Nathalie Kouta

Jacqueline Saliba

Nadia Saiyouri

Fracture behavior of flax fibers reinforced earth concrete

Keywords: Earth concrete, Flax fibers, Damage, Digital Image Correlation, Acoustic Emission technique

The mechanical behavior of earth concrete for sustainable construction has been studied in this paper. The effect of flax fibers on the fracture behavior has been monitored using digital image correlation (DIC) and acoustic emission (AE) techniques to better understand the fracture process and damage mechanism. Three-point bending tests have been conducted on prismatic specimens with different percentages (0%, 0.3% and 0.6%) and lengths (12 mm, 24 mm, 50 mm) of flax fibers. The AE activity and fracture energy increased with the percentage and length of fibers. Moreover, the AE signal parameters varied with the loading rate and different damage mechanisms have been distinguished. Crack opening resistance also increased due to the bridging effect of fibers along developed cracks. Those results show the benefits of adding flax fibers to enhance the ductility of earth concrete.

Introduction

The continuous increase of energy consumption, greenhouse gas emissions and natural resources depletion has created many problems that need to be addressed during the upcoming years [START_REF] Asdrubali | Sustainable Materials and Technologies A review of unconventional sustainable building insulation materials[END_REF].The construction field is responsible for 50% of the annual global carbon-dioxide emissions [START_REF] Dahlbo | Construction and demolition waste management -A holistic evaluation of environmental performance[END_REF]. Thus, the need for an alternative material that is eco-friendly and sustainable has become essential to reduce environmental impacts [START_REF] Pérez-Lombard | A review on buildings energy consumption information[END_REF][START_REF] Ramakrishna | Impact strength of a few natural fibre reinforced cement mortar slabs : a comparative study[END_REF][START_REF] Agarwal | Experimental investigation on chemically treated bamboo reinforced concrete beams and columns[END_REF][START_REF] Claramunt | Cellulosic fiber reinforced cement-based composites : A review of recent[END_REF][START_REF] Page | Multi-physical properties of a structural concrete incorporating short flax fibers[END_REF][START_REF] Llatas | A model for quantifying construction waste in projects according to the European waste list[END_REF]. The use of earth concrete can be an alternative to standard concrete in the construction of elements not requiring high mechanical performance. Earth concrete is constituted of soil stabilized with lime and cement and can be poured similar to classic concrete. It has a low energy consumption, especially during the production process, since it is constituted of local and natural raw material and presents good hygrothermal properties [START_REF] Van Damme | Earth concrete. Stabilization revisited[END_REF].

Natural fibers are widely used in earth construction. Using natural/vegetal fibers instead of artificial fibers can reduce the weight, cost and energy consumption of earth concrete [START_REF] Momoh | Behaviour of oil palm broom fibres ( OPBF ) reinforced concrete[END_REF].

Furthermore, these biodegradable and nontoxic fibers are known by their good thermal and acoustic insulation properties [START_REF] Momoh | Behaviour of oil palm broom fibres ( OPBF ) reinforced concrete[END_REF][START_REF] Benmansour | Thermal and mechanical performance of natural mortar reinforced with date palm fibers for use as insulating materials in building[END_REF][START_REF] Al-Oraimi | Mechanical characterisation and impact behaviour of concrete reinforced with natural fibres[END_REF][START_REF] Hill | A study of the potential of acetylation to improve the properties of plant fibres[END_REF]. The use of natural fibers can also improve the mechanical behavior and reduce the plastic shrinkage cracking of concrete. The presence of clay in earth concrete makes it sensitive to early age shrinkage cracking, hence adding fibers to the mixture can be beneficial [START_REF] Kouta | Effect of flax fibers on early age shrinkage and cracking of earth concrete[END_REF]. In addition, the brittle rupture and low mechanical performance of earth concrete subjected to tensile stress are the reasons behind the necessity of using fibers. The addition of fibers can lead to a higher tensile and flexural strength and significantly improve the ductility of concrete as the stress is transferred between the matrix and fibers during loading [START_REF] Sta | Manufacturing , fibre anisotropy and fracture of hybrid fibre concrete[END_REF]. The fracture process in fiber reinforced concrete presents different failure mechanisms such as the fracture of the mortar, the plastic deformation induced by the interface debonding between the matrix and the fibers due to fiber pull out and fiber rupture [START_REF] Pompo | Analysis of Steel Fibre Pull-Out from a Cement Matrix Using Video Photography[END_REF][START_REF] Pemberton | The fracture energy of metal fibre reinforced ceramic composites ( MFCs )[END_REF]. The fibers lead to bridge the crack sides and increase the crack opening resistance [START_REF] Sta | Manufacturing , fibre anisotropy and fracture of hybrid fibre concrete[END_REF][START_REF] Pompo | Analysis of Steel Fibre Pull-Out from a Cement Matrix Using Video Photography[END_REF]. However, one of the main disadvantages of using natural fibers is their degradation in alkaline environment due to the degradation of the main chemical component of these fibers, cellulose, by depolymerization known as the peeling off reaction [START_REF] Pavasars | Alkaline Degradation of Cellulose : Mechanisms and Kinetics[END_REF][START_REF] Dias | Durability of compression molded sisal fiber reinforced mortar laminates[END_REF]. Therefore, their mechanical properties and their effectiveness can be reduced [START_REF] Wei | Degradation mechanisms of natural fi ber in the matrix of cement composites[END_REF]. The comparison between different types of natural fibers and their impact on the mechanical behavior of earth concrete is difficult to quantify due to the large variety of natural fibers that could be sorted by type, properties, shape, length, strength, cohesion and water absorption in addition to their percentage and concrete mixtures [START_REF] Bouhicha | Performance of composite soil reinforced with barley straw[END_REF][START_REF] Ghavami | Behaviour of composite soil reinforced with natural fibres[END_REF][START_REF] Mohamed | Improvement of swelling clay properties using hay fibers[END_REF]. In this study, flax fibers have been used. Flax fibers are natural, ecological with no harmful effect on the environment even after their degradation and have an economic benefit compared to synthetic fibers. They are flexible due to the high length/diameter ratio and have good tensile strength and bond properties with concrete [START_REF] Page | Multi-physical properties of a structural concrete incorporating short flax fibers[END_REF].

The AE technique is a non-destructive technique widely used recently for damage monitoring [START_REF] Boniface | Evaluation of the acoustic emission 3D localisation accuracy for the mechanical damage monitoring in concrete[END_REF]. It offers a high sensitivity for damage detection and allows us to understand the fracture behavior of concrete [START_REF] Boniface | Evaluation of the acoustic emission 3D localisation accuracy for the mechanical damage monitoring in concrete[END_REF][START_REF] Dai | Damage investigation of single-edge notched beam tests with normal strength concrete and ultra high performance concrete specimens using acoustic emission techniques[END_REF][START_REF] Ohno | Fracture process zone in notched concrete beam under three-point bending by acoustic emission[END_REF][START_REF] Sutter | Fracture monitoring of lightweight composite-concrete beams[END_REF][START_REF] Ohno | Crack classification in concrete based on acoustic emission[END_REF][START_REF] Li | Development of fracture process zone in full-graded dam concrete under three-point bending by DIC and acoustic emission[END_REF][START_REF] Aggelis | Acoustic emission characterization of damage sources of lightweight hybrid concrete beams[END_REF]. It results from the creation of transient elastic waves due to the release of energy by different sources in the material as friction and micro-cracking. Elastic waves propagate from the source inside the material to the surface where they could be detected by piezoelectric sensors. The sensors record the response and convert the vibration into an electrical signal. The obtained signals are then amplified in order to be analyzed correctly. The AE parameters (amplitude, energy, average frequency…) can be correlated to the source, intensity and type of fracture [START_REF] Soulioti | Acoustic emission behavior of steel fibre reinforced concrete under bending[END_REF][START_REF] Saliba | Identification of damage mechanisms in concrete under high level creep by the acoustic emission technique[END_REF][START_REF] Saliba | Experimental and numerical analysis of crack evolution in concrete through acoustic emission technique and mesoscale modelling[END_REF]. The relationship between the average frequency (AF) and the RA value (the rise time over the maximum amplitude of the waveform) can be also used to classify the tensile and shear rupture modes [START_REF] Shiotani | Evaluation of long-term stability for rock slope by means of acoustic emission technique[END_REF][START_REF] Aggelis | Investigation of different fracture modes in cement-based materials by acoustic emission[END_REF]. The fracture behavior of fiber reinforced concrete using the AE technique have been evaluated in the literature [START_REF] Soulioti | Acoustic emission behavior of steel fibre reinforced concrete under bending[END_REF][START_REF] Rasheed | Fracture studies on synthetic fiber reinforced cellular concrete using acoustic emission technique[END_REF][START_REF] Aggelis | Influence of fiber chemical coating on the acoustic emission behavior of steel fiber reinforced concrete[END_REF][START_REF] Bhosale | A study on the efficiency of steel vs . synthetic vs . hybrid fibers on fracture behavior of concrete in flexure using acoustic emission[END_REF][START_REF] Xargay | Acoustic Emission behavior of thermally damaged Self-Compacting High Strength Fiber Reinforced Concrete[END_REF][START_REF] Kravchuk | Acoustic emission-based classification of energy dissipation mechanisms during fracture of fiber-reinforced ultra-high-performance concrete[END_REF][START_REF] Li | Effects of fiber type , volume fraction and aspect ratio on the flexural and acoustic emission behaviors of steel fiber reinforced concrete[END_REF][START_REF] Aggelis | Monitoring of the mechanical behavior of concrete with chemically treated steel fibers by acoustic emission[END_REF]. The results show that the addition of fibers leads to an increase of the AE activity [START_REF] Soulioti | Acoustic emission behavior of steel fibre reinforced concrete under bending[END_REF]. However, no previous investigation studied the effect of fibers on the mechanical behavior of earth concrete.

The DIC technique has been highly employed for crack monitoring due to its simplicity and high resolution [START_REF] Fayyad | Experimental investigation of crack propagation and crack branching in lightly reinforced concrete beams using digital image correlation[END_REF][START_REF] Ferreira | On the analysis of notched concrete beams: From measurement with digital image correlation to identification with boundary element method of a cohesive model[END_REF]. The displacement field of the specimen surface can be deduced due to the tracking of the speckle pattern applied on the surface. Based on the displacement field and using the finite element shape functions, the strain field can be calculated [START_REF] Zhao | Study on bending damage and failure of basalt fiber reinforced concrete under freeze-thaw cycles[END_REF][START_REF] Zhao | Experimental study on dynamic mechanical properties of the basalt fiber reinforced concrete after the freeze-thaw based on the digital image correlation method[END_REF][START_REF] Dong | An experimental study on crack propagation at rock-concrete interface using digital image correlation technique[END_REF]. This method has been also used in this study to investigate the cracking propagation [START_REF] Zhao | Experimental study on dynamic mechanical properties of the basalt fiber reinforced concrete after the freeze-thaw based on the digital image correlation method[END_REF][START_REF] Yasir | Fracture examination in concrete through combined digital image correlation and acoustic emission techniques[END_REF][START_REF] Wu | An experimental investigation on the FPZ properties in concrete using digital image correlation technique[END_REF][START_REF] Liu | Study on flexural behavior of carbon fibers reinforced coral concrete using digital image correlation[END_REF].

The main objective of this paper is to study the effect of different lengths and percentages of non-treated flax fibers on the fracture behavior of earth concrete during flexural tests. These tests were monitored in parallel with the DIC and the AE techniques. The materials and experimental techniques are first presented. Then, the AE and DIC results are exposed and the effect of fibers is analyzed.

Experimental program

2.1.Materials

Artificial soil has been used to overcome the discrepancies of natural soil. It is composed of 30% of clayey soil (which mineralogical composition is 75% of smectite, 15% of illite, 10% of Kaolinite) and 70% of sand. The clay is sourced from Lafaure and has a liquid and plastic limit of 84.6% and 29.2% respectively. The soil has been stabilized with two types of hydraulic binders: the natural hydraulic lime NHL5 in accordance with the European standard EN 459-1 and the cement CEM 1, 52.5 N PM-CP2 according to NF EN197-1 standard. The mixture composition of lime and cement is respectively 3% and 8% of the overall quantity of clay / sand mixture. These percentages have been set according to different studies concerning the effect of the percentage of stabilizer on the properties of earth concrete [START_REF] Bell | Lime stabilization of clay minerals and soils[END_REF][START_REF] Khattab | Long-term stability characteristics of a limetreated plastic soil[END_REF][START_REF] Anagnostopoulos | Strength properties of an epoxy resin and cement-stabilized silty clay soil[END_REF][START_REF] Ngo | Design of a soil concrete as a new building material -Effect of clay and hemp proportions[END_REF].

The mixture has been realized by first mixing clay and sand to ensure a certain homogeneity.

Then, lime and cement have been added to the dry mixture before the addition of water and superplasticizer (Tempo 10).

Natural flax fibers, cut at constant lengths of 12, 24 and 50 mm with diameters equal to 14.66 µm ±2.95, are used [START_REF] Page | Influence of different surface treatments on the water absorption capacity of flax fibres : Rheology of fresh reinforced-mortars and mechanical properties in the hardened state[END_REF]. These fibers are rich in cellulose and have good tensile strength properties [START_REF] Page | Multi-physical properties of a structural concrete incorporating short flax fibers[END_REF]. However, all natural fibers are hydrophilic which may affect the workability and the behavior of earth concrete [START_REF] Yan | Flax fibre and its composites -A review[END_REF][START_REF] Huner | Effect of Water Absorption on the Mechanical Properties \of Flax Fiber Reinforced Epoxy Composites[END_REF]. The coefficient of absorption of fibers is (105 ± 5%). Additional information about the constituents and mixtures properties at early age can be found in [START_REF] Kouta | Effect of flax fibers on early age shrinkage and cracking of earth concrete[END_REF].

2.2.Mixtures composition

Seven mixtures have been tested by varying the percentage (0; 0.3 and 0.6%) and length (12; 24 and 50 mm) of fibers. These mixtures are named following this method: [SA (%fibers) F (length)].

Table 1 presents the composition of these mixtures. The quantity of added fibers is subtracted from the artificial soil mass to maintain a constant solid mass. The effective water to binder ratio has been kept constant and equal to 0.45. Additional water has been added to account for the water absorbed by flax fibers.

Table 1. Mixtures composition

2.3.Three-point bending test

Flexural tests have been performed on prismatic specimens of 7 x 7 x 28 cm 3 (Figure 1).

Specimens have been removed from molds 24 hours after casting and stored at a relative humidity of 90% and a temperature of 20°C. Flexural tests have been conducted at the age of 28 days using an electromechanical machine with a capacity of 100 kN. The three-point bending test has been controlled with a constant loading displacement rate of 0.15 mm/min.

Digital image correlation technique

Flexural tests have been monitored with the DIC technique, which is a direct and simple method for fracture examination. This technique requires the surface preparation of earth concrete specimens prior to the test by using a white and black speckle (white paint at the bottom with small Components (kg/m black dots above). The displacement of the tested surface is obtained by the analysis of the random speckle at different times based on the pictures taken during the test. The photos of the specimen's surface have been captured using a digital camera with a resolution of 2452 × 2056 pixels. To improve the photo brightness, two lamps have been used in parallel. Images have been taken at a rate of one image per second during the test and stored directly. Image processing treatment has been realized using the commercial Vic 2D software with a resolution of 0.05 mm per pixel.

However, this technique does not give enough information about the microstructure damage as the obtained results remain at the surface of the specimen and cannot show damage volume evolution. Thus, the AE technique has been also used.

2.5.Acoustic emission technique

The used AE system consists of an eight channel AEWIN system with a general-purpose interface bus and a system for data storage analysis. A 3D localization algorithm has been used for the localization of AE events. 6 R15a piezoelectric sensors were used (Figure 2) with a frequency range between 50 and 200 kHz and a resonance frequency of 150 kHz. The sensors were placed on two opposite sides of the specimen using a thin layer of silicone grease expelled from air to ensure a good coupling between the specimen and the sensors. The transducers were placed around the cracking propagation area. The detected signals were amplified with a 40 dB gain differential amplifier. One of the main disadvantages of this technique is the lack of reproducibility due to the nature of the source signals (random cracks) and the presence of surrounding environment noise.

The detection threshold is set at 35 dB to avoid the background noise. The acquisition system was calibrated before each test using a pencil lead break procedure. The location accuracy is estimated to be ± 5 mm.

Results and discussion

Effect of the percentage and the length of fibers on the fracture behavior

Three specimens have been tested for each mixture under three-point bending test to evaluate the repeatability. Figure 3 presents the load versus the mid span deflection at the neutral axis for mixtures with three percentages and three lengths of flax fibers. The failure of earth concrete without fibers is brittle with an elastic phase followed by a high strength loss after reaching the peak indicating a fast propagation of the crack. For mixtures with fibers, an elastic phase has been also observed at the beginning. Then, the load drops after the peak due to the cracking of the matrix. This drop stops when the fibers, perpendicular to the crack path, are stretched and start to act in tension. This induces a resumption of stress and a nonlinear behavior is observed untill the second peak load. Note that, the load drop rate and strength recovery are highly dependent on the fibers distribution, orientation, length and quantity in earth concrete. The load recovery increases with the fiber's length and percentage. For SA03F50, SA06F24 and SA06F50, the strength drop is less important and specimens reached a second peak higher than the first one. This may be due to the efficiency of longer fibers to bridge the crack in comparison to the shorter ones that can be easily pulled out from earth concrete. Thus, long fibers allow the concrete to sustain higher loads and delay crack propagation in the specimen due to a higher anchor length. In addition, as the percentage of fibers increases, the probability of fibers presence at the crack level will increase which may also increase the fracture resistance due to a better stress redistribution and transfer between the matrix and the fibers.

In the softening part of specimens with flax fibers, the loss of stiffness is due to fibers being pull out from earth concrete and to micro cracking of concrete surrounding the initial crack and at the interface zone between the fibers and the matrix. This is responsible for the ductile failure and the high residual strength in the post peak region showing a good toughness and a post-crack resistance. In fact, the presence of flax fibers creates a bridge in the formed cracks and prevents any brittle rupture by redistributing the loads. where b is the thickness of the beam, h the height of the ligament, m is the weight of the specimen, l the span and Fmax the maximum strength.

The flexural strength and the deflection at the peak load increase with mixtures containing a higher percentage of fibers with longer length. In fact, earth concrete becomes less rigid with an additional nonlinear phase before the second peak due to stress redistribution between the fibers and the matrix. Moreover, longer fibers provide sufficient length for an adequate bonding surface with earth concrete matrix considering their position along the crack. A high variability has been observed for mixtures containing flax fibers with a length of 24 and 50 mm. This may be related to the random fiber distribution and orientation, to boundary effects, to the flexibility of flax fibers considering the shape and dimensions of earth concrete specimens, in addition to the possible segregation of fibers during mixing. Young's modulus (E) has been calculated using the load-deflection (Δ) curve according to the following formula:

! " $%& ' ∆)* +

Equation 3

The elastic modulus decreases slightly with the percentage of flax fibers; however, the fiber's length has no significant influence. The presence of fibers alters the pore distribution and total porosity of earth concrete as has been shown by mercury intrusion porosimetry, where the total porosity is equal to 35% for SA0F, 36% for the SA03F24 and 40% for the SA06F24 [START_REF] Kouta | Effect of flax fibers on early age shrinkage and cracking of earth concrete[END_REF]. The fracture energy is equal to 65 N.m -1 for SA0F and increases significantly with the percentage and length of flax fibers to reach a value of 770 N.m -1 for SA06F50 (approximately 10 times higher).

This indicates a higher energy absorption and a higher ductility due to different softening mechanisms induced by fibers.

Figure 5: Elastic modulus and fracture energy for specimens with different lengths and percentages of flax fibers

To have a better understanding of the effect of fibers on the fracture process, the AE activity and the strain fields have been also analyzed for different loading levels.

3.2.AE activity analysis

Figure 6 presents the correlation between the load, amplitude and cumulative number of AE hits during the flexural tests for SA0F, SA03F24 and SA06F24. For SA0F, the AE activity is very low in the elastic phase. The detected signals may be due to the crushing of earth concrete at the contact with the press. The rate of the AE activity increases quickly at the peak with signals of high amplitudes due to the unstable propagation of the macro crack.

For mixtures with 0.3% and 0.6% of fibers, the evolution of the cumulative number of AE hits in correlation with the loading level can be sorted into four phases. During the elastic phase, the acoustic activity is low. Then, from the first peak and till the nonlinear phase of the second peak, the AE activity begins to increase slightly indicating the development of micro cracking. The generated signals have low amplitude and energy. The rate of the AE activity increases quickly after the peak indicating the propagation of a macro-crack. In the last phase, the rate of the AE activity decreases indicating a stable propagation of the crack. The signals generated during last phase present higher energy and amplitude. This may be due to additional damage mechanisms such as crack bridging, tearing and friction at the fiber/matrix interface due to fibers pullout [START_REF] Saliba | Monitoring of steel-concrete bond with the acoustic emission technique[END_REF]. Note that, the cumulative number of AE hits increases with the percentage of fibers.

Figure 6: Correlation between the load, the amplitude and the cumulative number of AE hits for SA0F (a), SA03F24 (b) and SA06F24 (c)

The effect of the fiber's length on the AE activity has been also evaluated. Figure 7 presents the correlation between the load and the cumulative number of AE hits for mixtures with 0.3% and 0.6% of different flax fiber lengths. The same phases distinguished before are observed. However, the total number of AE hits and the rate of AE activity increase with the fiber's length. This may be due to a larger fracture process zone (FPZ) as longer fibers may transfer load to a further distance which is also responsible for the higher fracture energy.

Figure 7: Correlation between the load and the cumulative number of AE hits for (a) SA03F and (b) SA06F with different fiber lengths

The total number of AE hits and the generated absolute AE energy (Abs. energy) for the seven mixtures increase with the fiber's percentage and length (Figure 8) which may be due to additional damage mechanisms, a larger FPZ and a greater energy absorption that limits crack propagation [START_REF] Aymerich | Effect of reinforcing wool fibres on fracture and energy absorption properties of an earthen material[END_REF]. Note that, a higher variability has been also observed for specimens with fibers of higher length.

Figure 9: Correlation between the AE energy and the fracture energy for specimens with different lengths and percentages of flax fibers

In order to better evaluate the dimension of the FPZ, the AE localization maps and the strain fields have been also analyzed.

Correlation between AE and DIC results

A three-dimensional analysis is performed for the location of AE events representing the damage sources. The 3D localization algorithm is based on the difference between the arrival times at each transducer, the effective velocity, the geometry and the placement of sensors. For an AE event to be located in 3D, at least 4 waves must be detected.

Figure 10 presents the localization maps of AE events projected in 2D (m) and the 2D strain fields (mm/mm) along the X direction for SA0F, SA03F24 and SA06F24 at different loading levels. Four ranges of energy have been considered for the classification of AE events according to the rate of damage [START_REF] Muralidhara | Fracture process zone size and true fracture energy of concrete using acoustic emission[END_REF].

For SA0F, no AE events and no cracks have been detected before the peak. After the peak, the strain field shows a quick propagation of the crack till the rupture. However, only few AE events have been detected due to the brittle rupture of earth concrete. The detected AE events are due to the rupture of earth concrete matrix and present a good correlation with the position of the macro crack according to strain fields.

For mixtures with 0.3% and 0.6% of fibers, the crack initiated at the load drop. The DIC results

show a quick propagation of a macro crack. However, the number of the detected AE events is very low due to the brittle rupture. The number of AE events started to increase slowly after the first peak in the nonlinear phase when the load was recovered by the presence of fibers and till the second peak. AE events are mainly located at the bottom of the beam where the stress is the most significant. The crack length increased slowly in the post peak region due to load transfer between the matrix and flax fibers indicating good adhesion. The number of AE events increases with the loading level along the height of the beam indicating a continuous bond through the macro crack which is responsible for the slow crack propagation. This bond or fibers bridging prevented the brittle rupture of earth concrete.

The number of AE events increases with the percentage of flax fibers and reached 800 and 1000 events at 30% of the maximum strength in the post peak region for SA03F24 and SA06F24

respectively. This can be linked to the increase of the loaded contact points between flax fibers and earth concrete matrix.

Figure 10: AE events localization maps and strain fields for SA0F, SA03F24 and SA06F24

The same analysis has been conducted to study the effect of fiber's length on the FPZ. Figure 11 presents the 2D localization maps of AE events classified by their energy level and the strain fields for mixtures with 0.3% and 0.6% of fibers considering the three lengths at 30% of the maximum strength in the post peak region. For short fibers, AE events with high energy (the red points) are mainly located at the core of the FPZ. As the length of fibers increases, the width of AE events localization zone increases. In addition, AE events of higher energy are not only present near the crack but also along the width of the FPZ due to friction between flax fibers and earth concrete. For SA06F50, the AE localization map showed an important concentration of energetic events in the middle of the specimen. This may be related to the segregation of flax fibers at this level as can be seen also with the strain field. Those energetic events can be associated to friction during the pull out of fibers as it has been shown in [START_REF] Saliba | Monitoring of steel-concrete bond with the acoustic emission technique[END_REF]. The stress concentration and the higher density of AE events at this level may be due to fiber segregation as can be seen at the fractured surfaces (figure 12). Figure 13 presents the recorded number of AE events according to their energy for mixtures with different percentages of flax fibers. For SA0F, the generated AE events presented low energy.

For specimens with fibers, the number of energetic events increased with the loading level and the percentage of flax fibers indicating the advantage of using fibers for a better fracture resistance and energy absorption.

3.4.Effect of the fibers percentage and length on the fracture process zone

The evolution of the crack opening along the height of the specimens has been also analysed at different loading levels (Figure 14). It was obtained from the displacement across both sides of the crack [START_REF] Yasir | Fracture examination in concrete through combined digital image correlation and acoustic emission techniques[END_REF]. For SA0F, the crack opening started to increase after the peak and varied almost linearly throughout the height of the specimen causing its brittle rupture. The crack developed quickly along the height and reached an opening equal to 0.175 mm in its largest point at the bottom of the specimen.

For SA03F24, the crack started to propagate at the load drop where it reached a length of 40 mm and increased slowly in the nonlinear phase to reach 52 mm at the second peak. After the second peak, the length of the crack continued to slightly increase to reach 64 mm along the height of the specimen with a high crack opening. For SA06F24, the crack also initiated at the load drop and reached a length of 20 mm. The length of the crack continued to increase reaching 33 mm at the second highest peak and 60 mm at 30% of the maximum strength in the post peak region. In the post peak region, the crack opening increased significantly due mainly to flax fibers pull out from earth concrete. The crack opening increased with the loading level and the percentage of fibers reached 3 and 4.3 mm at the bottom of the beam for SA03F24 and SA06F24 respectively at 30% of the maximum load in the post peak region. Thus, the crack length at the load drop decreased with the percentage of fibers while the deformation capacity and the crack propagation resistance increased.

3.5.Damage source identification by AE analysis

The shape of AE signals can be used to define the crack mode [START_REF] Aggelis | Classification of cracking mode in concrete by acoustic emission parameters[END_REF]. RA-AF association diagram has been widely used to determine the shear and tensile failure modes [START_REF] Ohno | Crack classification in concrete based on acoustic emission[END_REF][START_REF] Zhang | AE monitoring of reinforced concrete squat wall subjected to cyclic loading with information entropy-based analysis[END_REF][START_REF] Xu | Experimental research on bond behavior of reinforced recycled aggregate concrete based on the acoustic emission technique[END_REF]. The RA parameter expressed in µs/V is defined as the ration between the rise time and amplitude. The AF in kHz is defined as the number of counts divided by the duration of the signal. The RA value of shear cracks failure is higher than the one obtained during tensile crack failure while the average frequency is lower [START_REF] Bhosale | A study on the efficiency of steel vs . synthetic vs . hybrid fibers on fracture behavior of concrete in flexure using acoustic emission[END_REF][START_REF] Chen | Experimental investigation of concrete fracture behavior with different loading rates based on acoustic emission[END_REF][START_REF] Ranjbar | Fracture evaluation of multilayered precast reinforced geopolymer-concrete composite beams by incorporating acoustic emission into mechanical analysis[END_REF].

Conclusions and perspectives

An experimental program has been conducted in this paper in order to study the effect of the percentage and length of flax fibers on the fracture behavior of earth concrete. Flexural tests have been monitored in parallel with the DIC and the AE techniques. Based on the obtained results, the following conclusions can be listed:

• Earth concrete presents a brittle failure and a low deformation capacity. The addition of flax fibers allows to enhance the ductility of earth concrete due to load redistribution and bridging effect of fibers.

•

The fracture properties as the tensile strength, the deflection at the peak and the fracture energy increased with the percentage and the length of flax fibers.

•

The cumulative number of AE hits increased with the percentage and the length of flax fibers due to additional damage mechanisms and a higher FPZ width.

•

The DIC validated the results obtained with the AE technique concerning the crack location and allowed to measure the crack opening that increased with the percentage of fibers.

• The AF-RA curves have been used to identify the shear and tensile failure modes during rupture. Shear cracks increased with fibers addition due to friction during fibers pull out.

An additional study will be realized in the future in order to better classify the various damage mechanisms based on a multivariable statistical analysis and evaluate the durability of the reinforced earth concrete.

Figure 1 :

 1 Figure 1: Flexural test setup

Figure 2 :

 2 Figure 2: 3D AE transducers position (black sensors-face 1, grey sensors-face 2)

Figure 3 :Figure 4

 34 Figure 3: Load -mid span deflection curves for SA0F (a) SA03F and (b) SA06F with different flax

Figure 4 : 5 . 2 where

 452 Figure 4: Tensile strength and deflection at the peak for specimens with different lengths and

Figure 8 :Figure 9

 89 Figure 8: Total number of AE hits and generated acoustic energy for specimens with different

Figure 11 :Figure 12 :

 1112 Figure 11: 2D localization maps of AE events and strain fields for mixtures with 0.3% and 0.6% of

Figure 13 :

 13 Figure 13: Number of AE events according to their energies for (a) SA0F, (b) SA03F24 and (c)

Figure 14 :Figure 15

 1415 Figure 14: Crack opening profile along the height of SA0F (a), SA03F24 (b) and SA06F24 (c) for

Figure 15 :Figure 16

 1516 Figure 15: Distribution of AE events in function of X for (a) SA0F, (b) SA03F24 and (c) SA06F24

Figure 16 :

 16 Figure 16: Fracture process zone width for mixtures with different percentages and lengths of flax

Figure 17

 17 Figure 17 presents the evolution of RA and AF during the loading process for SA0F,

Figure 17 :

 17 Figure 17: Evolution of AF and RA in correlation with the load for (a) SA0F, (b) SA03F24 and (c)

Figure 18

 18 Figure 18 presents the variation of RA-AF for SA0F, SA03F24 and SA06F24. The

Figure 18 :

 18 Figure 18: Evolution of AE in function of RA for (a) SA0F, (b) SA03F24 and (c) SA06F24.


M Sebastiano

W Jouanneau

P Blévin

Frédéric Angelier

Charline Parenteau

J Gernigon

J C Lemesle

F Robin

P Pardon

H Budzinski

High levels of fluoroalkyl substances and potential disruption of thyroid hormones in three gull species from South Western France

Introduction

Per-and Poly-fluoroalkyl substances (PFAS) raised increasing concerns over the past years due to their persistence and global distribution. Because of their high thermal and chemical stability, these synthetic substances have found an application in the manufacturing industry, mostly used as surfactants and additives [START_REF] Buck | Perfluoroalkyl and polyfluoroalkyl substances in the environment: terminology, classification, and origins[END_REF], and have been widely produced over the past 50 years [START_REF] Wang | A Never-Ending Story of Per-and Polyfluoroalkyl Substances (PFASs)?[END_REF]. Being extremely persistent in the environment, and due to their longrange transport via atmospheric and oceanic currents, they have been detected worldwide [START_REF] Giesy | Global distribution of perfluorooctane sulfonate in wildlife[END_REF]. Several studies have found PFAS to accumulate into living organisms (including invertebrates, fishes, amphibians, mammals, and birds) and to biomagnify through food webs [START_REF] Kannan | Perfluorinated compounds in aquatic organisms at various trophic levels in a Great Lakes food chain[END_REF][START_REF] Kelly | Perfluoroalkyl Contaminants in an Arctic Marine Food Web: Trophic Magnification and Wildlife Exposure[END_REF][START_REF] Simonnet-Laprade | Biomagnification of perfluoroalkyl acids (PFAAs) in the food web of an urban river: assessment of the trophic transfer of targeted and unknown precursors and implications[END_REF], and to date, PFAS exposure represents a global threat to human health and wildlife [START_REF] Sunderland | A review of the pathways of human exposure to poly-and perfluoroalkyl substances (PFASs) and present understanding of health effects[END_REF]. Documenting their occurrence in the environment and understanding their disruptive effect on the physiology of humans and wildlife remains a major challenge.

Seabirds are long-lived apex predators generally exposed to high levels of environmental contaminants [START_REF] Elliott | Tracking Marine Pollution[END_REF][START_REF] Furness | Seabirds as monitors of the marine environment[END_REF], thus they prove particularly valuable to investigate PFAS accumulation in marine food webs especially in northern areas. High levels of PFOS have been found in plasma samples of several seabird species from the Arctic including ivory gulls Pagophila eburnea (average concentration of 31 ng/g, [START_REF] Lucia | Contamination of ivory gulls (Pagophila eburnea) at four colonies in Svalbard in relation to their trophic behaviour[END_REF]; glaucous gulls Larus hyperboreus (average concentration of 47 ng/g, [START_REF] Melnes | Dissimilar effects of organohalogenated compounds on thyroid hormones in glaucous gulls[END_REF] and of 134 ng/g, [START_REF] Verreault | Perfluorinated Alkyl Substances in Plasma, Liver, Brain, and Eggs of Glaucous Gulls (Larus hyperboreus) from the Norwegian Arctic[END_REF]; in black-legged kittiwakes from Svalbard (average concentration of 10.2 ng/g, [START_REF] Tartu | Endocrine and Fitness Correlates of Long-Chain Perfluorinated Carboxylates Exposure in Arctic Breeding Black-Legged Kittiwakes[END_REF]; and in European shags Phalacrocorax aristotelis from Isle of May in Scotland (average concentration of 251 ng/g in females and 163 ng/g in males, [START_REF] Carravieri | Interactions between Environmental Contaminants and Gastrointestinal Parasites: Novel Insights from an Integrative Approach in a Marine Predator[END_REF]. High levels of PFOS were also found in egg samples of the European shag and common eider Somateria mollissima from Norway (average concentration of 36.8 ng/g and 37.4 ng/g, respectively, [START_REF] Herzke | Perfluorinated and other persistent halogenated organic compounds in European shag (Phalacrocorax aristotelis) and common eider (Somateria mollissima) from Norway: A suburban to remote pollutant gradient[END_REF]; in whole blood of the endangered lesser black backed gull Larus fuscus from Norway (average concentration of 33.5 ng/g, Bustnes et al., 2008a); and in several other seabird species. However, much work has been devoted to seabirds from the Arctic regions (i.e. considered a sink for environmental contaminants; [START_REF] Barrie | Arctic Contaminants: Sources, Occurrence and Pathways[END_REF][START_REF] Braune | A geographical comparison of chlorinated, brominated and fluorinated compounds in seabirds breeding in the eastern Canadian Arctic[END_REF][START_REF] Wong | Assessing temporal trends and source regions of per-and polyfluoroalkyl substances (PFASs) in air under the Arctic Monitoring and Assessment Programme (AMAP)[END_REF], or in highly contaminated areas (e.g. China; [START_REF] Xie | Industrial source identification and emission estimation of perfluorooctane sulfonate in China[END_REF], while fewer studies have focused on areas with not-known sources of PFAS (i.e. Antarctica, Munoz et al., 2017b). In France, most studies examining PFAS occurrence and exposure in aquatic ecosystems focused on water, sediments, invertebrates, and fishes [START_REF] Couderc | Occurrence of POPs and other persistent organic contaminants in the European eel (Anguilla anguilla) from the Loire estuary, France[END_REF][START_REF] Fernandes | Occurrence and spatial distribution of chemical contaminants in edible fish species collected from UK and proximate marine waters[END_REF][START_REF] Munoz | Temporal variations of perfluoroalkyl substances partitioning between surface water, suspended sediment, and biota in a macrotidal estuary[END_REF][START_REF] Simonnet-Laprade | Biomagnification of perfluoroalkyl acids (PFAAs) in the food web of an urban river: assessment of the trophic transfer of targeted and unknown precursors and implications[END_REF]. However, to the extent of our knowledge, no studies have been carried out on top predators including birds in this area, which may be exposed to concentrations of concern. It is therefore crucial to investigate PFAS exposure in French seabirds to document PFAS occurrence in marine biota and to provide early warning of its effects on their health status.

Over the past few years, there has been an increased body of evidence showing that PFAS may i) impact on adipogenesis thus with body condition [START_REF] Tartu | Endocrine and Fitness Correlates of Long-Chain Perfluorinated Carboxylates Exposure in Arctic Breeding Black-Legged Kittiwakes[END_REF], and ii) disrupt several physiological traits of seabirds. For instance, previous work found that PFAS exposure is associated with lower levels of the stress hormone corticosterone [START_REF] Tartu | Endocrine and Fitness Correlates of Long-Chain Perfluorinated Carboxylates Exposure in Arctic Breeding Black-Legged Kittiwakes[END_REF], higher oxidative stress [START_REF] Costantini | Higher plasma oxidative damage and lower plasma antioxidant defences in an Arctic seabird exposed to longer perfluoroalkyl acids[END_REF], longer telomeres (Blévin et al., 2017a;[START_REF] Sebastiano | Exposure to PFAS is associated with telomere length dynamics and demographic responses of an arctic top predator[END_REF], and a higher metabolic rate (Blévin et al., 2017b). Further studies found PFAS to be associated with higher levels of the parental hormone prolactin and altered incubation behaviours [START_REF] Blévin | Contaminants, prolactin and parental care in an Arctic seabird: Contrasted associations of perfluoroalkyl substances and organochlorine compounds with egg-turning behavior[END_REF], lower hatching success [START_REF] Tartu | Endocrine and Fitness Correlates of Long-Chain Perfluorinated Carboxylates Exposure in Arctic Breeding Black-Legged Kittiwakes[END_REF], and a higher survival rate [START_REF] Sebastiano | Exposure to PFAS is associated with telomere length dynamics and demographic responses of an arctic top predator[END_REF]. Specifically, one way through which PFAS may impact on organism function is by disrupting hormonal mechanisms. Previous work provided evidence that PFAS have a strong affinity for proteins and are known to bind to the thyroid hormone transport protein transthyretin [START_REF] Ren | Binding interactions of perfluoroalkyl substances with thyroid hormone transport proteins and potential toxicological implications[END_REF][START_REF] Weiss | Competitive Binding of Poly-and Perfluorinated Compounds to the Thyroid Hormone Transport Protein Transthyretin[END_REF]. In birds, the hypothalamic-pituitary-thyroid (HPT) axis controls the secretion of the thyroid hormone thyroxine (T4), which is then converted to triiodothyronine (T3), the active form of T4 [START_REF] Mcnabb | The Hypothalamic-Pituitary-Thyroid (HPT) Axis in Birds and Its Role in Bird Development and Reproduction[END_REF]. Although Blévin et al. (2017b) found no association between PFAS exposure and thyroid hormones in adult black-legged kittiwakes Rissa tridactyla, [START_REF] Braune | Biomarker responses associated with halogenated organic contaminants in northern fulmars (Fulmarus glacialis) breeding in the Canadian Arctic[END_REF] found a significant positive correlation between total triiodothyronine (TT3) levels and hepatic concentrations of PFAS in northern fulmars. [START_REF] Nøst | Halogenated organic contaminants and their correlations with circulating thyroid hormones in developing Arctic seabirds[END_REF] also found a positive association between PFAS levels and total thyroxine (TT4) in black-legged kittiwake and northern fulmar chicks, suggesting that PFAS may potentially act through an endocrine disrupting mechanism. More recently, [START_REF] Melnes | Dissimilar effects of organohalogenated compounds on thyroid hormones in glaucous gulls[END_REF] found that PFAS were positively associated with free triiodothyronine (FT3) in the glaucous gull. To date, further work is needed to understand the relationship between PFAS exposure and thyroid functioning in birds, especially considering that in birds, T3 and T4 are involved in a multitude of physiological pathways [START_REF] Mcnabb | The Hypothalamic-Pituitary-Thyroid (HPT) Axis in Birds and Its Role in Bird Development and Reproduction[END_REF]. A disruption of thyroid hormone levels may be detrimental to development, behaviour, and reproduction [START_REF] Mcnabb | The Hypothalamic-Pituitary-Thyroid (HPT) Axis in Birds and Its Role in Bird Development and Reproduction[END_REF].

The Lilleau des Niges Natural Reserve is an important site for breeding, wintering, and migration of several bird species. It is located north of Ile de Ré, an island off the west coast of France, in front of La Rochelle, in the Bay of Biscay. By hosting several seabird species during the breeding season, this island offers a unique opportunity to investigate the occurrence of PFAS in a French seabird community. Although most previous studies have been carried out on a single species (Blévin et al., 2017b;[START_REF] Costantini | Higher plasma oxidative damage and lower plasma antioxidant defences in an Arctic seabird exposed to longer perfluoroalkyl acids[END_REF][START_REF] Melnes | Dissimilar effects of organohalogenated compounds on thyroid hormones in glaucous gulls[END_REF][START_REF] Tartu | Endocrine and Fitness Correlates of Long-Chain Perfluorinated Carboxylates Exposure in Arctic Breeding Black-Legged Kittiwakes[END_REF], investigating several species simultaneously and from the same geographical area can help to better understand the mechanisms of exposure to PFAS and the potential physiological consequences of PFAS contamination. For instance, the Herring gull Larus argentatus, the lesser black-backed gull Larus fuscus graellsii, and the great black-backed gull Larus marinus, which breed sympatrically on the island, are characterized by different foraging and migratory strategies, thus potentially exposed to different concentrations of PFAS. The aims of this study were to i) assess to which extent French seabirds are contaminated by PFAS; ii) investigate the relationship between exposure to PFAS and body condition; and iii) determine the association between exposure to PFAS and plasma thyroid hormone T3 concentration in the three above mentioned seabirds from Ile de Ré. Data on PFAS occurrence and their potential adverse effects in seabirds from France are not yet available. To date, we are not aware of known point sources of PFAS in the region. But considering that diverse important rivers may discharge PFAS near the study area [START_REF] Simonnet-Laprade | Biomagnification of perfluoroalkyl acids (PFAAs) in the food web of an urban river: assessment of the trophic transfer of targeted and unknown precursors and implications[END_REF], and that PFAS may reach and accumulate in remote areas due to their long-range oceanic and atmospheric transport [START_REF] Munoz | Temporal variations of perfluoroalkyl substances partitioning between surface water, suspended sediment, and biota in a macrotidal estuary[END_REF], we expect comparable PFAS concentrations with seabirds from the Arctic. In addition, if PFAS have a stimulating effect on thyroid hormone production as found in previous work [START_REF] Dewitt | Toxicological Effects of Perfluoroalkyl and Polyfluoroalkyl Substances[END_REF][START_REF] Liu | The thyroid-disrupting effects of long-term perfluorononanoate exposure on zebrafish (Danio rerio)[END_REF][START_REF] Nøst | Halogenated organic contaminants and their correlations with circulating thyroid hormones in developing Arctic seabirds[END_REF], we expect a positive association between PFAS and the concentration of thyroid hormones. Furthermore, although some PFAS are listed as POPs by the Stockholm Convention and their production has subsequently been reduced over the past years, studies investigating temporal trends of PFAS in tissues of wildlife are limited. Although our data have been solely collected over three years of study, we further aim to describe the temporal variation in blood concentration of PFAS from 2016 to 2018 in local seabirds.

Materials and Methods

Sampling

Field work was performed in 2016, 2017, and 2018 at the Lilleau des Niges Natural Reserve (46° 13' 53" N, -1° 30' 22" W), managed by the Ligue pour la Protection des Oiseaux (LPO) located on the North side of Ile de Ré, France, as a part of a monitoring program for PFAS in the region. A total of 108 breeding adult birds from three species were captured during the incubation stage on their nests using a nest trap. Because out of the 108 observations, three were coming from the same individuals sampled at different years, one or the other observation was randomly excluded to perform statistical analyses. Therefore, the final dataset included a total of 105 birds (European herring gull, n=9 in 2016(European herring gull, n=9 in , n=16 in 2017(European herring gull, n=9 in , and n=9 in 2018;;lesser black-backed gull, n=11 in 2016, n=17 in 2017, and n=16 in 2018;great black-backed gull, n=9 in 2016, n=7 in 2017, and n=11 in 2018). After capture, 2mL of blood was collected from the alar vein using a heparinized syringe and a 25 gauge needle. Blood was kept in a cold container and centrifuged for 10 min at 8,000 x g at 20 °C at the laboratory within a few hours after collection; plasma and red blood cells were kept frozen at -20 °C until laboratory analyses. Skull and tarsus were measured with an accuracy of 0.1 mm using a caliper. Wing length was also measured with an accuracy of 1 mm using a ruler, and birds were weighted to the nearest 5 g using a Pesola spring balance. Birds were sexed from red blood cells by polymerase chain reaction amplification (PCR) of part of two highly conserved genes (CHD) of sexual chromosomes. Briefly, DNA was extracted from erythrocytes and the sex was determined by molecular sexing based on polymerase chain reaction (PCR) amplification of the CHD gene as described in [START_REF] Fridolfsson | A Simple and Universal Method for Molecular Sexing of Non-Ratite Birds[END_REF]. Amplification was performed in 20µl final volume with a Eppendorf Mastercycler using 0.5 U Taq DNA polymerase, 200µM dNTPs, 10mM Tris-HCl pH 8.3, 50mM KCl, 1.5mM MgCl2 and 0.4µM of primers 2550F (5'-GTTACTGATTCGTCTACGAGA-3') and 2718R (5'-ATTGAAATGATCCAGTGCTTG-3'). Female birds may deposit a significant amount of PFAS into their eggs. Therefore, to minimize the variation due to PFAS deposition in eggs, we have only sampled individuals having either two (17/105, 16% of birds) or three eggs (87/105, 83% of birds) except one sampled females that only laid one egg (1/105, 1%). Preliminary statistical analyses were carried out to test whether females with two (8/52, 15% of females) or three eggs (43/52, 83%) had different concentrations of PFAS. However, linear models showed that for any PFAS, concentrations were similar between females that laid two or three eggs (all t<1.14, all P>0.26), thus clutch size was not further included in the statistical analyses.

PFAS analyses

A total of 14 PFAS were analysed in each plasma sample, including eight carboxylates: branched-(Br-PFOA) and linear-perfluorooctanoate (L-PFOA), perfluorononanoate (PFNA), perfluorodecanoate (PFDA), perfluoroundecanoate (PFUnDA), perfluorododecanoate (PFDoDA), perfluorotridecanoate (PFTrDA), perfluorotetradecanoate (PFTeDA); and six sulfonates: perfluorohexanesulfonate (PFHxS), branched-(Br-PFHpS) and linear-perfluoroheptasulfonate (L-PFHpS), branched perfluoroctanesulfonate (Br-PFOS), linear perfluoroctanesulfonate (L-PFOS), and perfluorooctanesulfonamide (FOSA). Analytical standards of native PFAS along with a series of 10 13 C, 18 O or D mass-labelled internal standards used for quantification purposes were supplied by Wellington laboratories. All reagents were analytical grade or equivalent (see Munoz et al. (2017b) for full details). Briefly, in a 2 mL polypropylene Eppendorf tubes, a 25 µL aliquot of plasma was weighed (∼25 mg) and internal standards (ISs) were subsequently added under gravimetric control (∼15 mg of a 1 pg/µL IS mixture prepared in methanol). Following protein precipitation with 100 µL of acetonitrile (ACN), extracts were centrifuged for 10 min at 24,000 x g at 20 °C. The supernatant was then transferred to 2 mL polypropylene centrifuge tubes (0.22 µm nylon filter).

After centrifugation for 3 min at 7,000 x g at 20 °C, extracts were transferred to 2 mL auto sampler glass vials and diluted with 675 µL of HPLC-water. Extracts were briefly vortexed and then processed using an Agilent Technologies (Massy, France) on-line SPE platform which comprises a standard auto sampler (1260 Infinity ALS), a quaternary pump (1260 Infinity Quaternary Pump VL), a switch valve (1200 2 Position/6 Port Valve) and an on-line SPE column support (1200 6 Position Selection Valve), which were all automatically controlled via the Acquisition module of the Agilent Mass Hunter software as previously done (Munoz et al., 2017b). HPLC-water aliquots were run between each seabird plasma sample to eliminate any cross-contamination. Note that on-line extraction was performed with Waters Oasis HLB on-Line SPE columns (2 × 10 mm, dp= 25-35 µm) while analyte separation was carried out using an Agilent C18 Poroshell analytical column (2.1 × 100 mm, 2.7 µm).

Quality assurance/Quality control (QA/QC)

When analytes were detected in blanks, blank correction was performed and a limit of reporting (LOR) was defined as three times the maximum blank signal divided by the average mass of plasma used for analysis. A limit of detection (LOD) was also defined as the concentration yielding a signal to noise ratio of 3 in spiked plasma samples. Because laboratory analyses were performed in different years, a unique left-censoring threshold was set for each analyte, i.e. the maximum between LORs and LODs, all years combined. For those PFAS with concentrations below this threshold in less than 30% of samples, left-censored data were arbitrarily replaced with ½ x LOR or LOD to enable statistical analyses. Therefore, ten PFAS (PFNA, PFDA, PFUnDA, PFDoDA, PFTrDA, PFTeDA, PFHxS, L-PFHpS, Br-PFOS, and L-PFOS) could be further investigated (i.e. other analytes were excluded from statistical analyses). LORs, LODs and detection frequencies are presented in the supplementary information (Table S1). For each sample batch (20 samples), several QA/QC points were assessed by analyzing: i) two procedural blanks consisting of 25 µL of HPLC-water that went through the entire analytical procedure; ii) one human serum standard reference material (NIST SRM 1957, trueness assessment); iii) replicate spiked chicken plasma samples (target analytes added jointly with mass-labelled ISs at the beginning of the preparation procedure at 2 ng/g each, accuracy assessment); and iv) HPLC-water samples spiked at 2 ng/g, accuracy assessment) as previously described (Munoz et al. 2017b). Procedural blanks showed very limited contamination. The analysis of NIST SRM 1957 gave satisfactory results, i.e. within the specified uncertainty interval. For those compounds with a reference concentration (i.e. PFHpA, PFOA, PFNA, PFDA, PFUnDA, PFHxS and PFOS), levels deviated between 2 and 20% from the reference concentration (except for FOSA, which deviated 36%).

Thyroid hormone analyses

TT3 was determined by radioimmunoassay. Briefly, 25 µL of plasma was incubated for 24h at 4 °C with a known concentration (10000 cpm) of T3 marked with the radioisotope Iodine-125 (T3-125 I, Perkin Elmer, US, reference: NEX110X100UC) and an antibody Ab (polyclonal rabbit antiserum, Sigma-Aldrich, US, reference: T-2777). Because Ab is available in a limited concentration, T3 and T3-125 I compete for Ab, to which they bind. Therefore, after incubation, there is a bound fraction (T3 and T3-125 I bound to Ab) and a free fraction (T3 and T3-125 I unbound to Ab), which are separated by adding a sheep anti-rabbit antibody (whole anti-serum anti rabbit IgG produced in sheep), incubated for 12h at 4 °C followed by centrifugation at 4,300 x g at 18-20°C for 45 min. The bound fraction is then counted with a wizard 2 gamma counter (Perkin Elmer, US). Pooled plasma of diverse gull samples were serially diluted and produced a dose-response curve parallel to the T3 standard curve. The lowest TT3 detectable concentration was 0.07 ng/ml (LOD). Samples below this limit (n=3) were replaced with a value equal to ½ x LOD to enable statistical analyses. All samples were run in duplicates. Samples that had a coefficient of variation above 15% and could not be done in triplicates due to low plasma volume were not included in statistical analyses (n=11). An additional measurement of TT3 was excluded from statistical analyses since it was considered an outlier (the measurement exceeded the mean ± 3 times the standard deviation and was highly influential in statistical analyses). Therefore, for a total of 58 samples (n=6 in 2016 and n=12 in 2017 for the European herring gull; n=10 in 2016 and n=14 in 2017 in lesser black-backed gull; n=8 in 2016 and n=8 in 2017 in great black-backed gull), both TT3 and PFAS data were available. The intra-assay coefficient of variation was 9.73%, while the inter-assay coefficient of variation amounted to 15.13%.

Statistical analyses

After PFAS data were log-transformed to reduce the influence of extreme values (see below), linear models were used to investigate differences in PFAS concentrations among species and between genders (in samples collected from 2016 to 2018). In each model, each PFAS was considered as a dependent variable while the factors Species, Sex, and their interaction were considered as predictors. To simultaneously investigate species specific temporal trends in PFAS exposure from 2016 to 2018, the Year, the factor Species, and their interaction were also included in the model as explanatory variables. We used a similar model to test the difference in body condition between sexes. Briefly, the body condition has been calculated using the body mass adjusted by a linear body measurement (i.e. skull length) using the formula described in [START_REF] Peig | New perspectives for estimating body condition from mass/length data: the scaled mass index as an alternative method[END_REF].

Linear models were additionally used to study the association between TT3 and PFAS (in samples collected in 2016 and 2017). In these models, a three-way interaction between PFAS, the factors Species and Sex, was used to investigate sex-related responses to PFAS exposure. These models additionally included the Year (as a factor, to control for the temporal variation in TT3 and PFAS), and body condition (as a covariate, to control for the individual condition of birds). A similar model was built to test the association between the body condition and PFAS in all samples collected from 2016 to 2018.

All PFAS concentrations (except PFTeDA, which was normally distributed and assumptions listed below were respected without data transformation), were log-transformed when testing for time trends and when testing for inter-species and between sex differences. All PFAS concentrations were log-transformed when testing for the association between either TT3 or body condition and PFAS. Data transformation was done to meet model assumptions as homoscedasticity and normality of residuals, further confirmed by visually inspecting Q-Q plots. All data transformation and violation of models' assumptions are reported throughout the manuscript. Statistical significance was set to α=0.05 and 95% confidence intervals were used during data processing and data visualization. All statistical analyses were performed using R version 3.5.2.

Results

PFAS used in statistical analyses were detected in all samples (Table S1) and their concentrations are summarized in Table 1 and Figure 1. PFOS was the most abundant, followed by the odd-chain carboxylates PFTrDA and PFUnDA. Linear models showed statistically significant differences among species for all carboxylic and sulfonic PFAS (all F>5.15, all P<0.01), and all statistical outputs and post-hoc differences can be found in Table S2. Among carboxylates, PFNA levels were higher in herring gulls and lesser black-backed gulls than great black-backed gulls (both P<0.05, Figure 1), PFDA levels were higher in great black-backed gulls than herring gulls (P<0.01, Figure 1), PFUnA, PFDoDA, PFTrDA, and PFTeDA levels were higher in great and lesser black-backed gulls than herring gulls (all P<0.01, Figure 1), and PFTrDA levels were also higher in great than lesser blackbacked gulls (P<0.05, Figure 1). Among sulfonic acids, PFHxS was higher in both lesser blackbacked and herring gulls than great black-backed gulls (both P<0.01, Figure 1), PFHpS and L-PFOS were highest in lesser black-backed gulls (all P<0.001, Figure 1), and Br-PFOS was higher in lesser than great black-backed gulls (P<0.05, Figure 1).

All carboxylates showed significantly higher concentrations in males than in females for all species (all P<0.05, except for PFNA in the herring gull, for which P=0.07; Table 1). Among sulfonates, PFHxS showed similar concentrations between females and males in all species (all P>0.16, Table 1), while PFHpS, Br-and L-PFOS showed significantly higher concentrations in males than females (all P<0.05, Table 1). In all three species, there was no difference in the body condition between males and females (all t<1.27, all P>0.80). Finally, TT3 levels were similar between sexes in all three species (t=2.76, P=0.08).

In great black backed gulls, TT3 was positively associated with PFUnDA, PFDoDA, PFTrDA, PFTeDA and Br-PFOS in females (all t> 2.10, all P≤0.04; Figure 2a-d, 2f, Table S3), while TT3 was negatively associated with PFHxS in males (t=-2.69, P=0.01; Figure 2e, Table S3). There was no association between TT3 and any PFAS in herring gulls and lesser black-backed gulls (all t≤0.79, all P≥0.43, Table S3).

In great black backed gull females, increasing levels of PFNA, PFDA, PFHxS, PFHpS, Br-and L-PFOS were associated with a reduced body condition (all t≤-2.19, all p≤0.03; Figure S1, S2, Table S4), while increasing levels of PFNA and PFDA were associated with a reduced body condition (both t≤-2.18, both p=0.03; Figure S1, Table S4) in lesser black-backed gull males, but not females.

In great black backed gulls, there was a significant or marginally-significant increase in all PFAS from 2016 to 2018 (all t≥1.93 and all P≤0.056; Figure 3 and4, Table S5), while lesser black backed gulls showed an increase in PFTeDA and PFHxS, and a marginally significant decrease in PFUnDA levels from 2016 to 2018 (all t≥1.98, all P≤0.05; Figure 3, 4, Table S5). All PFAS showed similar concentrations among years in the herring gulls except for PFTrDA, which increased from 2016 to 2018 (t=2.30, P=0.02; Figure 3, Table S5). Body condition did not change in any of the species from one year to another (all t<1.14, all P>0.26). TT3 levels remained similar between 2016 and 2017 in all three species (all t<1.88, all P>0.42).

Discussion

Our study is the first to provide evidence that although not-known point sources of emission are present in the region, several PFAS were detected in seabird species from South Western France.

Great and lesser black-backed gulls show that both plasma carboxylate and sulfonate concentrations are comparable to highly contaminated seabird species from Arctic regions, while herring gulls are exposed to relatively lower levels of PFAS. We found that PFAS are negatively associated with the body condition of the birds. Furthermore, TT3 levels were associated with several PFAS in a contrasted manner between sexes in the great black-backed gull, suggesting a potential disrupting mechanism of PFAS exposure. Finally, the great black-backed gulls documented an increasing trend of plasma PFAS concentration from 2016 to 2018.

Our results show that among carboxylates, PFUnDA and PFTrDA are the most abundant congener in all three species, a pattern that is commonly found in seabird species (Bustnes et al., 2008b;[START_REF] Melnes | Dissimilar effects of organohalogenated compounds on thyroid hormones in glaucous gulls[END_REF][START_REF] Tartu | Endocrine and Fitness Correlates of Long-Chain Perfluorinated Carboxylates Exposure in Arctic Breeding Black-Legged Kittiwakes[END_REF]. Because of the strong winds and oceanic currents that characterize the Atlantic Ocean, and considering that, to the best of our knowledge, there are no point sources of PFAS in the region, perfluorinated compounds should occur at a lower concentration than in the Mediterranean and the Arctic regions, considered as sinks for pollutants [START_REF] Danovaro | Pollution threats in the Mediterranean Sea: An overview[END_REF][START_REF] Wong | Assessing temporal trends and source regions of per-and polyfluoroalkyl substances (PFASs) in air under the Arctic Monitoring and Assessment Programme (AMAP)[END_REF]. However, the levels of carboxylates found in this study (ranging from a median of 0.6 ng/g of PFTeDA in Herring gulls to 5.8 ng/g of PFTrDA in great blackbacked gulls) are similar to those reported for glaucous gulls (ranging from a median of 0.1 ng/g of PFOA to 3.8 ng/g of PFUnDA, [START_REF] Melnes | Dissimilar effects of organohalogenated compounds on thyroid hormones in glaucous gulls[END_REF] and lesser black-backed gulls (ranging from a median of 0.2 ng/g of PFDoDA to 5.9 ng/g of PFDA, Bustnes et al., 2008a) from Arctic regions, but lower than those found in other species (e.g. in black-legged kittiwakes from Svalbard; ranging from a mean of 1.0 ng/g of PFNA to 18.2 ng/g of PFTrDA, [START_REF] Tartu | Endocrine and Fitness Correlates of Long-Chain Perfluorinated Carboxylates Exposure in Arctic Breeding Black-Legged Kittiwakes[END_REF]. One possible explanation may be related to the continental input of PFAS through the Gironde, Loire, and Charente estuaries [START_REF] Munoz | Temporal variations of perfluoroalkyl substances partitioning between surface water, suspended sediment, and biota in a macrotidal estuary[END_REF]Munoz et al., 2017a;[START_REF] Munschy | Perfluoroalkyl substances (PFASs) in the marine environment: Spatial distribution and temporal profile shifts in shellfish from French coasts[END_REF], which may have contributed to the observed concentrations. Except for PFNA (which levels were higher in herring gulls and lesser black-backed gulls than great black-backed gulls), most carboxylates were higher in lesser and great black backed gulls than herring gulls. Although we cannot exclude that these three species differ in their ability to excrete PFAS from their body, our results suggest that the differences in exposure likely depend on the trophic niche occupied by the species. For instance, a recent study on PFAS in six seabird species from the Arctic regions showed that predatory birds (e.g. Great skua Stercorarius skua, 44.8 ng/g of sumPFAS) showed the highest contaminant load compared with species from a lower trophic level (e.g. Common eider Somateria mollissima, 1.3 ng/g of sumPFAS; [START_REF] Haarr | DNA damage in Arctic seabirds: Baseline, sensitivity to a genotoxic stressor, and association with organohalogen contaminants[END_REF]. Great black-backed gulls feed on higher trophic level preys and mainly forage along the shore [START_REF] Maynard | Inter-colony and interspecific differences in the isotopic niche of two sympatric gull species in Newfoundland[END_REF], while lesser black-backed and herring gulls are known to have a generalist diet which also includes food items from both terrestrial and marine origin [START_REF] Corman | Varying foraging patterns in response to competition? A multicolony approach in a generalist seabird[END_REF][START_REF] Maynard | Inter-colony and interspecific differences in the isotopic niche of two sympatric gull species in Newfoundland[END_REF].

Among sulfonates, L-PFOS was the most abundant, followed by Br-PFOS, PFHxS, and PFHpS and all occurred at very high concentrations. For instance, PFHxS ranged from a median value of 1.2 ng/g in great black-backed gulls to 2.2 ng/g in lesser black-backed gulls, while other studies from highly contaminated areas reported lower PFHxS plasma concentrations (a median below 1 ng/g in lesser black-backed gulls, Bustnes et al., 2008b; a median below 0.7 in glaucous gulls, [START_REF] Melnes | Dissimilar effects of organohalogenated compounds on thyroid hormones in glaucous gulls[END_REF] all samples below 0.2 ng/g in black-legged kittiwakes, [START_REF] Tartu | Endocrine and Fitness Correlates of Long-Chain Perfluorinated Carboxylates Exposure in Arctic Breeding Black-Legged Kittiwakes[END_REF]. Furthermore, L-PFOS ranged from a median of 11.6 ng/g in herring gull females to a median of 54.7 ng/g in lesser black-backed gull males. Thus, lesser black-backed gulls in our study showed very high sulfonate levels, even higher than Norwegian populations (a median of 40 ng/g of PFOS in males, Bustnes et al., 2008b), and in this species, sulfonate levels are significantly higher than those observed in great black-backed gulls. In addition, plasma concentrations of most PFAS showed significantly higher levels in males than in females, but this difference was not related to the body condition of the birds, with males showing a similar body condition than females.

Because females transfer contaminants in the eggs, it is thus possible that females have lower levels of circulating PFAS in plasma. However, previous work pointed out contrasting results between PFAS in eggs and plasma, suggesting that the extent of PFAS transfer to the eggs may significantly vary among the studied species (Bustnes et al., 2008a;[START_REF] Herzke | Perfluorinated and other persistent halogenated organic compounds in European shag (Phalacrocorax aristotelis) and common eider (Somateria mollissima) from Norway: A suburban to remote pollutant gradient[END_REF][START_REF] Verreault | Perfluorinated Alkyl Substances in Plasma, Liver, Brain, and Eggs of Glaucous Gulls (Larus hyperboreus) from the Norwegian Arctic[END_REF]. Given that in this study we did not analyse PFAS levels in eggs, it is not possible to clarify whether the species differ in terms of PFAS excreted in eggs. Although [START_REF] Verreault | Maternal transfer of organohalogen contaminants and metabolites to eggs of Arctic-breeding glaucous gulls[END_REF] found that the contaminant content in glaucous gull eggs fluctuated irrespectively of the laying order, other work suggests that the majority of PFAS are found in the first or the first two eggs, while negligible concentrations of PFAS are found in the third egg, as previously shown in Audouin's gulls Larus audouinii [START_REF] Vicente | Perfluoroalkyl and polyfluoroalkyl substances in entire clutches of Audouin's gulls from the ebro delta[END_REF]. Our results showed that females that laid three eggs had similar PFAS concentrations than females that laid two eggs, therefore our results should not be affected by the difference in PFAS deposition in eggs.

Because of the higher bioaccumulative properties and biomagnification and of longer-chain PFAS [START_REF] Boisvert | Bioaccumulation and biomagnification of perfluoroalkyl acids and precursors in East Greenland polar bears and their ringed seal prey[END_REF][START_REF] Simonnet-Laprade | Biomagnification of perfluoroalkyl acids (PFAAs) in the food web of an urban river: assessment of the trophic transfer of targeted and unknown precursors and implications[END_REF], these compounds tend to occur at higher concentrations in wildlife tissues [START_REF] Conder | Are PFCAs Bioaccumulative? A Critical Review and Comparison with Regulatory Criteria and Persistent Lipophilic Compounds[END_REF][START_REF] Muir | Levels and trends of poly-and perfluoroalkyl substances in the Arctic environment -An update[END_REF][START_REF] Muir | Trends of legacy and new persistent organic pollutants in the circumpolar arctic: Overview, conclusions, and recommendations[END_REF]. Therefore, individuals feeding at a higher trophic position are likely to be exposed to higher concentrations of long-chained carboxylates. Previous work also showed that longer chained PFAS are more likely to induce adverse health effects in seabirds compared to shorter chained PFAS. For instance, negative associations between PFAS and baseline corticosterone in black-legged kittiwakes were only found for PFTrDA and PFTeDA [START_REF] Tartu | Endocrine and Fitness Correlates of Long-Chain Perfluorinated Carboxylates Exposure in Arctic Breeding Black-Legged Kittiwakes[END_REF], while such association was not found for shorter-chain PFAS. Additional work on the same bird population found higher protein oxidative damage in those birds having higher concentrations of PFDoDA, PFTrDA and PFTeDA [START_REF] Costantini | Higher plasma oxidative damage and lower plasma antioxidant defences in an Arctic seabird exposed to longer perfluoroalkyl acids[END_REF]. Similarly, previous work found a positive association between PFTrDA and metabolic rate in the same species (Blévin et al., 2017b). These results were further corroborated by experimental work on rat Rattus sp. cell cultures, showing that the cytotoxicity of PFAS increases with increasing carbon chain length [START_REF] Berntsen | Time-dependent effects of perfluorinated compounds on viability in cerebellar granule neurons: Dependence on carbon chain length and functional group attached[END_REF], and that comparing molecules with a similar chain length, a sulfonate functional group may lead to greater toxicity than a carboxyl group [START_REF] Berntsen | Time-dependent effects of perfluorinated compounds on viability in cerebellar granule neurons: Dependence on carbon chain length and functional group attached[END_REF].

Our results are thus of particular interest as they suggest that birds feeding at a higher trophic position and showing a more marine diet (i.e. great black-backed gulls) should also be exposed to greater toxicological risks from carboxylates, while birds with a more generalist diet (i.e. lesser black-backed gulls) can be exposed to higher levels of sulfonates. This is likely the reason why we found associations between TT3 and PFAS only in great black-backed gulls, while no significant associations have been found in herring gulls and lesser black-backed gulls.

Interestingly, our results were dependent on the sex of the birds. Indeed, although we found that PFHxS was associated with TT3 in great black-backed gull males, all other associations were found in females only. Despite the lower concentration of circulating plasma PFAS levels, females can deposit PFAS in eggs therefore we cannot be certain that females were exposed to lower PFAS concentrations than males, and further work including other tissues (e.g. liver or muscle) would clarify whether PFAS intake differs between the sexes of sampled birds. Despite the absolute concentrations to which they are exposed, a possible explanation for the results in females may rely on the fact that incubation can be extremely costly for female birds [START_REF] Hanssen | Cost of reproduction in a long-lived bird: incubation effort reduces immune function and future reproduction[END_REF], thus they may be more susceptible to PFAS exposure. Previous work in birds suggest a modulation of thyroid function induced by exposure to various environmental contaminants. [START_REF] Smits | Thyroid hormone suppression and cell-mediated immunomodulation in American kestrels (Falco sparverius) exposed to PCBs[END_REF] reported decreased TT3 levels in American kestrels Falco sparverius experimentally exposed to PCBs, while [START_REF] Verreault | Effects of orgonochlorine contaminants on thyroid hormone levels in arctic breeding glaucous gulls, Larus hyperboreus[END_REF] reported a decrease in T4:T3 ratio in the glaucous gull. Similarly, exposure to organochlorines was associated with reduced TT3 in kittiwakes (Blévin et al., 2017b), and with reduced T3 and T4 in glaucous gulls [START_REF] Melnes | Dissimilar effects of organohalogenated compounds on thyroid hormones in glaucous gulls[END_REF][START_REF] Verreault | Effects of orgonochlorine contaminants on thyroid hormone levels in arctic breeding glaucous gulls, Larus hyperboreus[END_REF]. However, specifically related to PFAS, further work on seabirds found a positive association with thyroid functioning (i.e. between PFOS and FT3 in glaucous gulls, [START_REF] Melnes | Dissimilar effects of organohalogenated compounds on thyroid hormones in glaucous gulls[END_REF] between several PFAS and TT4 in black-legged kittiwakes and northern fulmars, [START_REF] Nøst | Halogenated organic contaminants and their correlations with circulating thyroid hormones in developing Arctic seabirds[END_REF].

Although being conducted in fish, an experimental approach showed that exposure to PFOS in zebra fish (Danio rerio) led to increased thyroid hormones secretion [START_REF] Liu | The thyroid-disrupting effects of long-term perfluorononanoate exposure on zebrafish (Danio rerio)[END_REF]. Thus, our results on great black backed gulls are in line with previous studies. In this study, increasing TT3 levels in this species were found with increasing concentrations of longer chain PFAS (PFUnDA, PFDoDA, PFTrDA, and PFTeDA) and Br-PFOS. This suggests that despite carboxylates and sulfonates are functionally different, their effect on TT3 is similar. However, this was not the case for PFHxS, which showed a decrease in TT3 levels with increasing concentrations. PFHxS is highly toxic and causes thyroid disruption by lowering thyroid hormone levels in rats [START_REF] Ramhøj | Evaluating thyroid hormone disruption: investigations of long-term neurodevelopmental effects in rats after perinatal exposure to perfluorohexane sulfonate (PFHxS)[END_REF], but it remains unclear why this effect was only found in great black-backed gull males. Indeed, females showed similar levels than males, and the other species exhibited higher PFHxS concentrations than those found in great black backed gulls, thus this result would strongly benefit from experimental support. Because the avian thyroid gland secretes almost exclusively T4 [START_REF] Darras | Thyroid hormone deiodination in birds[END_REF], most T3 is derived from the deiodination of T4 [START_REF] Darras | Thyroid hormone deiodination in birds[END_REF]. A possible explanation is that in great black-backed gulls, exposure to PFHxS may negatively impact either the transport of T4 (by reducing the activity of serum binding proteins) or deiodination processes. It is therefore strongly warranted to supplement in vitro experiments to verify the effect of PFHxS on T4 transformation. Our results do not provide evidence for a causal relationship PFAS exposure and circulating thyroid hormones. But the contrasting results found between sexes strongly call for further work to experimentally investigate the effect of PFAS exposure on thyroid functioning of birds.

Sex-related differences were also found while investigating the relationship between exposure to PFAS and the birds' body condition. In female great black-backed gulls, we found that increasing concentrations of PFNA, PFDA, and all four sulfonates were negatively associated with body condition, while in lesser black-backed gulls, a similar negative relationship between PFNA, PFDA, and body condition was only found in males. Previous work in humans and various animal models found that exposure to certain PFAS is suspected to disrupt fatty acids metabolism and promote adipogenesis [START_REF] Cheng | Chronic perfluorooctane sulfonate (PFOS) exposure induces hepatic steatosis in zebrafish[END_REF][START_REF] Wan | PFOS-induced hepatic steatosis, the mechanistic actions on β-oxidation and lipid transport[END_REF][START_REF] Xu | PFOS induces adipogenesis and glucose uptake in association with activation of Nrf2 signaling pathway[END_REF][START_REF] Yeung | Differential expression of chicken hepatic genes responsive to PFOA and PFOS[END_REF].

More specifically, these changes in lipid content are related to the capacity of PFAS to alter the expression of genes involved in the metabolism of lipids and fatty acids [START_REF] Jacobsen | Effects of perfluorooctane sulfonate on genes controlling hepatic fatty acid metabolism in livers of chicken embryos[END_REF][START_REF] Wan | PFOS-induced hepatic steatosis, the mechanistic actions on β-oxidation and lipid transport[END_REF]. To date, work on the effect of PFAS on lipid metabolism and body condition in birds remain extremely limited. The negative association we found is in contrast with a previous study on black-legged kittiwakes, showing that PFNA was positively associated with body condition in males [START_REF] Tartu | Endocrine and Fitness Correlates of Long-Chain Perfluorinated Carboxylates Exposure in Arctic Breeding Black-Legged Kittiwakes[END_REF]. One possible explanation for the negative relationship found in females may be related to the ability to deposit PFAS into the eggs. For instance, great blackbacked gull females in a better body condition may be more efficient in eliminating PFAS through egg-deposition, although evidences to support this statement are lacking. However, this would not explain why in a closely related species (i.e. lesser black-backed gull) a similar association has been found in males. Our study results are novel but emphasize the need to experimentally investigate the potential association between exposure to PFAS and body condition in birds.

Finally, not only does our work provide evidence of high PFAS levels in seabirds from metropolitan France, but our results clearly suggest increasing blood concentration of most PFAS over a relatively short period of time (i.e. from 2016 to 2018) in great and lesser black-backed gulls. A previous study showed that birds caught later on over the breeding season had lower concentration of PFAS (Bustnes et al., 2008a). However, all birds included in this study were sampled during the same period of the year (difference of a few days from one year to another), and, more importantly, all birds were sampled while incubating eggs, thus during the same reproductive state, which should not affect the results. Additionally, we cannot exclude that we unintentionally captured older birds in more recent years (assuming that PFAS levels increase with age in these species). However, considering that all individuals included in this study were adults, and assuming that PFAS concentrations in birds reach a steady level relatively early in life as previously shown for organochlorines [START_REF] Bustnes | Age and accumulation of persistent organochlorines: A study of arctic-breeding glaucous gulls (Larus hyperboreus)[END_REF], these trends should not be affected by the age of the bird. Information on temporal trends of PFAS in birds' tissues in recent years are scarce and do not exhibit any overall trend [START_REF] Jouanneau | Spatiotemporal Analysis of Perfluoroalkyl Substances in White-Tailed Eagle (Haliaeetus albicilla) Nestlings from Northern Norway-A Ten-Year Study[END_REF][START_REF] Land | What is the effect of phasing out long-chain per-and polyfluoroalkyl substances on the concentrations of perfluoroalkyl acids and their precursors in the environment? A systematic review[END_REF][START_REF] Muir | Levels and trends of poly-and perfluoroalkyl substances in the Arctic environment -An update[END_REF][START_REF] Sun | White-Tailed Eagle (Haliaeetus albicilla) Body Feathers Document Spatiotemporal Trends of Perfluoroalkyl Substances in the Northern Environment[END_REF]. Thus, although only three years of data could be included in the present study, our results provide valuable information on PFAS trends in South western France. Further work including several years of study is strongly warranted to corroborate these findings.

Conclusions

Our study provides the first evidence of the presence of high levels of PFAS in seabirds from South western France. Despite some PFAS showed similar levels of other seabird species, L-PFOS and some other PFAS showed either comparable or higher levels than highly contaminated seabird species, and may therefore pose a threat to long-lived seabirds. This hypothesis is further corroborated by our results showing an association between PFAS and the level of the thyroid hormone TT3. Similarly, we provide evidence that PFAS may interfere with lipid accumulation and body condition in birds, and we call for further work to experimentally test this hypothesis. Work on species-specific mechanisms of contaminant excretion and susceptibility to PFAS exposure would prove useful to understand the consequences of PFAS exposure in different species. Our results also document an increase in blood PFAS concentrations over time, particularly in great black-backed gulls, suggesting that PFAS concentrations may also be increasing in the investigated species. Because PFAS have detrimental effects on birds, these and other seabird populations should be monitored as an increase of PFAS exposure may impact on population viability both in the short-and long-term. 13.45 ± 1.52 10.53 (5.18, 25.92) 46.55 ± 13.74 27.62 (16.71, 194.72) <0.001

Figure 1 :

 1 Figure 1: Plasma concentrations of PFAS (expressed as ng/g of ww) in the three seabird species from Ile de Re. Statistically significant differences are indicated by the asterisk; *, **, ***, indicate a P-value <0.05, <0.01, and <0.001, respectively.

Figure 2 :

 2 Figure 2: Relationship between the concentration of the thyroid hormone TT3 (expressed as ng/mL) and log-transformed carboxylic (C-11 to C-14, panel a to d, respectively), and sulfonic PFAS (PFHxS and Br-PFOS, panel e and f, respectively) of the three seabird species from Ile de Re. The solid line represents the trend while the grey area represents 95% confidence intervals. Shapes of data points and three different grades of grey are used to distinguish the three species as explained in the figure legend. Only significant trends are shown. Data refer to the period 2016-2017 for which both TT3 and PFAS were available (n=58).

Figure 3 :

 3 Figure 3: Error bar plots mean ± standard error of carboxylate concentrations in 2016, 2017, and 2018 in the seabird species from Ile de Re. The shapes and the three different grades of grey are used to distinguish the three species as explained in the figure legend. Only significant trends are shown. Dashed lines indicate a trend close to significance.

Figure 4 :

 4 Figure 4: Error bar plots mean ± standard error of sulfonate concentrations in 2016, 2017, and 2018 in the seabird species from Ile de Re. The shapes and the three different grades of grey are used to distinguish the three species as explained in the figure legend. Only significant trends are shown. Dashed lines indicate a trend close to significance.

Table 1 :

 1 PFAS concentration (ng/g of ww) in females and males of the three seabird species from Ile de Re. P-values refer to the difference in PFAS between females and males. Significant P-values are bolded. SE refers to standard errors.

			Herring gull Larus argentatus
		Females (n=18)	Males (n=16)
		mean ± SE	median (range)	mean ± SE	median (range)	P-value
	PFNA	1.16 ± 0.11	1.06 (0.36, 2.16)	1.68 ± 0.17	1.63 (0.67, 2.85)	0.07
	PFDA	1.16 ± 0.19	0.94 (0.38, 4.01)	1.98 ± 0.30	1.79 (0.69, 6.04)	0.01
	PFUnDA	1.23 ± 0.17	1.01 (0.47, 3.45)	2.43 ± 0.34	2.17 (0.61, 6.49)	<0.001
	PFDoDA	0.95 ± 0.16	0.9 (0.3, 3.22)	1.82 ± 0.32	1.56 (0.55, 6.17)	<0.001
	PFTrDA	1.76 ± 0.27	1.42 (0.55, 5.25)	3.03 ± 0.29	3.05 (1.04, 4.81)	<0.001
	PFTeDA	0.61 ± 0.07	0.61 (0.25, 1.23)	1.23 ± 0.16	1.03 (0.33, 2.8)	0.04
	PFHxS	2.26 ± 0.21	2.16 (0.88, 3.5)	2.29 ± 0.36	1.89 (0.79, 6.92)	0.99
	PFHpS	0.27 ± 0.02	0.25 (0.11, 0.5)	0.49 ± 0.09	0.38 (0.11, 1.63)	0.045
	Br-PFOS	2.03 ± 0.34	1.71 (0.86, 6.8)	3.95 ± 0.45	3.75 (1.07, 7.9)	<0.001
	L-PFOS	13.82 ± 2.0	11.64 (5.57, 37.04)	30.44 ± 4.31	28.44 (6.6, 62.52)	<0.001

Lesser black-backed gull Larus fuscus graellsii

		Females (n=20)	Males (n=24)
	PFNA	1.14 ± 0.09	1.11 (0.46, 1.83)	2.04 ± 0.1	2.06 (0.95, 3.36)	<0.001
	PFDA	1.1 ± 0.08	1.15 (0.54, 1.64)	2.63 ± 0.15	2.58 (1.17, 4.16)	<0.001
	PFUnDA	1.88 ± 0.16	1.69 (0.66, 3.28)	5 ± 0.29	4.89 (1.98, 8.7)	<0.001
	PFDoDA	1.02 ± 0.07	1.02 (0.39, 1.87)	3.06 ± 0.2	2.86 (1.75, 5.52)	<0.001
	PFTrDA	1.97 ± 0.12	2.06 (0.66, 2.91)	6.38 ± 0.44	5.74 (3.53, 11.66)	<0.001
	PFTeDA	0.66 ± 0.07	0.61 (0.13, 1.39)	2.23 ± 0.2	1.87 (1.1, 4.35)	<0.001
	PFHxS	2.18 ± 0.25	1.95 (1.12, 6.01)	2.56 ± 0.26	2.24 (1.08, 5.56)	0.94
	PFHpS	0.37 ± 0.03	0.35 (0.16, 0.82)	0.79 ± 0.07	0.69 (0.34, 1.84)	<0.001
	Br-PFOS	1.86 ± 0.19	1.96 (0.58, 3.15)	5.06 ± 0.5	4.41 (2.38, 13.67)	<0.001
	L-PFOS	21.09 ± 2.06	22.76 (5.74, 34.49)	60.23 ± 4.44	54.68 (26.51, 119.69)	<0.001

Great black-backed gull Larus marinus Females (n=14) Males (n=13)

	PFNA	0.59 ± 0.06	0.59 (0.24, 0.95)	1.94 ± 0.29	1.72 (0.89, 4.85)	<0.001
	PFDA	1.41 ± 0.23	1.13 (0.32, 3.55)	3.83 ± 0.58	3.34 (1.52, 7.2)	<0.001
	PFUnDA	2.42 ± 0.35	2.23 (0.49, 4.93)	4.67 ± 0.65	4.08 (2.62, 11.46)	0.01
	PFDoDA	1.69 ± 0.24	1.63 (0.32, 3.12)	2.79 ± 0.29	2.37 (1.68, 5)	0.04
	PFTrDA	4.04 ± 0.72	3.55 (0.68, 9.61)	6.87 ± 0.83	5.79 (3.85, 14.39)	0.02
	PFTeDA	0.96 ± 0.17	0.92 (0.13, 2.4)	1.94 ± 0.32	1.42 (0.91, 4.17)	0.03
	PFHxS	1.2 ± 0.11	1.16 (0.62, 2.06)	2.01 ± 0.32	1.58 (0.93, 5.33)	0.16
	PFHpS	0.2 ± 0.03	0.15 (0.09, 0.47)	0.63 ± 0.21	0.42 (0.22, 3.01)	<0.001
	Br-PFOS	1.32 ± 0.17	1.1 (0.39, 2.53)	4.21 ± 0.89	3.21 (1.85, 13.01)	<0.001
	L-PFOS

Acknowledgements

This work was funded by the Région Nouvelle-Aquitaine, France (MULTISTRESS project to O. Chastel), the CPER ECONAT, and INSU-EC2CO Ecodyn 2014 program (to O. Chastel). This study has been carried out with financial support from the French National Research Agency (ANR) in the frame of the "Investments for the future" Program, within the Cluster of Excellence COTE (ANR-10-LABX-45). At the CEBC, we thank Marie Pallud and Cécile Ribout for their help in thyroids hormone assay and molecular sexing. The authors thank all the fieldworkers from Ligue Pour la Protection des Oiseaux (LPO) for their help in the long-term monitoring and ringing program (PP533) supported by the Centre de Recherches sur la Biologie des Populations d'Oiseaux (CRBPO). This study was approved by the French Animal Ethic Committee (authorisation number: APAFIS#15629-2019032823161213).