus we conclude that the spatial resolution of our imaging setup is better than 21.7 nm.

4.2

Holographic imaging of magnetic domains in the integrated mask/sample approach.:

For testing the permanent magnet setup of Fig. 4 we used a 40 repetitions Co/Pd multilayer, with an integrated gold mask for holography. The object hole was 2 microns in diameter. The energy of the circularly polarized photons was tuned to the Co edge (778 777? eV). The individual angles of the four permanent magnets were chosen in order to have a resultant magnetic field oriented normal to the sample surface (s direction, parallel to the beam). The gap was varied from 12 mm (500 mT) to 32 mm (80 mT). figure 8 shows the evolution of the magnetic domains with the strength of the magnetic field applied normal to the Co/Pd layers. These images are purely magnetic (difference of the two photon helicities). At high field values the magnetic layer is almost entirely saturated in the field direction (+s), with only few bubbles still remaining in the opposite direction (-s). While increasing the four magnets gap (i.e. decreasing the magnetic field strength at the sample position) the bubbles start to increase in size until the stable wormlike magnetic domain structure forms below ~100 mT. This is the equilibrium configuration with balanced "up" and "down" magnetic domains that do not evolve much with a further decrease of the field down to remanence.

Holographic imaging of magnetic domains in the separate mask/sample approach:

Preparing the holography mask and the sample on two separate x-ray transparent membranes allows one to image different parts of the sample, effectively extending the FTH field of view [ref-ref]. For testing this configuration in the COMET setup we used a continuous [Co(0.6nm)/Pt(1nm)]x20 multilayer sample, fabricated at the CNRS-Thales Laboratory [19]. This multilayer shows out of plane bubble-like magnetic domains at ambient temperature and represents a prototype system for skyrmion nucleation [START_REF] Chauleau | Chirality in Magnetic Multilayers Probed by the Symmetry and the Amplitude of Dichroism in X-Ray Resonant Magnetic Scattering[END_REF][START_REF] Chauleau | Chirality in magnetic multilayers probed by the symmetry and the amplitude of dichroism in Xray resonant magnetic scattering[END_REF].

The holography image was done using the separate mask and sample approach, in normal transmission geometry. The x-ray beam energy was tuned to 778 eV (Co L edge), in circular polarization. The mask consisted of a 1.8 µm diameter object hole and three reference holes with diameters around 80 nm. In figure 9 a) we show the 2D Fourier transform of the diffraction diagram. This corresponds to the square modulus of the autocorrelation of the mask and sample transmission function. The central disk is the autocorrelation of the object beam, while the six satellites are the cross-correlations between the object and the references (each of the three independent cross-correlations is accompanied by its corresponding complex conjugate). As the reference holes are much smaller than the object hole, these cross correlations represent the reconstructed transmission functions of the object, with a resolution given primarily by the size of the corresponding reference hole. The intensity (thus the contrast) of the reconstructed object goes with the size of the reference, being inversely proportional to the resolution.

One of the reconstructed objects from figure 9 a) is zoomed in figure 9b). This is the subtraction between images taken with different circular polarization helicities, thus the shown structures are purely magnetic. The quantitative profiles for the two separate polarizations, corresponding to the red line in figure 9 b) are displayed in figure 9 c) for both polarizations. As expected, they have inverted contrast and we can estimate a magnetic domain size around 90 nm. For this experiment the resolution was imposed by the size of the reference hole, i.e. 80 nm.

Ptychography :

The COMET end-station is adapted for the ptychography imaging technique [START_REF] Pfeiffer | X-ray ptychography[END_REF], were the sample position is scanned with respect to the incoming coherent beam (the probe) to create a sequential array of overlapping illuminated areas. For each illuminated area, the coherent diffraction pattern is recorded with a 2D detector in the far field region. To reconstruct the phase and the amplitude images, the Extended Ptychography Iterative Engine (EPIE) algorithm [START_REF] Maiden | An improved ptychographical phase retrieval algorithm for diffractive imaging[END_REF] is used. An improved ptychographical phase retrieval algorithm for diffractive imaging was implemented by Kadda Medjubi from the Nanoscopium beamline [24] into a Matlab script (The MathWorks, Natick, MA, USA) running on a 132 Gb RAM and 8 core processor personal computer.

A proof of principle experiment was performed on a patterned Co/Pd multilayer (40 repetitions) sample, as used in ref [START_REF] Spezzani | X-ray holographic imaging of magnetic order in patterned Co/Pd multilayers[END_REF], illuminated with a 778 eV coherent beam defined by a 1 µm diameter pinhole. The distance between the pinhole and the sample was several???? hundred microns. The sample to CCD distance was 42 cm, thus the size of the reconstruction pixel was 25 nm. figure 10 a) and b) show the reconstruction of a 1x3 µm 2 rectangle, done at the Co edge with circular left and right polarizations, respectively. For the sample positioning we used stepper motors with 150 nm encoded resolution. The curves in c) correspond to the intensity line profiles in a) and b). In this case the resolution was 300 nm, and it was measured as the distance corresponding to an intensity variation from 10% to 90% through the blurred edge of the rectangle. The local modulations of the two curves correspond to the magnetic domains and they are perfectly inverted as the images were taken with inverted photon helicities at resonance.

Figure 10: ptychography image reconstruction. a) and b): reconstructed image of a 1x3 µm 2 magnetic pattern done by scanning with stepper motors and using circular polarization, left and right, respectively. c): line profiles of a) and b). d): reconstructed image of a magnetic ring 2.5 microns in diameter and 170 nm wide, done by scanning with piezo motors and using left circular polarization. e): intensity profile along the red line in d) (red curve); the green curve is the convolution between a 170 nm wide box function and a Gaussian of 75 nm sigma radius. This comparison means the experiment resolution was 150 nm. f) Intensity profile along the magenta line in d) showing the contrast generated by the magnetic domains within the ring.

The image in figure 10 d) is the reconstruction of a magnetic ring of 2.5 microns in diameter and 170 nm lateral size. The experiment was done using piezo motors with 50 nm positioning resolution and one micron beam size on the sample. Gaussian blurring with different sigma radius were used on a 170 nm box function in order to compare with the experimental profile. The best fit was for a 75 nm sigma radius (figure 10 e)) thus we can conclude an imaging resolution of 150 nm.

The advantage of this technique is the possibility to image large fields of view without the need to put in contact the sample and the mask, as the case of holography with extended field on view (see 4.3). The final resolution depends on several parameters: the reconstructed pixel size, defined by the maximum scattering vector collected by the detector (in the examples above the reconstructed pixel size was 25 nm), the positioning precision of the sample (50 nm in Fig. 10d), the size of the beam on the sample and the efficiency of the retrieval algorithms. Further improvements can be done, like reducing the sample to detector distance, using nm precision piezo frames and reducing the size of the incident beam (zone plate).

Conclusion:

We have designed and built a new experimental chamber dedicated to soft x-ray coherent diffraction imaging in transmission. This station is already functional at the SEXTANTS beam-line of the SOLEIL Synchrotron and available to the user community. The main applications are in the field of solid-state physics and particularly of magnetic nano-structured materials. The state of the art resolution obtained with our instrument at the SEXTANTS beamline and employing a user custom mask is ~20 nm.

Our instrument is complementary to other existing magnetic imaging microscopes in the world, with some advantages and disadvantages. While the obtained spatial resolutions are about the same, the measuring conditions are different from one instrument to the other. For example a PEEM is dedicated to surface imaging only and is a good candidate for studying non-transparent samples e.g. epitaxially grown on solid substrates, not feasible with the transmission techniques. On the other hand the interaction with the electrons limits the applied external field to few tens of mT. A TXM/STXM works in transmission and allows fast recording of large fields of view, within seconds or tens of seconds, but the radiation dose on the sample can be very high. The focus distance of a zone plate, i.e. the space between the FZP and the sample is typically a few hundreds of microns, limiting the possibilities for the sample environment like coils or RF antennas. The holography in transmission with integrated mask permits high resolution imaging with a variety of experimental conditions applied to the sample, like temperature changes, external magnetic/electric fields or RF pulses. On the other hand the sample fabrication is more complex and the acquisition times are longer compared to STXM.

Thanks to the versatility of the COMET set-up, future developments are underway. For the present CCD, the read-out time in full-chip mode is around 4 seconds, for exposure times generally within half a second per acquisition. This means that the most of the time is actually spent for read-out, thus the development of future high frame-rate detectors is crucial. We planned to install shortly an additional 2D detector of the CMOS type, already successfully tested with soft x-rays, characterized by a much faster read-out time to improve drastically the efficiency of the data collection. Installing a piezo frame with nm positioning precision is also planned for improving the resolution of the ptychography experiments.

Another ongoing project is the setup at the beamline of a fs laser coupled to the x-ray beam to perform pump-probe time resolved experiments. The time structures available at the SOLEIL sychrotron will make it possible to image laser-induced magnetic transitions with time resolutions of either 50-100 ps (standard mode) or 5-10 ps (low-alpha mode ref.).. Considering the wide energy range available at the SEXTANTS beamline (50-1700eV) we would like to point out that this instrument may also be used for non-magnetic coherent scattering investigations in the soft X-rays range in order to benefit from the absorption edges available in this range, notably C, N and O Kedges.

Figure 2 :

 2 Figure 2: COMET chamber, with the cubic sample vessel on the left and the CCD vessel on the right.

Figure 3 :

 3 Figure 3: Left: internal mechanics seen from the beam, with the main rotation set for normal incidence. Right: internal mechanics seen from the side, with the main rotation set for 45° incidence. The four rods coming from the top are permanent magnets.

Figure 8 :

 8 Figure 8: Magnetic domains evolution as a function of the field strength, from few bubble-like domain nucleation (close to saturation) to homogenous worm domains (close to remanence).

Figure 9 :

 9 Figure 9: a) Fourier transform of the diffraction diagram, with six cross-correlation images produced by the three references. b) Zoom on one of the cross-correlations showing the magnetic image (subtraction between the two helicities). c) Intensity plot profiles for the two polarizations, separately, over the red line in b).

Acknowledgments:

The authors would like to thank W. Legrand, N. Reyren, V. Cros for providing the Co/Pt multilayer and M. Viret for discussion in the early conception stage of the magnetic device. We wish also like to thank K. Desjardins for his help in the test and commissioning of the CMOS detector.


Pasquale Lisena

Daniel Schwabe

Marieke Van Erp

Inger Leemans

William Tullett

Raphaël Troncy

Sofia Collette Ehrich

Thibault Ehrhart

An Ontology for Creating Hypermedia Stories Over Knowledge Graphs

Keywords: storylines, digital humanities, modelling cultural heritage data, navigating cultural heritage data

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

Introduction

Knowledge Graphs (KGs) have become one of the main tools used for Knowledge Management (KM) [START_REF] Hogan | Knowledge graphs[END_REF]. KGs are computational structures that are meant to be accessed programmatically using queries. Even so, it is also necessary for humans to consume KG contents directly. A typical approach for this has been to provide interfaces that allow users to explore the contents of KGs, for example through a faceted search. Examples of such tools, notably in the cultural heritage domain, include ADASilk [START_REF] Ehrhart | KG explorer: a customisable exploration tool for knowledge graphs[END_REF], 1 the Odeuropa Smell Explorer, 2 CultureSampo [START_REF] Mäkelä | How to deal with massively heterogeneous cultural heritage data-lessons learned in CultureSampo[END_REF], 3 and the Europeana Portal [START_REF] Petras | Europeana-a search engine for digitised cultural heritage material[END_REF]. 4 Storytelling has long been recognized as a knowledge management (KM) approach [START_REF] Dalkir | Organizational Storytelling and Knowledge Management: A Survey, Storytelling, Self[END_REF] and is used in many different settings to disseminate information: in journalism, SWODCH '23: International Workshop on Semantic Web and Ontology Design for Cultural Heritage, November 7, 2023, Athens, Greece Envelope pasquale.lisena@eurecom.fr (P. Lisena); dschwabe@gmail.com (D. Schwabe); marieke.van.erp@dh.huc.knaw.nl (M. v. Erp); inger.leemans@huc.knaw.nl (I. Leemans); will.tullett@york.ac.uk (W. Tullett); raphael.troncy@eurecom.fr (R. Troncy); sofia.ehrich@huc.knaw.nl (S. C. Ehrich); thibault.ehrhart@eurecom.fr (T. Ehrhart) Orcid 0000-0003-3094-5585 (P. Lisena); 0000-0003-4347-2940 (D. Schwabe); 0000-0001-9195-8203 (M. v. Erp); 0000-0003-1640-4109 (I. Leemans); 0000-0002-8975-8031 (W. Tullett); 0000-0003-0457-1436 (R. Troncy); 0009-0002-6841-6910 (S. C. Ehrich); 0000-0003-1377-8279 (T. Ehrhart) in books, TV-shows and movies, video games, exhibitions, virtual-and augmented-reality environments, etc. 5Accessing KG content via stories has been recognised as a complementary approach for knowledge capture and dissemination. This approach has been adopted in several cases such as the DLNarratives project [START_REF] Meghini | Representing Narratives in Digital Libraries: The Narrative Ontology[END_REF], 6 the Belgian Refugees project, 7 the Bletchley Park Text project, [START_REF] Mulholland | Bletchley Park Text: Using mobile and semantic web technologies to support the post-visit use of online museum resources[END_REF] the Paths project [START_REF] Fernie | PATHS: Personalising access to cultural heritage spaces[END_REF], and the Storyspace project [START_REF] Wolff | Storyspace: A Story-Driven Approach for Creating Museum Narratives[END_REF].

In this paper, we present an ontology for authoring and presenting hypermedia stories via knowledge graphs, and illustrate how it has been used in practice. The ontology was created in the context of the Odeuropa project, which is focused on the history and heritage of smell [START_REF] Lisena | Data Mining and Knowledge Graphs as a Backbone for Advanced Olfactory Experiences[END_REF], but its application generalises to any KG containing object descriptions for which one wants to generate linear and nonlinear stories. We show how existing KG connections can be enriched by story connections, providing contexts that express multiple points of view over a collection of objects in the KG.

The remainder of this paper is organised as follows. In Section 2, we discuss related work in modelling stories. In Section 3 we describe the requirements of the ontology, followed by its implementation in Section 4. We present examples in Section 5, a qualitative evaluation in Section 6 and we conclude with final remarks and directions for future work in Section 7.

The ontology in RDF/Turtle format can be found at https://github.com/Odeuropa/ontology, while its documentation is available at https://data.odeuropa.eu/ontology/story/.

Related Work

Prior ontologies for narrative and story structures include StoryTeller [START_REF] Yeh | StoryTeller: An event-based story ontology composition system for biographical history[END_REF], BBC News Storyline ontology [START_REF] Wilton | Storyline Ontology -An ontology to represent News Storylines[END_REF], Labyrinth data model, ODY-ONT [START_REF] Khan | Leveraging a Narrative Ontology to Query a Literary Text[END_REF], Curate ontology [START_REF] Mulholland | Curate and Storyspace: An Ontology and Web-Based Environment for Describing Curatorial Narratives[END_REF] and NIAO [START_REF] Xu | An ontology model for narrative image annotation in the field of cultural heritage[END_REF].

Most of these treat stories as linear sequences of events. For example, StoryTeller [START_REF] Yeh | StoryTeller: An event-based story ontology composition system for biographical history[END_REF] is an ontology for representing stories as collections of events, which are directly connected with the contains property. With this data model, the only way to reconstruct the order of story elements (events) is to compare their time, producing stories which are a pure linear concatenation of episodes, which excludes any descriptive or reflective part of a story. The BBC News Storyline ontology [START_REF] Wilton | Storyline Ontology -An ontology to represent News Storylines[END_REF] models news topics and events, mostly temporally focused. Similarly, in the Labyrinth data model [START_REF] Damiano | Ontological Representations of Narratives: a Case Study on Stories and Actions[END_REF] the Dynamics are the events which compose a Story. The ODY-ONT model [START_REF] Khan | Leveraging a Narrative Ontology to Query a Literary Text[END_REF] focuses on the representation of events, sub-events, and the temporal relation between them. Similar relations between events are represented in the Datasets of Mystery Stories [START_REF] Kozaki | Datasets of Mystery Stories for Knowledge Graph Reasoning Challenge[END_REF]. The chain of events is also at the foundation of other works [START_REF] Croft | Integrating Decompositional Event Structures into Storylines[END_REF][START_REF] Moreno-Schneider | Towards User Interfaces for Semantic Storytelling[END_REF].

Mulholland et al. [START_REF] Mulholland | Curate and Storyspace: An Ontology and Web-Based Environment for Describing Curatorial Narratives[END_REF] present an ontology and system to create unique stories, modeling narratives, plots, stories and events. The Storyspace API on top of the Curate ontology assists museum curators in manually creating stories, or importing them from Freebase 8 .

Automatic narrative generation from data has been explored in various projects. The tourist domain has been explored in [START_REF] Mauro | Exploring Semantically Interlaced Cultural Heritage Narratives[END_REF] in which a mobile guide generates walks along different cultural points of interest in Turin, Italy. Here, users do not generate the narrative themselves, similarly to [START_REF] Blin | Building a French Revolution Narrative from Wikidata[END_REF] in which a timeline of the French revolution is generated automatically from Wikidata. A different view on narratives is presented in [START_REF] Xu | An ontology model for narrative image annotation in the field of cultural heritage[END_REF], which is focused on annotating objects such as murals that describe a multiple-plots story across multiple times and spaces. Here, a narrative consists of one cultural heritage object, instead of multiple, as allowed in our ontology. Extracting storylines from texts has been addressed in the Events and Stories in the news workshops series held from 2015 to 2020 [START_REF] Bonial | Proceedings of the First Joint Workshop on Narrative Understanding, Storylines, and Events, Association for Computational Linguistics[END_REF] and a handbook compiled by a subset of organisers of this workshop series [START_REF] Caselli | Computational Analysis of Storylines: Making Sense of Events[END_REF]. Here, events are often expressed as verbs, and tend to be quite fine-grained.

Problem Overview

In this section, we give the basic definition and foundation of our work, from which we define the modelling requirements.

Background concepts

To understand the various layers that make up a storyline, it is useful to refer to concepts based on those proposed by Bal and van Boheemen in [START_REF] Bal | Narratology: Introduction to the Theory of Narrative -4th[END_REF]. There, a narrative text (our storyline) is the production (physical rendition) of a story, which in turn is a rhetorical arrangement of elements of a fabula. The fabula is a representation of the actual "meaning" of the story, i.e., the conceptualizations that the author intends to create in the reader's mind. A fabula is formed by logically or chronologically related events, and may contain references to external contents. In our ontology, we generalize this notion, considering elements that are not necessarily events.

A story can be seen as a collection of atomic elements variously referred to in the literature as passages, chunks, fragments, scenes, cards or nodes. A story element has some textual (and, more generally, multimedia) content, and references elements of the underlying KG. It may also reference other sources, e.g., Wikipedia. A story in our definition corresponds to a story in Bal and van Boheemen's terminology, but may have a richer (graph) structure in our case.

In addition, a story establishes structure over its elements, expressed through one or more types of relations between them. These structural relations dictate the order of consumption by the reader. The user starts at an element, and proceeds by consuming (navigating to) any related element according to the story's structure.

From this point of view, the semantics of a KG element is complemented by the semantics of all story elements that reference it, according to the point of view of the story author. In Bal's terminology, the content of the story elements correspond to the fabula.

Once the story is specified, it is necessary to create a presentation, which is the surface form of the story that is effectively consumed by humans. We will discuss this in Section 5.

The most common story topology is a linear structure -the elements form a sequence. This structure is also very common in story representations in the history and cultural heritage domains, specifically chronologically ordered knowledge. However, these structures can be be limited when there is a wish to accommodate more complex narratives, such as the integration of multimedial heritage objects or interactive elements (tests, images, artefacts). Increasingly history and cultural heritage require narratives that incorporate multiple points of view, multiple time periods, and multiple geographies (multi-perspectivity; pluritemporality). In addition, event-based models struggle to capture more abstract notions (such as complex cultural concepts, collective imaginations, symbolism, or cultural practices) which often form the basis of a historical or cultural heritage storyline.

To illustrate this, we present an example from the history of smell. Consider, for instance the civet cat, a mammal native to the tropical climates of Africa and Asia [START_REF] Dannenfeldt | Europe discovers civet cats and civet[END_REF][START_REF] Dugan | The ephemeral history of perfume: Scent and sense in early modern England[END_REF][START_REF] Fudge | Renaissance animal things[END_REF]. At some point, humans discovered and started to appreciate the olfactory qualities of the substance the cat secretes through its perineal gland. The secretion was regarded as having medicinal functions and desirable hedonic qualities. The civet cats 'story' thus changed from a biological story to a story in which the cat also assumed an important role in the history of perfumery, where the role of the cat changed from 'animal' to 'economic product'. From here, 'civet' enters the story of musk, ambergris, and other animal fragrances. Now, the story invites more interest about the human use and evaluation of the civet paste as olfactory substance. With the evolution of social norms and values, its initial hedonic qualities became associated with "lower" social circles such as prostitutes, which also changed its economic value. Civet's use in many perfumes produced from the eighteenth through the early twentieth centuries means that many heritage perfumes being recreated today include civet. Perfumes with a rich heritage, such as Chanel No.5, continue to include a civet note. Meanwhile, a synthetic substitute for its odor element (civetone) was discovered, based on palm oil. This brief description highlights how one can create a narrative about the civet and civetone from a medicinal perspective (use); from a social perspective (appreciation, symbolism); and from the perspective of abstract concepts such as capitalism. In each perspective (expressed as a storyline), it can be associated with different elements and further storylines, e.g., a story about the economic relevance of palm oil to the perfume industry. Civet's meanings are multi-temporal: the decline in the fashion for civet meant that it obtained an air of pastness, the continuing use of civet in contemporary perfumery ensures its persistence in the present. The attempt to recreate historical perfumes in which civet is used points to its place as a future heritage scent. Smells -and the historical and heritage stories we tell about them -are palimpsests that blend past, present, and future into complex experiences that challenge linear chronologies [START_REF] Tullett | Smell and the Past: Noses, Archives, Narratives[END_REF].

From this example, we note that each point of view can be captured in its own linear storyline, with the end result that these various storylines have one or more elements in common. This means that, while consuming the contents of a particular element, the reader will have the option to continue by following along more than one storyline. As a result, the overall story structure effectively becomes a graph.

The next level of complexity in the story structure is when it is natively a graph, regardless of the existence of linear paths through it. This type of structure has been used in hypertext literature 9 , games (e.g. Telltale Games10 ) and more recently in interactive film (e.g, the Black Mirror: Bandersnatch episode 11 ). In these stories, it is left to the reader to choose the available links without a suggested sequence by the author. Here the fabula is only apprehended by the reader after a more extensive exploration of the story elements.

Modelling Requirements

In light of the previous discussion, we define the requirements for our proposed ontology: Req 1: Given the need to accommodate multiple perspectives, and the variety of possible association between elements, it should allow to represent hypertexual (graph-structured) stories, In other words, its elements can be connected through multiple alternative paths; Req 2: Users can consume stories via navigation links present in each element, in hypertext fashion; Req 3: To express their particular point-of-view about the elements in a story, authors should be able to define a suggested order of consumption of elements, effectively defining paths (storylines) through the story. Req 4: An element may be part of different storylines, as the different points-of-view may share common elements. Consequently, while consuming an element, the reader has the option of continuing along the storyline s/he came from or continuing along a different storyline. Req 5: Stories, while building narratives about different sets of elements, may refer to each other, by sharing common elements -for example, "War" and "Religion" stories. Thus, while consuming one story, the reader should be able to follow a reference to another story. Req 6: Stories should be able to connect to KGs, and to other sources in general, therefore its elements can refer to external resources via direct links. Req 7: Since stories reflect points-of-view of its authors, it should be possible to include provenance metadata about the story, namely authors, creation and modification dates, etc. Req 8: Whereas the semantics of individual concepts referred to in a story element can be given by the underlying KG, there will be additional content in story elements whose semantics are left to the reader to apprehend, as they are expressed through textual and multimedia content. Consequently, the ontology should be independent of (i.e, not make assumptions about) the definition of elements in any underlying KG. Req 9: A story can be rendered in multiple delivery formats and in multiple environments. It should therefore be neutral in this respect, accommodating this multiplicity.

Ontology

We distinguish between the concepts of Story and Storyline. The basic idea is that the same Story can be told -actually, consumed -in different ways by following different paths throughout the story. Each path (or linearisation, or narrative) is a Storyline which can be chosen in a Story. According to this definition, for each Story there can be multiple Storylines that can be proposed by the storyteller to offer a particular perspective of the Story. The core part of the StoryLine Ontology is represented in Figure 1. The main building block is the Story Element. It is the basic and atomic element of a Story. The contents of the element are envisaged to actually be of text or multimedia types, e.g, in HTML, or some structured format to be interpreted by the rendering engine (see Section 5). It is expected that it contains anchors, which can be rendered as hypertext links to related (linked) elements which can be navigated by the user. These links are represented in the graph using RDF-Star [START_REF] Hartig | RDF-star and SPARQL-star: Final Community Group Report[END_REF], qualifying (annotating) an RDF statements such as <element1, connects to, element2> with the class Story Connection. This class has properties for representing its association to a Story and a Storyline, and for tracking the text of the anchor in the content to the linked element. These links may have types, which can be used for automatic generation of stories.

Story

These two classes are defined in the following way, keeping in mind that each element can have multiple anchors and, consequently, multiple Story Connections linking to successor elements:

• a Story is a collection of interlinked elements, with a starting Story Element and a number of succeeding ones; • a StoryLine identifies a selection of elements that constitute a particular path (set of Story Connection of a Story, one per element) representing one possible linearisation.

The presence of both Story and StoryLine allows to represent both an hypertextual and a linear story, depending on the balance between freedom and guidance that one wants to give to the user. Finally, the ontology allows for an External Connection to other entities which may have some relation with the Story Element. We decided to not limit the range of this relation, to enable linking a Story element with knowledge graph entities, but also encyclopaedic articles (e.g., Wikipedia), etc. Also for this connection, it is possible to define an anchor and an additional type, as seen in Figure 2.

We purposefully did not include specific properties besides dc:creator for the metadata of Stories. Given the existence of ontologies that encompass a large number of metadata fieldssuch as the Dublin Core (dc, as in Figure 1) or the PROV Ontology [START_REF] Lebo | PROV-O: The PROV ontology[END_REF]. We encourage the use of the StoryLine ontology in combination with other schemas, according to the specific needs of the application.

Story Element has reference

External Connection

Application Examples

In this section, we present two application examples of our ontology: a hypermedia storyline and a guided city tour application.

Hypermedia storyline

In the Odeuropa project, historians are experimenting with hypermedia narratives to present their syntheses in a data-driven and interactive manner as a complement to traditional monographs. For this, we need to map a story that is represented as an instance of our ontology into a form suitable for human consumption. We refer to this process as "rendering". At the moment, we have taken the approach that the contents of story elements contain presentation specifications that can be directly used by a rendering engine.

In the realm of interactive storytelling tools, Twine 12 is a popular open-source tool which empowers users to create interactive narratives through a visual, node-based interface. In this tool, individual passages or nodes represents story elements, each connected through hyperlinks. These hyperlinks establish the flow of the storyline, allowing readers to make choices that determine the progression of the narrative.

Our Storyline ontology is designed to work well with interactive stories created using tools like Twine, which present stories structured in a graph format -see Figure 3. Moreover, our ontology facilitates bidirectional conversion, enabling the seamless translation of ontologymodeled stories back into Twine-compatible formats.

As an example, a Twine story with the title War has always been an olfactory affair is available at https://data.odeuropa.eu/ontology/story/war.html. An excerpt of the RDF version of this story is shown in Listing 1, while a more extended version is available online 13 .

City Sniffers Guided Tour

The City Sniffers application 14 is a smell tour of Amsterdam's eco-history. It consists of a rub and sniff card and a mobile phone application. Users can follow a path of six selected stops through Amsterdam, the Netherlands. At each stop, the participant scratches the corresponding spot on the card and can sniff a scent while reading a text explaining the smell and its cultural-historical context and relevance in the application. Figure 4 shows the rub and sniff card alongside two screenshots of the applications. This application also links to the Odeuropa KG and story elements are the texts associated with each stop. The storyline structure is the linear path connecting each spot to its successor.

Evaluation

We present a qualitative evaluation of the ontology, by examining -together with researchers from the humanities domain -each of the requirements given in Section 3.2 and showing how they are satisfied. To facilitate reading, each item mentions its corresponding requirement.

Req 1: The graph-like structure is enabled by the "connects-to" relation, and allows an element to participate in several relation instances. Req 2: Hypertext navigation by the user is enabled by the "anchor" property, which is used by the rendering environment to allow navigation;

Req 3: Storylines are enabled via the association of a link to the "StoryLine" class in the ontology. Req 4: An element may be part of different storylines, since "connects-to" relation can be qualified by more than one "Storyline" instance in the ontology. Req 5: Stories may refer to each other since the source and the destination elements in a "connectsto" relation may each also be part of a different "connects-to" relation that are respectively qualified with different instances of "Story". Req 6: Stories are be able to connect to KGs, and to other sources, since the "Story Element" class has a "has reference" property that points an external URL. Req 7: Stories can have provenance meta-data, although only the "dc:creator" property of the "Story" class is explicitly included in the ontology. Req 8: The ontology is independent of the definition of elements in any underlying KG since the content of the "Story Element" class has a "content" property which is a string. This string can be interpreted in innumerable ways by the rendering environment. Req 9: A story can be rendered in multiple formats and multiple environments, since the "content"

and "anchor" attributes may contain formatting instructions to be interpreted by the rendering environment, such as Twine (which uses a markup language) .

Conclusion and Future Work

In this paper, we have presented an ontology for creating hypermedia stories over knowledge graphs using RDF-Star and shown its use in two example applications. Our ontology effectively enables a narrative layer over large datasets. Our ontology allows smooth integration with underlying KGs, and enables stories with rich hypermedia structure, encompassing non-eventbased elements as well.

In future work, we aim to further expand the ontology to include provisions for expressing rhetorical structures such as the ones enabled by the Curate ontology [START_REF] Mulholland | Curate and Storyspace: An Ontology and Web-Based Environment for Describing Curatorial Narratives[END_REF], and others. Furthermore, we will refine the provenance requirements to aid users in tracing their information sources and narrative development. Additionally, we plan to realise an automatic converter between the Twine format and the Storyline ontology representation, and vice versa to empower humanities and GLAM professionals to create storylines to expose their archival searches and collections. We also plan to extend the implementation to allow users to record, resume and possibly share navigation histories over a set of stories.

Figure 1 :

 1 Figure 1: Schema of the StoryLine ontology, with the main elements and the Story Connection

Figure 2 :

 2 Figure 2: Schema of the StoryLine ontology, with a focus on the External Connection

Figure 3 :

 3 Figure 3: An example of story in Twine, with the story overview on the left and a story element in editing mode on the right. The connections between cards (the story elements) naturally give to the story a graph structure.

Listing 1 :

 1 The representation in RDF of the War story :War a story:Story; story:title "War has always been an olfactory affair"; story:start :Introduction .

  (a) 'Rub'n'Sniff' map created for Odeuropa's, City Sniffers-A smell tour of Amsterdam's ecohistory, printed for the event by Scent the Brand. Designed by Liam R. Findlay. Note: the numbers indicate the order of stops. (b) Screen shot of the City Sniffers application

Figure 4 :

 4 Figure 4: City Sniffers 'Rub and Sniff' card and smartphone application screenshots.

https://blog.mused.org/an-overview-of-digital-storytelling-platforms-in-2021/

https://dlnarratives.eu/narratives.html

https://tour.belgianrefugees14-18.be/en

Freebase was a large collaborative knowledge base that was shut down in 2016. It now powers part of Google's Knowledge Graph.

https://en.wikipedia.org/wiki/Hypertext_fiction

https://en.wikipedia.org/wiki/Telltale_Games

https://en.wikipedia.org/wiki/Black_Mirror:_Bandersnatch

https://twinery.org/

https://github.com/Odeuropa/ontology/blob/master/storylines_examples/e1.ttl

https://citysniffers.odeuropa.eu/

Acknowledgments

This work is supported by the European Union's Horizon 2020 research and innovation program within the Odeuropa project (grant agreement no. 101004469) and by the French National Research Agency (ANR) within the kFLOW project (Grant n°ANR-21-CE23-0028). Like King Frederick II of Prussia, the Odeuropa team also dislikes the smell of the pipe.


Kaouthar Kessabi
email: kaoutharkessabi@yahoo.fr

Ali Annabi

Aziza Ibn

Hadj Hassine

Ingrid Bazin

Wissem Mnif

Khaled Said

Imed Messaoudi

Possible chemical causes of skeletal deformities in natural populations of Aphanius fasciatus collected from the Tunisian coast

Keywords:

Patterns of skeletal deformities were detected in natural population of Aphanius fasciatus. " The high incidence of spinal deformities was observed in the most polluted site. " The most polluted site is contaminated mainly by heavy metals and organic compounds. " Relation between the observed deformities and chemical pollution was suggested.

Introduction

Spinal deformities have been reported in natural fish populations as well as in farmed fish [START_REF] Aunsmo | Association of spinal deformity and vaccine-induced abdominal lesions in harvest-sized Atlantic salmon, Salmo salar[END_REF][START_REF] Ayed | Incidence of kyphosis deformities in natural population of Atherina lagunae (Trabelsi et al., 2002) from the Tunis North Lake, Tunisia[END_REF][START_REF] Kessabi | Involvement of Cd bioaccumulation in spinal deformities occurrence in natural populations of Mediterranean Killifish[END_REF][START_REF] Bardon | What is the heritable component of spinal deformities in the European sea bass (Dicentrarchus labrax)[END_REF]Messaoudi et al., 2009a;[START_REF] Sun | Morphological deformities as biomarkers in fish from contaminated rivers in Taiwan[END_REF]. However, skeletal malformations in farmed fish are very common and are a major problem in aquaculture (Koumoundourous et al., 2001a,b). Whereas, the observation of spinal deformities in wild specimens of teleost fish is relatively rare [START_REF] Dahlberg | Frequencies of abnormalities in Georgia estuarine fishes[END_REF][START_REF] Da Chuna | Occurrence of vertebral deformities in Gobiidae (Pisces) from the Tagus estuary[END_REF].

While, skeletal malformations in aquaculture appear to be related to a complex interaction of genetic, procedural and infectious/immunological factors [START_REF] Lall | Role of nutrients in skeletal metabolism and pathology in fish, an overview[END_REF][START_REF] Aunsmo | Association of spinal deformity and vaccine-induced abdominal lesions in harvest-sized Atlantic salmon, Salmo salar[END_REF][START_REF] Aunsmo | Modelling sources of variation and risk factors for spinal deformity in farmed Atlantic salmon using hierarchical-and cross-classified multilevel models[END_REF][START_REF] Bardon | What is the heritable component of spinal deformities in the European sea bass (Dicentrarchus labrax)[END_REF], the presence of deformities in natural populations are most likely related to pollution [START_REF] Lemly | Symptoms and implications of selenium toxicity in fish: the Belews Lake case example[END_REF][START_REF] Kessabi | Involvement of Cd bioaccumulation in spinal deformities occurrence in natural populations of Mediterranean Killifish[END_REF]Messaoudi et al., 2009a). Indeed, it has been suggested that spinal deformities in natural populations of fish serve as a biomarkers of environmental pollution [START_REF] De | Lordosis in topsmelt Atherinops affinis (Ayers, 1860) (Teleostei: Atherinopsidae)[END_REF][START_REF] Antunes | Skeletal anomalies in Gobius niger (Gobiidae) from Sado Estuary, Portugal[END_REF][START_REF] Yang | Use of fish biomarkers to assess the contaminant exposure and effects in Lake Erie tributaries[END_REF].

It is well known that a wide variety of chemicals can cause skeletal deformities through such mechanisms by altering the development and bone formation. These include heavy metals such as lead (Pb) [START_REF] Bengtsson | Vertebral deformities and physiological effects in fourhorn sculpin (Myoxocephalus quadricornis) after long term exposure to a simulated heavy metal-containing effluent[END_REF], cadmium (Cd), many organophosphorus pesticides, polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), toxaphene [START_REF] Cheng | Cellular and molecular basis of cadmium-induced deformities in zebrafish embryos[END_REF][START_REF] Teraoka | 2,3,7,8-Tetrachlorodibenzo-pdioxin toxicity in the zebrafish embryo: altered regional blood flow and impaired lower jaw development[END_REF] and natural estrogenic compounds such as ethinylestradiol (EE2) [START_REF] Boudreau | Utility of morphological abnormalities during early-life development of the estuarine mummichog, Fundulus heteroclitus, as an indicator of estrogenic and antiestrogenic endocrine disruption[END_REF] and 17 beta-oestradiol (E2) [START_REF] Urushitani | Early estrogen exposure induces abnormal development of Fundulus heteroclitus[END_REF].

The Mediterranean killifish, Aphanius fasciatus (Valenciennes, 1821) is a small cyprinodont fish inhabiting brackish waters in coastal habitats of the central and eastern Mediterranean Sea. It tolerates a wide range of physico-chemical parameters, such as temperature (5-39 °C) and salinity (0-180 g L À1 ). We have recently described, in natural populations of this species collected from the Gulf of Gabes in Tunisia, a complex spinal deformities consisting of a consecutive repetition of scoliosis, lordosis, and kyphosis from the head to the caudal fin. Deformed fish were eight times more frequent in the industrialized coast of Sfax than in the coast of Luza which is unaffected by human activities, possibly indicating a relationship between pollution levels and spinal deformities (Messaoudi et al., 2009b). However, a direct relationship between observed spinal deformities and pollution was not determined in this species.

Therefore, this study attempt to quantify and identify skeletal deformities in A. fasciatus populations which is naturally exposed to several types of pollutants. For this purpose fish were collected from three polluted localities: S2, S3 and S4, and from a reference site S1. In addition, heavy metals (Cd, Cu and Zn), PAHs, estrogenic activities and estrogenic compounds were also determined in water and sediment of these sites in order to establish a relationship between these pollutants and the high incidence of spinal deformities detected in A. fasciatus.

Material and methods

Study sites and sampling

Sites characterization

Sampling sites were selected in the Tunisian coast (Fig. 1), three polluted sites and a reference one. The S2, surrounded by important industrial activities, mainly crude phosphate treatments and chemical industries, was chosen as a most polluted site. This site is contaminated with heavy metals, essentially Cd. The S1, located 50 km north of S2 apparently unaffected by human activities, was used as reference sample site [START_REF] Barhoumi | Cadmium bioaccumulation in three benthic fish species, Salaria basilisca, Zosterisessor ophiocephalus and Solea vulgaris collected from the Gulf of Gabes in Tunisia[END_REF]Messaoudi et al., 2009b). Pollution in S3 caused by human activities is related to the proximity of the industries in Monastir South, aquaculture activities, the penetration of the water weir South Bay loaded Khniss of pollutants from the village and neighboring areas of culture, and, particularly through the wadi and the activity of washing the wool. The discharge of hot water by Power station via S4 used for overflow sewage, contributes to a sizeable rise in water temperature, this temperature can reach 36 °C. It also contributes to environmental enrichment in nutrients especially ammonium which is an indicator of urban pollution.

Sample collection

Fish were caught with a hand nets. Caught fish were counted and the percentage of deformed fish was calculated from 2007 to 2010. 75 normal specimens (mean total length 3.14 ± 0.36 cm; mean total weight 0.54 ± 0.17 g) and 77 deformed specimens (mean total length 2.79 ± 0.34 cm; mean total weight 0.45 ± 0.15 g) were collected for the morphological study.

Water samples were collected from all localities at a depth of 0.5 m in clean bottles from the different areas. The upper 5 cm of sediments were also collected. Sampling bottles were previously cleaned by soaking with 10% nitric acid (HNO 3 ) and rinsed with ultrapure water.

Analytical procedures 2.2.1. Skeletal deformities analysis

The double staining method was used to evaluate anomalies observed in specimens. Normal fish and fish showing a morphological deformity were fixed in buffered 10% formalin and stained using the ''Alcian Blue-Alizarin Red'' double staining method [START_REF] Park | A procedure for staining cartilage and bone of whole vertebrate larvae while rendering all other tissues transparent[END_REF]. Alcian Blue 8 GX (Sigma) was used to stain cartilage in blue followed by Alizarin RedS (Sigma) to stain calcified structures in red. The combination of KOH and peroxide treatment tends to increase the transparency of soft tissues and allow clear observation of skeletal structure.

Metal analysis

Water samples were stabilized at pH 2 with 1 M nitric acid prior to direct determination of total metal concentrations. Sediment samples were oven-dried for 48 h at 100 °C and 100 mg of each sample was mineralized at 250 °C with a set of acids composed of 1 ml of nitric acid, 2 ml of fluorhydric acid and 0.5 ml of perchloric acid and then adjusted to 10 ml with deionised water. Samples were analyzed to determine Cd, Cu and Zn concentrations using a graphite furnace AAS technique (ZEEnit 700). Samples were analyzed in triplicate. The variation coefficient was usually less than 10%. Metal concentrations are reported as lg g À1 of dry weight for sediments and as lg L À1 for water samples.

PAHs (polycyclic aromatic hydrocarbons) determination

The assays performed were carried out according to the protocol of CEAEQ (www.ceaeq.gouv.qc.ca). PAHs levels in sediment were determined by gas chromatography coupled with mass spectrometry. The following compounds were analyzed: acenaphthylene, acenaphthene, naphthalene, phenanthrene, anthracene, fluoranthene, pyrene, benzo(a) anthracene, chrysene, benzo(b) fluoranthene, benzo (e, a)pyrene, benzo(g, h, i) perylene, and dibenzo [a, h] anthracene. Data are reported as lg of PAHs/ g dry weight.

YES assay

Both agonist and antagonist activities of chemical dyes were examined using the Yeast strain BY4741 [START_REF] García-Reyero | Monitoring of endocrine disruptors in surface waters by the yeast recombinant assay[END_REF]. This recombinant yeast strain carries the b-galactosidase reporter gene under the control of the estrogen responsive element ERE. The test consisted on the measurement of the b-galactosidase activity (fluorescence at 460 nm after excitation at 355 nm; Fluoroskan, Twinkle LB 970, BERTHOLD Technologies) after 6 h of exposure to the solutions to be analyzed.

Within each experiment, an internal control was set up for the reference estrogen, 17b-estradiol on one plate. The upper part of each plate was filled with the solvent control (methanol), nine concentrations of E2 (1.0 Â 10 À12 , 1.0 Â 10 À11 , 1.0 Â 10 À10 , 1.0 Â 10 À9 , 1.0 Â 10 À8 , 1.0 Â 10 À7 , 1.0 Â 10 À6 , 1.0 Â 10 À5 and 1.0 Â 10 À4 M).

For the effluent samples, dilution series (1-1000) of concentrated extract were prepared in distillated water. Each test for effluent samples was evaluated at least three times by independent experiments. Agonist activities of effluent samples were measured as % of maximal fluorescence intensity produced by 17b-estradiol.

Estrogens detection

The derived estrogens were separated on a waters 2695 HPLC machine, coupled with UV detector DAD996. The injection of 40 ll of samples was performed at 25 °C. The column was a C18 Ascentis C18, 5 cm  4.6 mm I.D., 5 lm; the mobile phase was a mixture of water: acetonitrile (55:45); and the flow rate was set at 0.4 ml min À1 . A UV detector was used to detect estrogens at 220 nm.

Statistical analysis

For the skeletal deformities, results were reported as percentage of fish affected and comparisons were performed with chi-square test. For metal concentrations, data are given as means ± SE, and differences were assessed by one-way ANOVA followed by protected least significant difference Fisher's test. Values were considered statistically significant when p < 0.01.

Results

Incidence of spinal deformities in A. fasciatus

Spinal deformities were visible on the fish body immediately upon catching. The results of spatial distribution among different sites show that the percentage of deformed fish was significantly higher (p < 0.0001) in S2 from 2007 to 2010 compared to the three other sites (Table 1).

Skeletal anomalies: description and frequencies

Skeletal anomalies of adult A. fasciatus were grouped into 13 categories that described abnormalities on spines, vertebrae arcs and mandibles. A total of 1025 anomalies were observed: 460 in the vertebrae, 478 in the spines, 73 in the arcs and 14 in mandibles. The number of anomalies recorded in normal fish was 31 (3.02%) and 994 in deformed fish (96.97%). The most common abnormalities described are shown in Fig. 2. The frequencies of fish affected with different skeletal anomalies are reported in Table 2.

Chemical analysis

The water physico-chemical parameters of sampling sites are summarized in Table 3. Our results showed that the industrialized coast of Sfax (S2) appeared as the most polluted site. In fact, PAHs and heavy metals, especially Cd and Cu, were predominant in S2 compared to the three other sites (S1, S3 and S4). Physico-chemical parameters such as pH, conductivity and dissolved O 2 are similar in all studied sites, while the highest temperature was recorded in the Hamdoun river (S4).

Results presented in Fig. 3 showed that spatial variations of incidence of spinal deformities in A. fasciatus populations and CdLire phonétique that spinal deformities that contents in water and sediment of studied sites were very similar.

Estrogenic activities

Our results show that the high level of estrogenic activity was observed only in S4 (60.5% in water and 99.65% of relative estrogenic activity in suspended mater). A light difference was observed between S1, S2 and S3 (Table 4). The results related to the estrogenic compounds, detected by HPLC analysis in water and suspended mater (SM) of different areas of study; indicate the presence of estrone in water and 17b-estradiol and estriol in SM collected from S4 (Table 4).

Discussion

The aim of this study was to diagnose skeletal deformities and found a possible relationship between these deformities and several types of pollutants present in the environment.

Skeletal deformities, one of the most serious pathological effects in fishes, are well known for many fish species from different areas of the world [START_REF] Tutman | Incidence of spinal deformities in natural populations of sandsmelt, Atherina boyeri (Risso, 1810) in the Neretva river estuary, middle Adriatic[END_REF][START_REF] Ayed | Incidence of kyphosis deformities in natural population of Atherina lagunae (Trabelsi et al., 2002) from the Tunis North Lake, Tunisia[END_REF][START_REF] Sun | Morphological deformities as biomarkers in fish from contaminated rivers in Taiwan[END_REF]. Assessing morphological deformities represent one of the most straight forward methods to study the effects of contamination on fish because of the ease of recognition and examination when compared with other types of biomarkers.

In our previous study, we have showed that CYP1A mRNA levels in deformed A. fasciatus collected from the industrial coast of Sfax (S2) were 10 times higher than the normal ones, suggesting a link between the extent of deformity of impacted fish and their exposure to CYP1A-inducing pollutants (dioxins, dioxin-like PCBs, PAHs) in the environment [START_REF] Kessabi | Evaluation of environmental impact on natural populations of the Mediterranean killifish Aphanius fasciatus by quantitative RNA biomarkers[END_REF]. In the present study, population from the industrialized coast of Sfax (S2) was found to have higher frequency of abnormal fishes as compared to A. fasciatus from the other sites. PAHs and heavy metals have been found in water and sediments of S2 with high levels as compared to other sites, suggesting a combined effect of these pollutants leading to different types of skeletal deformities observed in A. fasciatus. This ecological situation is closely connected with the promoted industrial development and intense human activities. This region differs greatly in many environmental aspects and observed population differences in malformation frequency might be explained by influence of environmental pollution. In fact, the pollution in this coast is due essentially to the presence of continuous discharge of pollutants from local industrial activities [START_REF] Hamza-Chaffai | Health assessment of a marine bivalves Ruditapes decussatus from the Gulf of Gabès (Tunisia)[END_REF] and from the phosphogypsum. Several reports have also indicated high metal concentrations in soft tissues of Reditapes decussatus [START_REF] Hamza-Chaffai | Health assessment of a marine bivalves Ruditapes decussatus from the Gulf of Gabès (Tunisia)[END_REF][START_REF] Smaoui-Damak | Preliminary study of the clam Ruditapes decussatus exposed in situ to metal contamination and originating from the gulf of Gabès, Tunisia[END_REF] and in the fish Scorpaena porcus [START_REF] Hamza-Chaffai | Physicochemical forms of storage of metals (Cd-Cu and Zn) and metallothionein-like proteins in gills and liver of marine fish from the Tunisian coast: ecotoxicological consequences[END_REF] from this coast.

The present study showed that spatial variations of incidence of spinal deformities in A. fasciatus populations and CdLire phonétique that spinal deformities that contents in water and sediment of studied sites were very similar. An increase in spinal deformities rate induced by exposures of fish to Cd were reported by many authors [START_REF] Bervoets | Metal concentrations in water, sediment and gudgeon (Gobio gobio) from a pollution gradient: relationship with fish condition factor[END_REF][START_REF] Sassi | Influence of high temperature on cadmium-induced skeletal deformities in juvenile mosquitofish (Gambusia affinis)[END_REF]. Myoxocephalus quadricornis exposed for 1 year to an artificial heavy metalcontaining effluent displayed spinal curvature and increased frequency of vertebral deformities [START_REF] Bengtsson | Vertebral deformities and physiological effects in fourhorn sculpin (Myoxocephalus quadricornis) after long term exposure to a simulated heavy metal-containing effluent[END_REF].

A causal relationship between high environmental levels of heavy metals, especially Cd, and skeletal anomalies in fish has been suggested by [START_REF] Bengtsson | Vertebral defects in fourhorn sculpin, Myoxocephalus quadricornis L., exposed to heavy metal pollution in the Gulf of Bothnia[END_REF]. [START_REF] Buhringer | Spinal shortening (osteosclerosis) in spawners of the rainbow trout[END_REF] found highest Cd levels in the bones in rainbow trout, Oncorhynchus mykiss suffering from deformities compared to healthy individuals. Most of the evidence on Cd influence on induction of spinal deformities in fish is from laboratory studies. Resorption of Ca from the bone to compensate for disturbed Ca homeostasis, under Cd influence, is suggested as the main mechanism behind the observed deformities in these studies [START_REF] Bengtsson | Vertebral deformities and physiological effects in fourhorn sculpin (Myoxocephalus quadricornis) after long term exposure to a simulated heavy metal-containing effluent[END_REF][START_REF] Bervoets | Metal concentrations in water, sediment and gudgeon (Gobio gobio) from a pollution gradient: relationship with fish condition factor[END_REF][START_REF] Sassi | Influence of high temperature on cadmium-induced skeletal deformities in juvenile mosquitofish (Gambusia affinis)[END_REF]. [START_REF] Cheng | Cellular and molecular basis of cadmium-induced deformities in zebrafish embryos[END_REF] showed that the frequency of malformations in zebrafish embryos increased with Cd concentration in culture media and demonstrated a reduced myotome formation in Cd-induced spinal deformity. These authors suggested that fish embryonic malformations induced by Cd might be mediated through ectopic expression of developmental regulatory genes. PAHs comprise a large class of widespread environmental contaminants produced by both natural and anthropogenic sources. Due to their persistence and accumulation in soils and sediments, these compounds are now widely dispersed in the biosphere, notably in sediments of estuaries and coastal marine areas throughout the world. It was frequently reported that spinal deformities can be experimentally induced in fish upon exposure to different organic and inorganic substances, including metals, metalloids and organic substances like PAHs, organochlorine compounds OC and dioxins [START_REF] Yamauchi | Toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in developing red seabream (Pagrus major) embryo: an association of morphological deformities with AHR1, AHR2 and CYP1A expressions[END_REF][START_REF] Cachot | Characterization of toxic effects of sediment-associated organic pollutants using the lambda transgenic medaka[END_REF][START_REF] Miller | Effects of acute and subchronic exposures to waterborne selenite on the physiological stress response and oxidative stress indicators in juvenile rainbow trout[END_REF][START_REF] Holth | Differential gene expression and biomarkers in zebrafish (Danio rerio) following exposure to produced water components[END_REF][START_REF] Gagnon | Diuron increases spinal deformity in earlylifestage pink snapper Pagrus auratus[END_REF].

Given their high estrogenic potency and their high prevalence in the aquatic environment, the natural estrogens such as E1 (estrone), E2 (17 beta-estradiol), E3 (estriol) and the synthetic estrogens, are of particular concern in the risk assessment of endocrine disrupting compounds for wildlife [START_REF] Kramer | Reproductive impairment and induction of alkaline-labile phosphate, a biomarker of estrogen exposure, in fathead minnows (Pimephales promelas) exposed to waterborne 17b-estradiol[END_REF][START_REF] Bjerselius | Male goldfish reproductive behaviour and physiology are severely affected by exogenous exposure to 17b-estradiol[END_REF][START_REF] Van Den Belt | Reproductive effects of ethynylestradiol and 4t-octylphenol on the zebrafish[END_REF][START_REF] Lemly | Symptoms and implications of selenium toxicity in fish: the Belews Lake case example[END_REF]. Estrogens are potent chemicals of which the estrogenic effects can be observed in laboratory studies at 1 ng L À1 [START_REF] Routeledge | Some alkyl hydroxy benzoate preservatives (parabens) are estrogenic[END_REF]. In addition, length of the exposure period influenced the incidence of morphological abnormalities as abnormalities worsened as exposure progressed. This temporally cumulative effect was also observed in response to EE2 treatment, in which a 60-d exposure of Fundulus heteroclitus produced abnormalities at lower concentrations than a 25-d exposure [START_REF] Boudreau | Utility of morphological abnormalities during early-life development of the estuarine mummichog, Fundulus heteroclitus, as an indicator of estrogenic and antiestrogenic endocrine disruption[END_REF]. Although S3 and S4 are polluted and show high values of PAHs, and S4 shows an estrogenic activity up to 100%, the percentage of spinal deformities is low compared to S2. This confirms that estrogenic compounds or PAHs alone are not able to give a high rate of malformations in A. fasciatus.

The causes of the occurrence of spinal deformities in natural populations of fish are complicated and, to a large extent, unknown. They may be a response to a number of biotic factors and abiotic factors which are not related to pollution. Nevertheless, the observed association of the relatively high prevalence of spinal deformity in A. fasciatus and the important accumulation of heavy metals and PAHs in the industrialized coast of Sfax, seems to be very interesting and suggest that these mixture of chemical pollutants may represent a potential risk to induce spinal deformities in natural population of A. fasciatus. However, clear and confirmed association of skeletal abnormalities in A. fasciatus from the industrialized coast of Sfax with contaminants still requires additional research.

Fig. 1 .

 1 Fig. 1. Location of the sampling sites. S1: coast of Luza: S2: industrial coast of Sfax; S3: Khniss; S4: Hamdoun'Oued.

Fig. 2 .

 2 Fig. 2. Examples of skeletal anomalies in A. fasciatus. (a): Normal specimen, (b): deformed specimen, (c): normal vertebral column profile, (d): the black arrow points to absence of spine, the white arrow points to fused spines. (e): The black arrow points to bifurcated spine, the white arrow points to broken spine. (f): The black arrow points to short spine, the white arrows point to detachment of spines. (g): Abnormal shape of three consecutive spines, (h): fused vertebrae, (i): hypertrophied vertebrae, (j): the black arrow points to atrophied vertebrae, the white arrows point to a Hypertrophied arcs, (k): detached arcs and (l): prognathe.

Fig. 3 .

 3 Fig. 3. Cd levels and distribution of spinal deformities frequencies in A. fasciatus collected from studied sites.

Table 1

 1 Spatio-temporal distribution of spinal deformities (percentages) in A. fasciatus populations from studied sites.

	Year	2007			2008			2009			2010
	Sites	T	D	%	T	D	%	T	D	%	T	D	%
	S1	341	9	2.63	477	20	4.19	440	13	2.95	500	20	4
	S2	263	46	17.49 *	518	80	15.44 *	394	72	18.27 *	500	71	14.2 *
	S3	225	4	1.77	250	7	2.8	263	5	1.9	219	3	1.36
	S4	220	5	2.27	268	3	1.12	228	4	1.75	264	5	1.89

T: Total number of fish examined. D: Number of deformed fish. S1: Luza; S2: industrialized coast of Sfax; S3: Khniss and S4: Hamdoun river. * p < 0.0001: Significance from samples of the three other sites throughout the year.

Table 2

 2 Number (N) and frequencies (F) of normal and deformed A. fasciatus affected with different skeletal anomalies.

		Normal (n = 75)	Deformed (n = 77)
		N	F	N	F
	Anomalies of spines
	Fused spines	3	0.63	46	59.74 *
	Bifurcation	6	8	77	100 *
	Detachment	8	10.66	77	100 *
	Broken spines	0	0	39	50.64 *
	Absence of spines	0	0	20	25.97 *
	Short spines	0	0	19	24.67 *
	Abnormal shape	0	0	49	63.63 *
	Anomalies of vertebrae
	Fused vertebrae	4	5.33	77	100 *
	Atrophy	3	4	50	64.93 *
	Hypertrophy	2	2.66	65	84.41 *
	Anomalies of arcs
	Arc detachment	0	0	27	35.06
	Arc hypertrophy	0	0	15	19.48
	Prognathe	0	0	14	18.18
	n: Number of specimens examined

* p < 0.0001: Significance from normal fish.

Table 3

 3 Sites description.

	Parameter	S1	S2	S3	S4
	pH	8.5	8.4	8.5	8.4
	Temperature (°C)	20.3	21.4	24	29.6
	Conductivity (mS cm À1 )	54.5	54.6	56	56.3
	O 2 (ppm)	8.4	8.5	8.5	8.4
	Heavy metals
	Water (lg L À1 )
	Cd	0.03 ± 0.01	0.62 ± 0.02 *	0.02 ± 0.004	0.03 ± 0.01
	Cu	3.06 ± 1.54	15.29 ± 3.39 *	2.73 ± 0.79	2.43 ± 0.49
	Zn	12.28 ± 1.47	67,25 ± 7.96 *	12.85 ± 1.5	11.62 ± 1.78
	Heavy metals sediment
	(lg g À1 dry weight)
	Cd	0.05 ± 0.01	1.78 ± 0.15 *	0.04 ± 0.01	0.05 ± 0.01
	Cu	3.05 ± 1.19	36.84 ± 2.44 *	11.98 ± 2.06	4.74 ± 1.21
	Zn	32.00 ± 9.57	143.66 ± 31.08 *	20.35 ± 1.86	18.38 ± 3.00
	PAHs sediment
	(lg g À1 dry weight)
	Acenaphtylene	<DL	<DL	<DL	<DL
	Acenaphtene	<DL	<DL	<DL	<DL
	Naphthalene	<DL	0.12	0.07	0.06
	Phenanthrene	<DL	0.1	0.09	0.05
	Anthracene	<DL	<DL	<DL	<DL
	Fluoranthene	0.05	0.29	0.21	0.18
	Pyrene	0.05	0.28	0.22	0.25
	Benzo(a)anthracene	0.06	0.23	0.13	0.2
	Chrysene	<DL	0.25	0.09	0.15
	Benzo(b)fluoranthene	<DL	0.17	0.11	0.22
	Benzo(e)pyrene	<DL	0.09	0.07	0.09
	Benzo(a)pyrene	<DL	0.2	0.17	0.23
	Benzo(g,h,i)perylene	<DL	0.26	0.18	0.22
	Dibenzo[a,h]anthracene	<DL	<DL	<DL	<DL
	R PAHs	0.16	1.99	1.34	1.65

PAHs: total concentration of PAHs presents in each site; <DL: inferior to limit of detection; S1: Luza; S2: industrialized coast of Sfax; S3: Khniss and S4: Hamdoun river. * p < 0.0001 (Significance from samples of S1, S3 and S4).

Table 4

 4 Estrogenic

	Sites	Estrogenic activity (%)	Estrogenic compounds
		W	S M	W	S M
	S1	17.18	6.85	E2	E2, EE2
	S2	22.46	5.16	<LD or NF	E1, E3
	S3	20.39	6.68	<LD or NF	E2, EE2
	S4	60.5	99.65	E1	E2, E3

activities (%) and estrogenic compounds detected in water (W) and suspended mater (SM) of studied sites. NF: compound not found. <LD: inferior to limit of detection. (E1: estrone; E2: 17b-estradiol; E3: estriol; EE2: ethynilestradiol). S1: Luza; S2: industrialized coast of Sfax; S3: Khniss and S4: Hamdoun river.