Hamzé Karaky
email: hamze.karaky@etudiant.univ-reims.fr

Chadi Maalouf
email: chadi.maalouf@univ-reims.fr

Christophe Bliard
email: christophe.bliard@univ-reims.fr

Alexandre Gacoin

Mohammed Lachi
email: mohammed.lachi@univ-reims.fr

Nadim El Wakil

Guillaume Polidori

Characterization of beet-pulp fiber reinforced potato starch biopolymer composites for building applications

Keywords: Sugar beet pulp, Potato starch, Building insulation, Bio-sourced composite, Hygrothermal properties, Mechanical properties, Sound absorption, Porosity Moisture buffering value

This study shows the mechanical performance of the beet pulp composite materials.

The binding strength of the beet pulp composite depends on the material density.

The beet pulp composite shows a good thermal and acoustical properties.

Introduction

Energy consumption is rapidly increasing in the world. It doubled in the period 1970-2000, and according to some studies it might double again in 2020. The building sector in Europe is responsible for 25% of the total energy consumption and 11% of all greenhouse gas emissions. In this context, developing innovative building material from locally available renewable resources is of primary importance. It allows to upgrade the energy characteristics of buildings and induce changes in energy consuming behavior [1].

Several studies have already been published on the use of crop by-products for building applications. Boussetoua et al. [START_REF] Boussetoua | Mechanical and hygrothermal characteri s ation of cork concrete composite: experimental and modelling study[END_REF] studied the elaboration and the characterization of a cork-cement composite. The results show that increasing the amount of cork granule increases the moisture buffering value but decreases the thermal conductivity and mechanical properties. In addition, Bourdot et al. [START_REF] Bourdot | Characterization of a hemp-based agro-material: Influence of starch ratio and hemp shive size on physical, mechanical, and hygrothermal properties[END_REF] and Umurigirwa et al. [START_REF] Umurigirwa-Vasseur | Elaboration et Caractérisation d ' un Agromatériau Chanvre-amidon Pour le Bâtiment[END_REF] studied the characterization of hempstarch composite.

The results show that hemp-starch is an excellent hygric regulator, and that increasing hemp content tends to increase the moisture buffering value and decreases slightly the mechanical properties. However, Le et al [START_REF] Le | Influence of various starch/hemp mixtures on mechanical and acoustical behavior of starch-hemp composite materials[END_REF] deduced that the mechanical properties depends strongly on the hemp/starch ratio and the hemp shives sizes. Furthermore [START_REF] Mazhoud | Development and hygric and thermal characterization of hemp-clay composite[END_REF], worked on the development of hemp-clay composite. The measurement of moisture buffering value (MBV = 2.68 g/(m.%RH) shows that hemp-clay material is an excellent hygric regulator.

Sugar beet (Beta vulgaris var. saccharifera) is widely grown in France (44 million tons in 2017). The process of producing beet sugar generates enormous quantities of by-products [START_REF] Roge | Extraction Du Sucre[END_REF]. One of these by-products is sugar beet pulp. In the factory, clean beets emerge from the washhouse and fall into the root cutter, where knives, driven by a large diameter disk, cut the roots into thin, rigid strips called Cossettes. After extraction of the sugar from the Cossettes, the remaining pulps shreds are generally pressed to 20-30% dry matter called Fresh pulp [START_REF] Roge | Extraction Du Sucre[END_REF]. The sugar beet pulp is then mechanically compressed and extruded at elevated temperature to produce the extruded sugar beet pulp pellet (EBPP) (Fig. 1a).

EBPP is recognized as an excellent feed for livestock due to its high nutritional value and palatability [START_REF] Nasielski | Le bon usage de la pulpe surpressée Tienen[END_REF]. In recent years, beet pulp has become an important source of gelling pectin. The pectin is extracted from the pulp of the sugar beet from various extraction procedures [START_REF] Sun | Extraction and physico-chemical characterization of pectins from sugar beet pulp[END_REF][START_REF] Phatak | Isolation and characterization of pectin in sugar-beet pulp[END_REF][START_REF] Chen | Properties and extraction of pectin-enriched materials from sugar beet pulp by ultrasonic-assisted treatment combined with subcritical water[END_REF], and used in food and cosmetics industries. It has also been used as a bioadsorbent for the removal of heavy metals [START_REF] Dronnet | Binding of divalent metal cations by sugar-beet pulp[END_REF]. The extruded pulps are composed mainly of pectin, cellulose and hemicellulose. The different EBP components are shown in Fig. 2. Fig. 2. The components of the extruded sugar beet pulp [START_REF] Belhamri | Extraction des macromolécules pariétales des eaux de presse de betteraves sucrières : Etude de leur composition, de leurs propriétés physicochimiques et de leur effet sur le process sucrier[END_REF].

One study has already been published on beet pulp based concrete using cement as a binder. In this study, authors present various chemical and physical treatments carried out on EBP to modify its properties, one treatment using cement in association with linseed oil showed the best results in reducing the potential of pulps to absorb enormous quantities of water and swell [START_REF] Monreal | Effects of aggregate coating on the hygral properties of lignocellulosic composites[END_REF].

Starch is the main carbohydrate reserve of higher plants. It represents a significant weight fraction in many agricultural raw materials such as cereals (30-70%), tubers (60-90%) and legumes (25-50%). Starch is composed of two polymers with a different primary structure: Amylose, (linear), and amylopectin (branched) [START_REF] Boursier | Amidons natifs et amidons modifiés alimentaires[END_REF].

Starch is already widely used in many industrial sectors, mainly paper, textile and also food via the beverage, confectionery and baked goods industries. The chemical industry uses starch in fermentation processes to produce bioethanol, surface treatment products, formulation of adhesives, encapsulation of pharmaceutical products, cosmetics, and biodegradable plastics [START_REF] Wertz | PLA: Deux Biopolymères Sur le Marché[END_REF]. Starch was used to elaborate an agro-material for building applications such as starch-hemp composite, starch-date palm fiber and Cassava/sugar palm fiber reinforced cassava starch [START_REF] Bourdot | Characterization of a hemp-based agro-material: Influence of starch ratio and hemp shive size on physical, mechanical, and hygrothermal properties[END_REF][START_REF] Edhirej | Cassava/sugar palm fiber reinforced cassava starch hybrid composites: physical, thermal and structural properties[END_REF][START_REF] Jumaidin | Effect of seaweed on mechanical, thermal, and biodegradation properties of thermoplastic sugar palm starch/agar composites[END_REF][START_REF] Belakroum | Hygric buffer and acoustic absorption of new building insulation materials based on date palm fibers[END_REF].

Based on the above-mentioned literature review, the present work describes the elaboration of a new agro-composite based on sugar beet pulp and potato starch as a bio-sourced binder. Physical mechanical and hygrothermal characteristics are measured with different compositions designed to be used in the building sector for walls and floor insulation applications.

Materials and methods

Materials

2.1.1.

Extruded beet pulp Due to the fast biodegradability of fresh pulp-dried extruded beet pulp (EBP) was used in this study exclusively. The 8-10 mm diameter extruded beet pulp pellets EBPP (18% humidity) (Fig. 1a) were provided by Cristal Union factory (Pomacle, France). Before the use of EBP, they were immersed in water until their saturation. Then, EBP were dried at 50 C (Fig. 1b). To ensure proper conservation in the lab the pulps were kept at 20 C until use.

2.1.2.

Potato starch Potato starch was purchased from ROQUETTE, Lestrem, France. Potato starch has a high polymerization degree which gives a viscous binder and provides a good mechanical property for the EBPstarch formulas.

2.2.

Beet pulp characterization

The properties of the elaborated composites were conditioned by the EBP morphology and their adhesion with the binder within the matrix. This morphology was studied by a microscopic analysis and image analysis software.

2.2.1.

Microscopic analysis A KEYENCE microscope was used to analyze the pulp particles at microscopiclevel. The microscope was equipped with a camera that allowed to capture of microscopic photos with a very high depth of field (x50).

Image analysis

The computer analysis program (ImageJ, National Institutes of Health) was used to process and analyze plain EBP macroscopic images. The geometrical characteristics of individual centimeter size pulp chunks and model regular geometrical shapes such as rectangle and ellipse were determined. The experim were spread out. More than 2000 particles were dispersed in such a way that they did not touch each other. The resulting image were processed and analyzed using the ImageJ software.

EBP granulometric analysis

The pulp was sieved using a vibrating sieve (Controlab) to analyze the particle size distribution. The sizes of sieves were between 0.125 mm and 8 mm. After the sieving process, the aggregates retained in each sieve were weighed and the cumulative masses were then calculated with their mass percentages. The granulometric curve was obtained by representing the cumulative mass percentages on the ordinate as a function of sieve size in logarithmic scale.

Porosity

The porosity is an essential characteristic for evaluating insulation properties. To evaluate the porosity of the EBP composite, the bulk density was measured using the Eq. ( 1):

 bulk = (mass of the sample)/(volume of the container) (1) The method used for the measurement of absolute density was the one already described by Bourdot et al. [START_REF] Bourdot | Characterization of a hemp-based agro-material: Influence of starch ratio and hemp shive size on physical, mechanical, and hygrothermal properties[END_REF]. In summary, the pycnometer was filled with a given mass of crushed sample and half its volume with cyclohexane, which is a non-polar solvent and does not affect EBP composition and mass (M). The system was kept under reflux (see Fig. 3) for 6 x 10 min. boiling then cooling cycles, during which air was released from the pulps voids and replaced by cyclohexane. During the 6 th cycle the system is kept under Argon atmosphere to avoid humidity from being reabsorbed in EBP. At ambient temperature (20°C) the flask was filled up to the top and sealed with a dedicated glass top, allowing the excess solvent to overflow and avoiding the retention of bubbles, in order to have a constant volume inside the measuring system. The system was then weighed to a 10 -3 g accuracy. The absolute density was calculated according to this formula (2):

 abs = (M1  cyc ) / (M1 -(M2 -M3)) (2)
where  abs is the absolute density,  cyc the density of cyclohexane, M1 the mass of dried sample, M2 the mass of the pycnometer containing a cyclohexane and the dried sample, and M3 the mass of the pycnometer containing only the cyclohexane. Care was taken to avoid the reflux of condensed humidity back in the mixture. Each measurement was carried out at least three times to be representative. The porosity was calculated using the following Eq. ( 3):

 1 -(bulk / abs) (3)
where  is the porosity of composite, bulk the bulk density in kg/m3 and abs the absolute density in kg/m3.

Composite characterization

Composite preparation

The EBP pellets were immersed to swell in distilled water until saturation (350%). The wet pulp was mixed with the appropriate amount of dry potato starch powder. The obtained mixture was then placed in an autoclave and heated for 30 min to jellify the starch granules. The obtained formulas were poured into the wooden molds.

The molds were used with a fitted wood piston on the upper part to compact the mix and provide a flat surface on each side. To keep the compaction level steady during drying the piston was kept in a custom-made clamp system consisting of two long screws holding two pieces of wood pressing onto the piston. A layer of 5 mm thick cardboard was placed between the piston and the mixture to help the water vapor released to diffuse from the sample (see Fig. 4). The filled-up molds were then frozen at -80°C and the obtained frozen discs were dried using a Freeze dryer under a pressure of 2 mbar. Different drying methods were carried out and several molds were constructed in such a way to accelerate the permeation of the moisture from the sample during the drying process by drilling a series of 1 mm bit holes spaced at 1 cm from each other.

Four compositions were prepared with four EBP-starch mass ratio S/EBP 0.1, 0.2, 0.3 and 0.4. The samples were compacted under a pressure of 44 kPa.

Sound absorption coefficient

The sound absorption coefficient is defined as the ratio of the sound energy absorbed by a composite to the sound energy incident upon its surface.

Sound absorption coefficient was measured by BandK Type 4206 impedance tube with two microphones according to the standard ISO (10534-2) procedure. Acoustic samples were fixed in the impedance tube with two different diameters 2.9 and 9.9 cm (see Fig. 5). The samples were prepared previously and equilibrated at ambient conditions. The acoustic parameter of 9.9 cm diameter cylindrical samples was measured in the 50 Hz-1.6 kHz frequency range, whereas the samples with a 2.9 cm diameter were measured in the 500 Hz-6.4 kHz frequency range. The device determines the sound absorption coefficient by measuring the pressure at two fixed points caused by a stationary random sound propagating as plane wave in the tube.

The sound absorption coefficient was measured in the frequency range from 50 Hz to 6.4 kHz. Three 9.9 cm diameter and 4 cm thick samples and three 2.9 cm diameter and 4 cm thick samples were tested for each formula. Compression test and bending test in four-point (see Fig. 6) were recorded by using Instron 8801 machine with a constant rate of 0.1 mm/s for the bending test and 0.2 mm/s for the compression test. Compression and bending test were performed in the pressure direction. The tensile strength was determined by using the Navier Eq. ( 4):

 t = (M f y) / I Navier equation(4)
with Mf = (F/2) a, F: the maximum load, y = h/2, h: the sample height, I: the sample inertia I = b h 3 /12, a and b are shown in the Fig. 6d. The transversal and longitudinal strain respectively ( xx ,  yy ) were determined using an Aramis optical system (see Fig. 6a). This system is a high performance optical system which determines both sample displacement and strain during loading through digital image correlation methods [START_REF] Le | Influence of various starch/hemp mixtures on mechanical and acoustical behavior of starch-hemp composite materials[END_REF][START_REF] Bourdot | Characterization of a hemp-based agro-material: Influence of starch ratio and hemp shive size on physical, mechanical, and hygrothermal properties[END_REF]. The elasticity modulus (E) and Poisson's ratio (m) were calculated using the Eqs. ( 5) and ( 6) were  c is the compressive strength,  yy is the longitudinal strain and  xx is the transversal strain.

E =  c /  yy (5) V =  xx /  yy (6)
ARAMIS stereo cameras were rigidly connected to measure the displacement fields and the 2D deformations from one pair of images corresponding to several instants of strain of samples. Before measuring, all the samples were placed at the same distance to the camera (34 cm) to obtain a sharpness image.

Moisture buffering value (MBV)

The Moisture Buffering Value [START_REF] Rode | Moisture Buffering of Building Materials Department of Civil Engineering Technical[END_REF] measures the capacity of the composite to regulate the relative humidity of the medium [START_REF] Moussa | Development and performance evaluation of a hemp-starch composite[END_REF][START_REF] Bourdot | Characterization of a hemp-based agro-material: Influence of starch ratio and hemp shive size on physical, mechanical, and hygrothermal properties[END_REF][START_REF] Boussetoua | Mechanical and hygrothermal characteri s ation of cork concrete composite: experimental and modelling study[END_REF][START_REF] Mazhoud | Development and hygric and thermal characterization of hemp-clay composite[END_REF]. The Nordtest protocol defines cyclic step-changes in relative humidity after stabilization, between high (75%) and low (33%) values for 8 and 16 h, respectively. Four 9.8 cm diameter and 4 cm thick cylindrical samples were tested for each formula. The edges and the back-sides of samples were sealed with duct tape to obtain a one-dimensional moisture flow. Samples were dried at 50°C and 10% R in a climatic chamber and then stabilized at 23°C and 50% RH. They were weighted until they reached equilibrium (see Fig. 7). During the periodic exposure, samples were weighted five times during the adsorption phase and twice during desorption. The velocity is given by the climatic chamber manufacturer and it is about 0.5 m/s. When the mass variations between three consecutive days were under 5%, the experiment was stopped and the moisture buffering value (MBV) was calculated according to the Eq. ( 7): MBV = m / (A (RH high -RH low )) [START_REF] Roge | Extraction Du Sucre[END_REF] A (m2) is the area of the sample that is in contact with air. The RH high and RH low respectively represent high relative humidity (75% RH) and low relative humidity (33% RH) and m represents the change in mass during the absorption/desorption phase (g).

Thermal conductivity

A series of cubic samples (10 10 10 cm) were made to study the thermal behavior of the starch-Beet Pulp composite. Before the measurement, all samples were dried in a climatic chamber at 50°C and 10% RH. The thermal conductivities were measured using a Isomet 2114 (Applied Precision) device by which applies a dynamic measurement method to reduce the measurement time in comparison with steady state measurement methods. This measurement analyzes the temperature response of the measured composite to heat flow impulses. The heat flow is produced by an electrical heating of resistor heating in direct contact with the tested sample. The thermal conductivity was evaluated by periodically recording the sampled temperature as function of the time. Built-in menu system on color graphic display and the alphanumeric keypad provides a good utilization of the device. The measurement reproducibility of the device was 3% + 0.001 W/(m•K), the measurement was done at ambient temperature (20°C) and 10 %RH. The thermal conductivity of the Starch-Beet Pulp was measured on three samples for each formula [START_REF] Haba | Hygric properties and thermal conductivity of a new insulation material for building based on date palm concrete[END_REF].

Results and discussion

Aggregate morphology

The extruded pulps have a cavernous shape and an irregular rough surface, both characteristics provide a good adhesion within the matrix (see Fig. 8). The air-filled cavities in EBP reduce the thermal conductivity of the elaborated composite. In addition, the resulting bulk density is very low, which results in a lightweight composite with good acoustic and thermal insulation properties.

Image analysis

From a macroscopic point of view, the image software was used to calculate the surfaces and perimeters of each pulp, the lengths and widths of the aggregates (see Figs. 9 and 10), as well as the major and minor axis of the ellipse associated to each particle. The surface has an average of 7.9 mm 2 , and a perimeter 11.37 mm. The surfaces and the perimeters of the geometric shapes were calculated, as well as the quadratic error of each parameter. The best calculated model for the pulp shape was the rectangular form.

Particle size analysis by sieving

The results presented in Fig. 11, show that the particle size is continuous, that no granulometric fraction is missing. The largest fraction found was between 2 and 4 mm with 62% of the beet particles. The EBP particles have a specific long shape originating from the root cutter knives design, which offers a larger perimeter and good adhesion with the matrix.

Density and porosity

The bulk density was measured for EBP before and after extrusion. Table 1 shows the bulk and the absolute density of sugar beet pulp. In both cases the extruded EBP appears denser, probably due to the strong compression during the extrusion process.

Composite analysis

Results of dried samples

During oven drying of the samples, microscopic cracks appear on the composite surface due to extensive shrinkage. As both the beet pulp and starch are highly hydrophilic, water gradients inside and anisotropy of the drying within the composite sample probably cause irregular shrinkage of the composite. Large dimensional variations were observed after classic oven drying at 100°C, the sample underwent a (33%) irregular retraction, which caused large millimeter size cracks. Drying under vacuum at 40°C resulted in much more regular shapes, the composite retracted, there were no macroscopic cracks observed and the surface was flatter.

When dried under the freeze dryer, the EBP-starch composites release water more regularly, and consequently retract less, which results in rigid and light composite blocks. The blocks present a uniform volume shrinkage (8%). Therefore, freeze drying provided the best results (see Fig. 12).

Density and porosity

Fig. 13 shows the variation in the density of the completely dried composite material in climatic room at 50°C and 10 %RH, as a function of the S/EBP weight ratio. It can be observed that the composite material density increases linearly with S/EBP weight ratio. The bulk density increases from 271.4 kg/m3 to 360 kg/m3, noting that the average density of the Cement-Beet Pulp concrete ranged between 570 kg/m3 and 770 kg/m3 [START_REF] Monreal | Effects of aggregate coating on the hygral properties of lignocellulosic composites[END_REF]. The bulk density of the samples can be expressed as function as S/EBP weight ratio between 0.1 and 0.4 by the following Eq. ( 8):

 = 295.42 (S/ EBP) + 242.8 (8)
Results of density and porosity are shown in Table 2. The absolute and bulk density increase with S/EBP weight ratio. However, the porosity of the composite decreases logarithmically. Therefore, the sample having the lowest amount of starch (S/EBP = 0.1), has the higher porosity (79.75%) and the lowest absolute density (qabs = 1222 kg/m3). The total porosity was between 70.60% and 79.75% and decreasing the S/EBP weight ratio seems to increase the total porosity. The starch influence is consistent with results obtained by Rahim et al. [START_REF] Rahim | Effect of moisture and temperature on thermal properties of three bio-based materials[END_REF] and Bourdot et al. [START_REF] Bourdot | Characterization of a hemp-based agro-material: Influence of starch ratio and hemp shive size on physical, mechanical, and hygrothermal properties[END_REF]. The presence of starch gel increases the bulk density and decreases the total porosity by filling the inter-particular space between pulp particles and filling or sealing the pores. Therefore, the composite would contain more or less accessible closed and open pores. Sound absorption coefficient In this section, the effect of the binder amount in the formula on the sound absorption of the composite material is analyzed. The porosity and the pore size influence the dissipation of the sound waves. The sound absorption of the sugar beet pulp-starch composite depends on the origin of the pulp and the starch/sugar beet pulp mass ratio. Fig. 14 shows the sound absorption variation as a function of sound frequency and for different S/EBP ratios. The increase of the binder content in the mixture decreased the porosity and increased mass density. Therefore, the sound absorption of the composite material decreased slightly. The sound absorption curve was also shifted to the lower frequencies. This result is in correlation with Glé study [START_REF] Glé | Acoustical properties of materials made of vegetable particles with several scales of porosity[END_REF]. Fig. 15 shows the comparative sound absorption coefficient of a hemp-starch material, two samples manufactured from starch and date palm fiber (F and WF) and four samples from starch-EBP. At 500 Hz, samples of sugar beet pulp fiber (S/EBP 0.2 and 0.3) and the samples composed of 20% palm fiber and 80% starch solution absorbed 40% of sound waves. Hemp-based material only absorbs 40% of the sound waves at this frequency [START_REF] Belakroum | Hygric buffer and acoustic absorption of new building insulation materials based on date palm fibers[END_REF][START_REF] Le | Influence of various starch/hemp mixtures on mechanical and acoustical behavior of starch-hemp composite materials[END_REF]. These results show interesting acoustical absorption performances of these materials at high frequencies. At 1000 Hz, the hemp composite absorbs 70%, of the soundwave, whereas the sound absorption of sugar EBP samples (S/EBP = 0.1) is only 55%, and 50% for the (F 20% -St 80%) palm fiber samples. We can observe that when the frequency decreases the sound absorption capacity of the considered samples decreases to very low values. The superior performance of EBP-based material as compared to date palm composite and hemp-starch material, for medium and low frequencies, is due to the thickness of the samples used by [START_REF] Belakroum | Hygric buffer and acoustic absorption of new building insulation materials based on date palm fibers[END_REF] and [START_REF] Le | Influence of various starch/hemp mixtures on mechanical and acoustical behavior of starch-hemp composite materials[END_REF] which was 4 cm; while in the other studies, it was 3 and 3.5 cm thickness. For frequencies lower than 500 Hz, the starch-EBP showed higher acoustical absorption efficiency as compared to the other composites. It absorbs 60% of sound waves. Increasing the starch mass increases the sound absorption coefficient of sugar beet pulp-starch composite. However, for frequencies higher than 500 Hz, this composite absorbs 50% of sound waves whereas the hempstarch composite absorbs 70% of the sound waves [START_REF] Belakroum | Hygric buffer and acoustic absorption of new building insulation materials based on date palm fibers[END_REF]. Increasing the starch mass decreases the sound absorption coefficient of EBPstarch composites. Therefore, by increasing the starch content in the sugar beet pulp-starch composite to 40% the material becomes denser, the binder fills the EBP pores and consequently the sound absorption coefficient of the composite decreases.

Mechanical properties

All mechanical properties shown in this section: tensile strength, compressive strength, elasticity modulus and Poisson's ratio were measured on four S/EBP weight ratio between 0.1 and 0.4. The compression test led to the failure of the sample. The compressive strength variation of the aforementioned samples is shown in Fig. 16. At 10% of the strain of the sample for the samples of 10 10 10 cm with S/EBP = 0.1, 0.2, 0.3 and 0.4, the corresponding stress is 0.20, 0.23, 0.24 and 0.32 MPa respectively. It is useful to indicate that the Starch-beet pulp composite is deformable and compressible. However, the sample can be crushed with load greater than load limit. The compressive strength was obtained for longitudinal strain  between 22% and 27%. The compressive strength of the Starch-Beet Pulp increased from 0.37 MPa to 0.52 MPa. For the starch-hemp composites, the mechanical behavior was also studied [START_REF] Bourdot | Characterization of a hemp-based agro-material: Influence of starch ratio and hemp shive size on physical, mechanical, and hygrothermal properties[END_REF][START_REF] Le | Influence of various starch/hemp mixtures on mechanical and acoustical behavior of starch-hemp composite materials[END_REF][START_REF] Le | Experimental investigation on the mechanical performance of starch-hemp composite materials[END_REF][START_REF] Arnaud | Experimental study of parameters influencing mechanical properties of hemp concretes[END_REF]. The average compressive strength is ranged between 0.40 and 0.63 MPa [START_REF] Bourdot | Characterization of a hemp-based agro-material: Influence of starch ratio and hemp shive size on physical, mechanical, and hygrothermal properties[END_REF][START_REF] Le | Influence of various starch/hemp mixtures on mechanical and acoustical behavior of starch-hemp composite materials[END_REF][START_REF] Le | Experimental investigation on the mechanical performance of starch-hemp composite materials[END_REF]. It can be seen that the compressive strength of the Starch-Beet Pulp is comparable. It seems to indicate that the compressive strength of the Starch-Beet Pulp increases with increasing the starch amount. However, the influence of the variation in the density on the compressive strength of the Starch-Hemp composites is not significant [START_REF] Bourdot | Characterization of a hemp-based agro-material: Influence of starch ratio and hemp shive size on physical, mechanical, and hygrothermal properties[END_REF][START_REF] Le | Influence of various starch/hemp mixtures on mechanical and acoustical behavior of starch-hemp composite materials[END_REF][START_REF] Le | Experimental investigation on the mechanical performance of starch-hemp composite materials[END_REF]. These values are greater than those measured on the Starch-Hemp composites 0.08-0.11 MPa [START_REF] Bourdot | Characterization of a hemp-based agro-material: Influence of starch ratio and hemp shive size on physical, mechanical, and hygrothermal properties[END_REF]. These results are probably due to a better coating of the EBP by the starch binder during manufacturing of the material. The tensile strength of the Starch-Beet Pulp composite ( t ) can be expressed as function as the S/EBP mass ratio by the Eq. ( 9).  t =0.79 (S/EBP) 2 -0.06 ( S/ EBP) + 0.11 [START_REF] Sun | Extraction and physico-chemical characterization of pectins from sugar beet pulp[END_REF] Fig. 18a shows the elasticity modulus curve measured by compression test. The average elasticity modulus of the Starch-Beet Pulp is ranged between 2.06 MPa and 3.17 MPa.

The average Poisson's ratio of the studied samples is shown in Fig. 18b. The results show that the Poisson's ratio increases linearly with S/EBP weight ratio from 0.11 to 0.18. This can be partially explained by the decrease of the composite porosity. Increasing the starch amount will fill the pores and decreases the porosity of the composite material, consequently the Poisson's ratio increases. The average measured Poisson's ratios are comparable with those of Starch-Hemp composites [START_REF] Bourdot | Characterization of a hemp-based agro-material: Influence of starch ratio and hemp shive size on physical, mechanical, and hygrothermal properties[END_REF][START_REF] Le | Influence of various starch/hemp mixtures on mechanical and acoustical behavior of starch-hemp composite materials[END_REF][START_REF] Le | Experimental investigation on the mechanical performance of starch-hemp composite materials[END_REF]. The relation between the Poisson's ratio and S/EBP weight ratio is linear and can be represented by the Eq. ( 11).

V = 0.21 (S/EBP) + 0.09 (11)
The Aramis method used gives the distribution of longitudinal and transversal strain ( xx and  yy ). Fig. 19 shows the typical cartographies of the strain for several samples studied with different S/EBP weight ratio for  =  max /3.

The iso-strain value ( max - max ) was more important in the sample with a greater starch amount (S/EBP = 0.4) (see Table 3). It can be seen that the iso-strain tends to increase when the S/EBP weight ratio increases from 0.1 to 0.4. It seems to indicate the iso-strain of the Starch-Beet Pulp is less than that of the Starch-Hemp [START_REF] Bourdot | Characterization of a hemp-based agro-material: Influence of starch ratio and hemp shive size on physical, mechanical, and hygrothermal properties[END_REF], which can confirm that when the particles used in the composite materials is finer, the particles (Beet Pulp) are well distributed in all directions of the composite and more homogenous.

Fig. 1 (

 1 Fig.1 (a)-Extruded beet pulp pellet (EBPP), (b)-Beet pulp (EBP). Fig. 1. (a)-Extruded beet pulp pellet (EBPP), (b)-Beet pulp (EBP).

Fig. 3 .

 3 Fig. 3. The system of absolute density measurement.

Fig. 4 .

 4 Fig. 4. Presentation of wooden molds and piston used.

Fig. 5 .

 5 Fig. 5. Kundt tube.

2. 3

 3 .3. Mechanical properties 2.3.3.1. Experimental procedure. For this test, a series of samples (12 samples 10 10 10 cm and 12 samples 16 4 4 cm) were prepared by varying the Starch/Beet pulp (S/EBP) ratio (S/EBP = 0.1, 0.2, 0.3 and 0.4). The samples were made by the procedure in the paragraph 2.3.1.

Fig. 6 .

 6 Fig. 6. (a) Aramis stereo optimal system, (b) Compressive test, (c) and (d) Bending test.

Fig. 7 .

 7 Fig. 7. The MBV samples in climatic room.

Fig. 8 .

 8 Fig. 8. Microscopic photos of EBP.

Fig. 9 .

 9 Fig. 9. Length distribution of the pulps.

Fig. 10 .

 10 Fig. 10. Width distribution of the pulps.

Fig. 11 .

 11 Fig. 11. Curve of retained particle size during sieving.

Fig. 12 .

 12 Fig. 12. Freeze-dried composite blocks.

Fig. 13 .

 13 Fig. 13. Density of the composite material with different Starch/pulp ratio.

Fig. 14 .

 14 Fig. 14. Sound absorption according to the starch content.

Fig. 15 .

 15 Fig. 15. The sound absorption coefficient of several green materials.

Fig. 16 .

 16 Fig. 16. (a) Compressive strain-stress curves for different S/EBP weight ratios, (b) Compressive strength-S/EBP weight ratio curves.

Fig. 17 .

 17 Fig. 17. (a) Tensile stress-displacement curves for different S/EBP weight ratios, (b) Flexural strength-S/EBP weight ratio curves.

Fig. 18 .

 18 Fig. 18. (a) Elasticity modulus, (b) Poisson's ratio.

Fig. 19 .

 19 Fig.[START_REF] Belhamri | Extraction des macromolécules pariétales des eaux de presse de betteraves sucrières : Etude de leur composition, de leurs propriétés physicochimiques et de leur effet sur le process sucrier[END_REF]. Cartographies of compressive strain (transversal and longitudinal strain) under loading obtained by ARAMIS method for 30% =  max /3 for different studied formula.

3. 2 . 5 .Fig. 20 .

 2520 Fig.20shows mass variation of beet pulp-starch composites under variations in relative humidity between 33% and 75% at 23°C. It can be seen that the composite absorbs moisture at 75% RH and releases moisture at 33% RH. For the last three cycles, the mass variation of the samples appears to have stabilized. The results presented in Fig.21clearly show that the composite is an excellent regulator of the relative humidity of the environment (MBV > 2 g/(%RH. m 2 )) according to the classification proposed by Rode[START_REF] Rode | Moisture Buffering of Building Materials Department of Civil Engineering Technical[END_REF]. The samples present MBV values between 2.6 and 2.8 g/(%RH.m 2 ), which are comparable with the MBV values of hemp-starch composites[START_REF] Bourdot | Characterization of a hemp-based agro-material: Influence of starch ratio and hemp shive size on physical, mechanical, and hygrothermal properties[END_REF] It can be observed that the variation in moisture buffering value as a function of the starch/sugar beet pulp is linear and can be presented by the Eq. (12). Besides increasing the mass of starch, increases the MBV value, because of starch high moisture buffering. The samples with more starch, have the highest MBV value, around 2.8 g/(%RH.m2).

Fig. 21 .

 21 Fig. 21. Moisture Buffering Value as function of S/EBP ratio.

Fig. 22 .

 22 Fig. 22. Variation of the thermal conductivity as function of the S/EBP weight ratio.

Table 1

 1 Bulk and absolute density of different aggregates.

	Aggregates	 bulk (kg/m3)	 abs (kg/m3)	Porosity (%)
	Fresh beet pulp	134 ± 6.7	911.61 ± 45.6	85.30
	Extruded beet pulp	194 ± 7.9	1073.38 ± 53.7	81.93
	Hemp shive 5 mm (a)	135 ± 6.75	1271 ± 63.5	91.35
	Hemp shive 20 mm (a)	110 ± 5.5	1266 ± 63.3	89.34
	(a) Bourdot et al. [3].

Table 2

 2 The density and the porosity of EBP composite.

	S/EBP	 abs (kg/m3)	 app (kg/m3)	Porosity (%)
	0.1	1222.18 ± 44.83	271.44 ± 13.19	77.8
	0.2	1240.25 ± 52.94	302.77 ± 10.18	75.6
	0.3	1284.92 ± 68.87	332.38 ± 13.95	74.1
	0.4	1293.42 ± 53.17	360.04 ± 14.32	72.1
	3.2.3.

Acknowledgment: This research was supported by the Ministry of Interior and Municipalities (Lebanon) through a PhD grant (Grant number: 69/ S). We thank G. Stockton who checked the paper.

Conclusions

In this paper, the physical properties of the extruded sugar beet pulp were studied (morphology, porosity, densities and particles size). Several methods and designs were used to elaborate and dry the EBP-starch composites.

The properties of a new agro-composite based on EBP and potato starch are presented. Different formulations were studied with EBP-starch ratios varying between 0.1 and 0.4.

The bulk density of the Starch-Beet Pulp composite depends of the S/EBP weight ratio. The bulk density of the composite increases when the amount of the potato starch increases. The results show that the starch-beet pulp is heavier than starch-hemp composites.

The starch-beet pulp composite absorbs up to 60% of the sound waves for the high frequencies depending of the starch amount of the composite.

The average of the tensile strength evolved from 0.11 MPa to 0.21 MPa when the S/EBP weight ratio increased from 0.1 to 0.4. These values were greater than those measured on the Starch-Hemp composites 0.03 MPa 0.13 MPa. The elasticity modulus increased from 2.06 MPa to 3.17 MPa when the S/EBP weight ratio increased from 0.1 to 0.4. These results were comparable with those obtained with the Starch-Hemp composites. The difference can be explained by the better adhesion that the finer particles used (Beet pulp) provide to the composites and consequently increased elasticity. The Poisson's ratio from 0.11 to 0.18 increases linearly with the S/EBP weight ratios.

The cartographies obtained by ARAMIS method show that the Starch-Beet Pulp is more homogenous that Starch-Hemp composites, but the cracks already existing in the samples increased the strain.

The studied EBP-starch composites showed interesting hygrothermal properties. The Moisture Buffering Values (MBV) was 2.62-2.80 g/(m2 %RH) classifying them as excellent hygric regulators. The increase of starch content increases the moisture buffering value of the composites. Starch-beet pulp composite have a low thermal conductivity to around 0.070 W/(m•K). The thermal conductivity increases when the S/EBP weight ratio increases, which can be, explained the reduction of the pores filled by the starch binder.

Finally, the EBP-starch composite S/EBP = 0.4 can be considered as the optimal composition in terms of the hygrothermal properties. However, the S/EBP 0.1 presents the optimal composition for acoustical properties.

Conflict of interest:

None.


Morgane Touzot
email: morgane.touzot@univ-lyon1.fr

Thierry Lengagne

Jean Secondi

Emmanuel Desouhant

Marc Thery

Adeline Dumet

Claude H B Duchamp

Nathalie Mondy

Marc Théry

Artificial light at night alters the sexual behaviour and fertilisation success of the common toad

Keywords: Fitness, reproduction, amphibian, anthropogenic light pollution, behaviour

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

Introduction

In recent decades, the rapid growth in the world's population has led to a sharp increase in human activities necessary to support this growth. One consequence of the expansion of urban areas along with the development of transport infrastructures [START_REF] Grimm | Global Change and the Ecology of Cities[END_REF][START_REF] Gaston | The ecological impacts of nighttime light pollution: a mechanistic appraisal[END_REF] is the major increase of light levels at night. In 2016, nearly 23% of the Earth's surface, 88% of Europe and almost half of the United States experienced brightness levels higher than light levels at night in natural ecosystems [START_REF] Falchi | The new world atlas of artificial night sky brightness[END_REF]. Artificial Light At Night (ALAN) dramatically expanded. From 2012 to 2016, Earth's artificially lit outdoor area grew by 2.2% per year, with a total radiance increase of 1.8% per year [START_REF] Kyba | Artificially lit surface of Earth at night increasing in radiance and extent[END_REF]).

One of the major effects of ALAN is the disruption of the natural photoperiod, which is one of the most important cues for biological timing [START_REF] Bradshaw | Light, Time, and the Physiology of Biotic Responses to Rapid Climate Change in Animals[END_REF]). Among all organisms, nocturnal species, which represent a large proportion of biodiversity, 28% of vertebrates and more than 60% of invertebrates [START_REF] Hölker | Light pollution as a biodiversity threat[END_REF], are most likely to experience and to be affected by ALAN [START_REF] Buchanan | Observed and potential effects of artificial night lighting on anuran amphibians[END_REF]Duffy et al. 2015;[START_REF] Desouhant | Mechanistic, ecological and evolutionary consequences of artificial light at night for insects: review and prospective[END_REF]. Nocturnal artificial light is known to affect a wide range of physiological and behavioural phenomena, such as migration [START_REF] Van Doren | Highintensity urban light installation dramatically alters nocturnal bird migration[END_REF], orientation [START_REF] Tuxbury | Competitive interactions between artificial lighting and natural cues during seafinding by hatchling marine turtles[END_REF], activity (e.g. [START_REF] Tallec | Light pollution modifies the expression of daily rhythms and behavior patterns in a nocturnal primate[END_REF][START_REF] Pulgar | Endogenous cycles, activity patterns and energy expenditure of an intertidal fish is modified by artificial light pollution at night ALAN[END_REF][START_REF] Touzot | Artificial light at night disturbs the activity and energy allocation of the common toad during the breeding period[END_REF], foraging [START_REF] Czarnecka | Combined effects of nocturnal exposure to artificial light and habitat complexity on fish foraging[END_REF], energy balance (e.g. [START_REF] Welbers | Artificial light at night reduces daily energy expenditure in breeding great tits (Parus major)[END_REF][START_REF] Touzot | Artificial light at night disturbs the activity and energy allocation of the common toad during the breeding period[END_REF] and hormonal synthesis (e.g. [START_REF] Brüning | Spotlight on fish: Light pollution affects circadian rhythms of European perch but does not cause stress[END_REF][START_REF] Newman | Using novel methodologies to examine the impacts of artificial light at night on the cortisol stress response in dispersing Atlantic salmon Salmo salas L. fry[END_REF]. These effects on individuals have the potential to alter population dynamics (e.g. Gaston et al. 2014a;[START_REF] Grubisic | Artificial light at night decreases biomass and alters community composition of benthic primary producers in a sub-alpine stream[END_REF][START_REF] Sanders | Low levels of artificial light at night strengthen Top-Down control in insect food web[END_REF].

Despite the evidence of profound effects of ALAN on life history traits across different taxonomic groups with important ecological consequences (e.g. Gaston et al. 2014b; [START_REF] Knop | Artificial light at night as a new threat to pollination[END_REF][START_REF] Bennie | Artificial light at night causes topdown and bottom-up trophic effects on invertebrate populations[END_REF], we still have little information on the direct effects of ALAN on the fitness of individuals although its assessments are key elements in conservation. Fitness, i.e. the average contribution to the gene pool of the next generation that is made by individuals of a specified genotype or phenotype in a given environment, is often estimated through measurements of mortality and breeding success. Most of the studies conducted to date focused on the effects of ALAN on the mortality component of fitness (e.g. [START_REF] Rodríguez | Fatal attraction of Short-Tailed Shearwaters to Artificial lights[END_REF][START_REF] Willmott | Artificial light at night alters life history in a nocturnal orb-web spider[END_REF]. The breeding component of fitness has been less examined, even if However, observation-based studies did not control for other environmental factors that are often correlated with ALAN in the wild (temperature of urban environment, habitat fragmentation, prey availability…), while most experiment-based studies used higher intensities than those actually encountered by individuals in their habitat [START_REF] May | The effect of intensified illuminance and artificial light at night on fitness and susceptibility to abiotic and biotic stressors[END_REF]). a growing number of studies (reviewed in [START_REF] Ouyang | Hormonally mediated effects of artificial light at night on behavior and fitness: linking endocrine mechanism with function[END_REF] reported that the timing and the physiology of seasonal reproductive processes differ between individuals living in lit areas and their conspecifics living in darker areas. Alterations of the period of functional development of reproductive organs, reproductive hormonal synthesis (estradiol, ketotestosterone), number of eggs produced by females, egg hatchling success, number of offspring and birth schedule in response to ALAN exposure were found in mammals (e.g. Le [START_REF] Tallec | Effects of light pollution on seasonal estrus and daily rhythms in a nocturnal primate[END_REF][START_REF] Robert | Artificial light at night desynchronizes strictly seasonal reproduction in a wild mammal[END_REF], birds (e.g. [START_REF] Dominoni | Artificial light at night advances avian reproductive physiology[END_REF][START_REF] De Jong | Effects of nocturnal illumination on life-history decisions and fitness in two wild songbird species[END_REF], fishes (e.g. [START_REF] Brüning | Influence of artificially induced light pollution on the hormone system of two common fish species, perch and roach, in a rural habitat[END_REF][START_REF] Fobert | Artificial light at night causes reproductive failure in clownfish[END_REF] and insects (e.g. [START_REF] Willmott | Artificial light at night alters life history in a nocturnal orb-web spider[END_REF][START_REF] Mclay | Dim artificial light at night affects mating, reproductive output and reactive oxygen species in Drosophila melanogaster[END_REF]. Photoperiod modifications have also been shown to affect sexual maturation and sex steroids synthesis, especially in fish (e.g. [START_REF] Norberg | Photoperiod regulates the timing of sexual maturation, spawing, sex steroid and thyroid hormones profiles in the Atlantic cod (Gadus morhua)[END_REF]). The suppression of the dark period triggered a decrease of spermatocytes in male Asian toads, Bufo melanostictus, [START_REF] Biswas | Effect of continuous Light and Darkness on the testocular histology of toad (Bufo melanostictus)[END_REF]) and a severe reduction of sexual calls in male green frogs, Rana clamitans melanota, [START_REF] Baker | The effect of artificial light on male breeding-season behaviour in green frogs, Rana clamitans melanota[END_REF]). On contrary, other studies have found no effect of ALAN on the time for copulation or between mating and laying egg, on the number of eggs or egg sacs laid and on sperm viability (e.g. Durrant et al. 2018;[START_REF] Mclay | Dim artificial light at night affects mating, reproductive output and reactive oxygen species in Drosophila melanogaster[END_REF][START_REF] Willmott | Artificial light at night alters life history in a nocturnal orb-web spider[END_REF]. A recent study investigating the influence of ALAN on the fertilization success of fish, ultimately showed no effect [START_REF] Fobert | Artificial light at night causes reproductive failure in clownfish[END_REF]. In view of the consequences of ALAN on the breeding behaviour and physiology, it is necessary to investigate the direct influence of ALAN on the breeding component of fitness in amphibians.

In this context, we experimentally studied the effect of three ecologically relevant light intensities at night (0.01, 0.1 or 5 lux), which correspond to light levels measured in areas hosting amphibians [START_REF] Secondi | Variability of surface and underwater nocturnal spectral irradiance with the presence of clouds in urban and peri-urban wetlands[END_REF], on both the reproductive behaviour and the fertilisation success of male common toads, Bufo bufo. This nocturnal amphibian is one of the most common and ubiquitous amphibians in France, and as a result is often a useful indicator of ecosystem health and function [START_REF] Hilty | Faunal indicator taxa selection for monitoring ecosystem health[END_REF]. Moreover, the common toad is an explosive breeding species with a breeding period lasting only a few days, thus limiting the number of pairing opportunities [START_REF] Wells | The social behaviour of anuran amphibians[END_REF]. The operational sex ratio of this species is biased towards males, leading to scrambling competition among males to mate with a female.

During the breeding season, common toads are frequently found in urban and peri-urban areas with wetlands [START_REF] Beebee | Habitats of the british amphibians (2): suburban parks and gardens[END_REF], especially small ones (ponds for instances) which are subjected to ALAN [START_REF] Secondi | Variability of surface and underwater nocturnal spectral irradiance with the presence of clouds in urban and peri-urban wetlands[END_REF]. In addition, due to their high nocturnal visual sensitivity, amphibian activities, such as foraging and breeding, are expected to be affected by changes in night brightness [START_REF] Buchanan | Observed and potential effects of artificial night lighting on anuran amphibians[END_REF][START_REF] Grant | The lunar cycle: a cue for amphibian reproductive phenology?[END_REF][START_REF] Yovanovich | The dual rod system of amphibians supports colour discrimination at the absolute visual threshold[END_REF]. We have previously shown that ALAN exposure decreased common toad activity during the night and increased allocation of energy to maintenance [START_REF] Touzot | Artificial light at night disturbs the activity and energy allocation of the common toad during the breeding period[END_REF]. This suggested that breeding activities occurring at dusk or at night may be influenced by ALAN. Considering this, we predicted that (i) ALAN exposure would alter male breeding behaviour, particularly their ability to maintain pairing with a female, which is a costly activity for this species [START_REF] Lengagne | Cost of sexually embracing a large female offset by the number of eggs fertilized for small male Bufo bufo L[END_REF]), (ii) these behavioural alterations could be due to changes in male testosterone concentrations, and (iii) ALAN exposure would reduce male fertilisation success.

Methods

Animal collection and housing conditions

A total of 60 male common toads were collected during the breeding season (8-9 March 2018) in La Burbanche, France (45°N, 5°E). This site was chosen for its low levels of ALAN regardless of weather conditions and the lunar phase (≤0.01 lux). Upon arrival at the animal care facility (EcoAquatron, University of Lyon), males were weighed (LAB 800-3000, precision: 0.1 g, B3C pesage, Sérénité) and housed individually in boxes (47 x 36 x 25 cm) containing a 15 cm section of PVC tubing (diameter 10 cm) for shelter and 4 cm of litter that was moistened daily. Ambient temperature and relative humidity were kept during the whole experiment at 15.9 ± 0.6°C and 55.2 ± 2.9%, respectively. In these conditions, males were active during the night. Toads were fed ad libitum with live domestic crickets, Acheta domesticus, except during the mating trials. Sixty females of common toad collected at the same breeding site were grouped by 5 in large boxes (60 x 42 x 39 cm) with 10 cm of litter that was moistened daily. The ambient temperature was kept at 5.0 ± 1.0°C. At this temperature, females were inactive which prevented early spawning. Females were not exposed to ALAN. Two days before the end of male ALAN exposure, the temperature of the compartment containing females was increased to reach 15°C, a temperature which turns them active during the night.

Light treatments

During 7-8 days of acclimation, toads were exposed during the daytime to 254 ± 4 lux (mean ± SEM) provided by light tubes (Philips Master TL-D 58W/865 and Exo Terra Repti Glo 2.0, 40W T8) and remained in the dark (< 0.01 lux) during the scotophase. After the acclimation period, under the natural photoperiod at the date of the experiment, male toads were assigned to one of the three treatments (n=20 for each treatment) and exposed to their respective light treatment at night. To reproduce artificial light by night, we used white light-emitting diode (LED) ribbons (white cold Light Plus, 6000-6500 K, 14W, 60 LED/m). White LEDs were chosen because they are increasingly used for street lighting worldwide [START_REF] Falchi | The new world atlas of artificial night sky brightness[END_REF]. A LED ribbon of 95 cm (57 LED) was fixed 20 cm above the bottom of each box for each light treatment (see Appendix Supplementary Material S1 for details). All the ribbons in a given light treatment were connected to a dimmer (manual dimmer, 12V max, 8A) and a laboratory power supply (15V/DC max, 3A), which allowed to finely adjust the light intensity of each light treatment. The boxes assigned to one light treatment were isolated from each other, so that the light intensities did not interfere with each other. Under the natural photoperiod at the date of the experiment, during the night, the control group was exposed to 0.01 lux, corresponding to the illuminance of a sky under clear conditions with a quarter moon [START_REF] Gaston | The ecological impacts of nighttime light pollution: a mechanistic appraisal[END_REF]). The first experimental group was exposed to 0.10 lux (hereafter called the "0.1 lux-group"), corresponding to the illuminance of urban skyglow [START_REF] Gaston | The ecological impacts of nighttime light pollution: a mechanistic appraisal[END_REF]). The second experimental group was exposed to 5 lux (hereafter called the "5 lux-group"), which corresponds to the light level of a residential street [START_REF] Gaston | The ecological impacts of nighttime light pollution: a mechanistic appraisal[END_REF]). The daylight and 5 lux-group intensities were measured with a luxmeter (Illuminance meter T-10A, Konica Minolta). Intensities for the 0.10 lux and control groups were measured with a highly sensitive light meter (Sky Quality Meter-SQM-L, Unihedron). Consequently, to compare intensities, SQM measured values were converted into lux (see Appendix Supplementary Material S2 for details). Light intensities were measured at the bottom of the boxes and checked every week (0.01 ± 0.001 lux for the control group, 0.05 ± 0.01 lux for the 0.10 luxgroup and 5.01 ± 0.06 lux for the 5 lux-group, mean ± SEM). The exposure period lasted twelve days (D1 to D12), which corresponds to the average duration of a breeding period for this species in the wild [START_REF] Wells | The social behaviour of anuran amphibians[END_REF][START_REF] Reading | Male breeding behaviour and mate acquisition in the Common toad, Bufo bufo[END_REF].

Reproductive behaviours

On D13, exposure to nocturnal light was stopped, and the litter contained in the box was replaced with 21 l of water (one-third of dechlorinated water and two-third of pond water) and tree branches for oviposition. Females that had not been exposed to ALAN were assigned to a partner (we have checked that both female body mass and the ratio between male and female size did not statistically differ between light treatments). During the following 27 hours, we recorded hourly males that amplexed a female. Amplexus can be either successful (i.e., the male maintains the female until egg laying) or not (i.e., the male stops maintaining the female in amplexus). As parameter we recorded the latency to obtain successful amplexus, i.e., the duration required for males to form an amplexus leading to egg laying, and the separation rate, i.e., the percentage of pairs that broke amplexus before egg laying. The following days (between 1 and 7 days), we determined the duration of amplexus before the clutch was laid.

After laying, animals were removed, weighed and measured (Snout-to-Vent length, SVL) because previous studied showed that male pairing success was linked to their size [START_REF] Davies | Deep croaks and fighting assessments in toads Bufo bufo[END_REF][START_REF] Lengagne | Cost of sexually embracing a large female offset by the number of eggs fertilized for small male Bufo bufo L[END_REF]) and fertilisation rate was linked to the ratio between male and female size [START_REF] Davies | Optimal mate selection in the toad Bufo bufo[END_REF]. Four days after laying, we quantified the number of unfertilised eggs at the blastulation stage [START_REF] Lengagne | Cost of sexually embracing a large female offset by the number of eggs fertilized for small male Bufo bufo L[END_REF]) and the total number of laid eggs. These data allowed us to calculate the fertilisation rate, i.e., the proportion of fertilised eggs to the total number of laid eggs by a female.

Hormonal assay

On D13, males were weighed and measured, and a saliva sample was taken to measure their testosterone level. Sampling was carried out between 01:00 a.m. and 03:00 a.m., when testosterone concentration was the highest (see Appendix Supplementary Material S3 for details). Within the 2 min after capture, a dry cotton ball of known weight was introduced into the toad's mouth for 30 s to collect saliva. Each cotton ball was immediately weighed, placed into a microtube containing a filter to retain the cotton ball fibre during centrifugation and stored at -80°C. We investigated testosterone levels in saliva, which is a non-invasive method commonly used for the detection of steroids in wild species, including amphibians [START_REF] Kutsukake | Validation of salivary cortisol and testosterone assays in chimpanzees by liquid chromatography-tandem mass spectrometry[END_REF][START_REF] Janin | Use of stress-Hormone levels and habitat selection to assess functional connectivity of a landscape for an amphibian[END_REF][START_REF] Desprat | Immunocompetence handicap hypothesis in tree frog: trade-off between sexual signals and immunity?[END_REF]. Saliva was extracted from the cotton ball by the addition of 500 µl of phosphate buffer (1 M phosphate solution containing 1% bovine serum albumin, 4 M sodium chloride, 10 mM ethylenediaminetetraacetic acid, and 0.1% sodium azide) and centrifugation (10 000 g at 4°C for 5 min). We used a testosterone Enzyme Linked ImmunoSorbent Assay (ELISA) kit (number 582701, Cayman Chemical Company, Ann Arbor, MI, U.S.A.), validated for amphibians [START_REF] Desprat | Immunocompetence handicap hypothesis in tree frog: trade-off between sexual signals and immunity?[END_REF]) and previously optimized for common toads, to analyzed testosterone from saliva samples. Concentrations were calculated using a standard curve run on each plate and were estimated in 1 mg of saliva and expressed in pg mg -1 of saliva.

Statistical Analysis

We performed a linear model (LM) to test the effects of the light treatment (0.01, 0.1, 5 lux) on male body mass gain during ALAN exposure and on male body mass on D13. To confirm that there were no confounding differences in body size between light treatments, we performed a LM to test the effect of light treatment (0.01, 0.1, 5 lux) on male SVL measurement and on the ratio between male and female size on D13. Successful amplexus latency data were analysed as survival data following a Weibull distribution. Light treatment (0.01, 0.1, 5 lux), male body mass, female body mass, male salivary testosterone concentrations, and the interactions between light treatment and male body mass and between light treatment and male salivary testosterone concentrations were included as explanatory variables. The effect of light treatment (0.01, 0.1, 5 lux), male body mass, female body mass, male salivary testosterone concentrations and the interactions between light treatment and male body mass and between light treatment and male salivary testosterone concentrations on the probability of separation before egg laying (separation = 1; no separation = 0) was analysed through a generalized linear model (GLM) with a binomial distribution (logit link).

As the results of the model indicated a marginal effect for the chosen significance threshold (5%), we calculated the effect size (Cohen's h for proportions). We performed a GLM with a Gamma distribution to test the effect of the light treatment (0.01, 0.1, 5 lux), male body mass, female body mass and the interaction between light treatment and male body mass on amplexus duration before egg laying. For this analysis, one value is missing in the 5 luxgroup, as one female paired to a 5 lux-exposed male did not lay eggs. We performed a LM to test the effects of the light treatment (0.01, 0.1, 5 lux) and male body mass and their interaction on male salivary testosterone concentrations. For male salivary testosterone concentrations, one data point was excluded because the value was below the colour range of the ELISA kit. Male fertilisation rate was analysed through a GLM with a binomial distribution (logit link) using male body mass, female body mass, light treatment and the interaction between light treatment and male body mass as predictors. Because clutches are synchronous and, given the number of eggs to be counted, we counted fertilised eggs on a sub-sample of pairs (n = 10 for control, n = 9 for 0.1 lux and n = 8 for 5 lux), which corresponds to 94 102 eggs counted. For the analyses, the fitted models were simplified by a backward procedure to keep only the significant variables. For all models, multiple comparisons were performed using a Tukey adjusted post hoc test. All data were analysed using the software R 3.5.1 (R Core Team 2018) and the packages lsmeans (for successful amplexus latency and male fertilisation rate analysis), pwr (for the probability of separation before egg laying) and survival (for successful amplexus latency analysis).

Ethical note

The capture of common toads was authorized by the Préfecture de l'Ain (DDPP01-16-145) and by the French government (APAFIS#3655-2016011914372094) in accordance with the ethical committee of Lyon 1 University. The animal care structure "EcoAquatron" (University of Lyon) received an agreement of veterinary services (approval DSV 692661201). At the end of the experiments, all the toads and the egg clutches were released to their original site.

Results

ALAN affected reproductive behaviours of males (Fig. 1)

Male body mass gain during ALAN exposure was on average of 10.66 ± 1.27 g in the control group (mean ± SEM) and did not significantly differ between the light treatments (see Appendix Supplementary Material S4 for details). At mating, on D13, male body mass, male SVL measurement and the ratio between male and female size did not significantly differed between the light treatments (see Appendix Supplementary Material S4 for details). For all light treatments, each male performed an amplexus with the female. However, ALAN strongly affected the reproductive behaviours of male common toads. First, successful amplexus latency significantly differed between pairs with an exposed male and pairs with a control male (Fig. 1; X 2 2 = 14.01, P < 0.001). Males exposed to 0.1 or 5 lux took much longer to achieve successful amplexus as shown by the 3-fold increase (Tukey post hoc test P < 0.001) and the 1.5-fold increase in latency (Tukey post hoc test P = 0.004), respectively, compared with control males. No significant difference was found between males exposed to 0.1 or 5 lux (Tukey post hoc test P = 0.911). Male body mass negatively affected successful amplexus latency (X 2 1 = 4.58, P = 0.032). However, this variable was not affected by female body mass, by male salivary testosterone concentrations, by the interaction between light treatment and male body mass, nor by the interaction between light treatment and male salivary testosterone concentrations. Second, pairs with a control male never split after the amplexus was initiated, whereas the separation rate was by 20% and 10% for males exposed to 0.1 or 5 lux, respectively (X 2 2 = 5.99, P = 0.050). Even if the model indicated a marginal effect for the chosen significance threshold, the effect size was strong (Cohen's test |h| = 0.795), reflecting a clear biological difference. The marginal effect may likely due to low statistical power. A stronger effect may have been observed with more replicates. We did not observe an effect of male body mass, of female body mass, of male salivary testosterone concentrations, of the interaction between light treatment and male body mass or of the interaction between light treatment and male salivary testosterone concentrations on the separation rate. The time spent in amplexus before egg laying was not altered by light treatment, as pairs spent on average 55 ± 4 hours (mean ± SEM) in amplexus before egg laying. We neither observed an effect of male body mass, female body mass, nor of the interaction between male body mass and light treatment on the time spent in amplexus before egg laying (see Appendix Supplementary Material S4 for details).

ALAN did not modify male salivary testosterone concentrations

After 12 days of exposure, the mean salivary testosterone concentration of the control group was 3.7 ± 0.69 pg mg -1 of saliva (mean ± SEM). Exposure to ALAN did not modify male salivary testosterone concentrations. We neither observed an effect of male body mass nor of the interaction between male body mass and light treatment on testosterone levels (see Appendix Supplementary Material S4 for details).

Exposure to ALAN reduced male fertilisation success (Fig. 2) When all light treatments were combined, the average clutch size laid by a female was 3347 ± 272 eggs (mean ± SEM). Male fertilisation success was affected by the interaction between the male body mass and light treatment (Fig. 2; X 2 2 = 248.5, P < 0.001). By visually interpreting the difference of slopes, we noticed that in the control and 5 lux groups, the relationship between the fertilisation rate and male body mass was weakly negative, whereas in the 0.1 lux group, this relationship was weakly positive. Moreover, control males had an average fertilisation rate of 75.14 ± 6.23% (mean ± SEM), while a 25%-decrease was highlighted in the 5 lux-group compared with control males (Tukey post hoc test P < 0.001).

A difference between the 0.1 lux-and 5 lux-exposed males was also observed (Tukey post hoc test P < 0.001).

Discussion

Here, we demonstrated that exposure to low light intensities at night during the breeding period alters both the mating behaviours and the fertilisation success of common toad males.

Indeed, ALAN affected mating behaviour as male common toads previously exposed to ALAN needed several attempts to maintain a female in amplexus until clutch laying, although all males finally paired in our experimental setup. In line with this first result, the latency to pair successfully with a female was strongly increased by previous exposure to ALAN as well as male body mass. Similarly, Drosophila melanogaster reared under 10 lux took longer to initiate mating than control flies [START_REF] Mclay | Dim artificial light at night affects mating, reproductive output and reactive oxygen species in Drosophila melanogaster[END_REF]). In the wild, male-male competition is strong in common toad with values of operational sex ratio as high as 4 or 5 males to one female [START_REF] Davies | Competitive mate searching in male common toads, Bufo bufo[END_REF]. Therefore, ALAN might increase male-male competition as females will be caught and released several times before one male manages to stay in amplexus until egg laying is complete. The strong operational sex ratio should probably prevent males that release females from having another possibility to gain a female during the breeding season. In accordance with the important role of photoperiod as a cue to initiate the reproductive season in many species, most studies have shown that ALAN exposure advances the date of reproduction [START_REF] Ikeno | Dim light at night disrupt the short-day response in Siberian hamsters[END_REF][START_REF] Dominoni | Light at night, clocks and health: from humans to wild organisms[END_REF][START_REF] Tallec | Melatonin concentration and timing of seasonal reproduction in male mouse lemurs Microcebus murinus exposed to light pollution[END_REF]. Other direct effects of artificial light on sexual behaviour have also been highlighted in various species. For example, [START_REF] Botha | Effects of lifetime exposure to artificial light at night on cricket Teleogryllus commodus courtship and mating behavior[END_REF] reported that Australian black field crickets, Teleogryllus commodus, reared under a very high light intensity (100 lux) were more likely to mate than individuals reared under lower light (0, 1 and 10 lux). Further, male Blue tits, Cyanistes caeruleus, occupying territories exposed to streetlights (> 0.06 lux) were twice as successful in obtaining extra-pair mates as males occupying territories further away inside the forest, but no information is given about their breeding success with their social partners [START_REF] Kempenaers | Artificial night lighting affects dawn song, extra-pair siring success and lay date in songbirds[END_REF]). In addition, ALAN reduced the proportion of mated females Winter moth, Operophtera brumata, compared with control females, and the reduction was strongest under green ALAN than red ALAN (Van Greffen et al. 2015). These observations suggest that ALAN may differentially affect mating behaviour depending on animal species and on spectral composition. In our study, we used white cold LED, that emit a prominent peak of blue wavelength. It is well known that many species, including amphibians [START_REF] Hailman | Phototactic responses to spectrally dominant stimuli and use of colour vision by adult anuran amphibians: a comparative survey[END_REF], are sensitive to blue wavelength light, which provokes behavioural and physiological modifications [START_REF] Musters | The effects of coloured light on nature: A literature study of the effects of part of the spectrum of artificial light on species and communities[END_REF]). It would therefore, be highly interesting to study the effects of low colour temperature and other spectra on the fitness of amphibians.

ALAN negatively affected male fertilisation success, a direct measurement of the breeding component of male fitness that does not provide information on offspring quality but instead informs on their potential number. Present data showed that large males may be more sensitive to high light intensity since under 5 lux, the fertilisation rate was 25% lower than in controls and decreased as body mass rose. The weak negative relationship between fertilisation rate and male body mass in the controls contrasts with earlier studies showing either a positive or no relationship between fertilisation rate and the ratio between male and female size in wild common toads [START_REF] Davies | Optimal mate selection in the toad Bufo bufo[END_REF][START_REF] Lengagne | Cost of sexually embracing a large female offset by the number of eggs fertilized for small male Bufo bufo L[END_REF]). The mechanism underlying the deleterious effect of ALAN in large males is unclear. Interestingly, it was suggested that the energetic cost of amplexus increases with size in an anuran species [START_REF] Mclister | The metabolic cost of amplexus in the grey tree frog (Hyla versicolor): assessing the energetics of male mating success[END_REF]. Furthermore, we also noticed an effect of body mass on the ALAN-induced rise in basal metabolic rate of common male toads. In males exposed to 5 lux during the night, the largest males showed the highest increase in metabolic rate (Touzot, unpublished data).

Further investigations are needed to clarify the link between amplexus cost and fertilisation rate. In amphibian species, large males often have more opportunities to access reproduction than small males [START_REF] Davies | Optimal mate selection in the toad Bufo bufo[END_REF]. By altering behaviour and fertilisation success, especially in larger males, ALAN may lead to an increase in the genetic contribution of poorquality males in populations naturally exposed to ALAN. Another scenario could be that larger males would win the competition for female access, but as being the most affected by ALAN, fail at fertilisation, and thus contribute to the reduction in the overall reproductive success of the population.

The physiological mechanisms responsible for the 25% reduction in fertilisation rate in male exposed to 5 lux at night as compared with control are unknown but may be linked to spermatogenesis. Effectively, several studies have reported that exposure to continuous light causes spermatocyte degeneration [START_REF] Rastogi | Seasonal cycle in anuran amphibia testis: the endocrine and environmental controls[END_REF][START_REF] Biswas | Effect of continuous Light and Darkness on the testocular histology of toad (Bufo melanostictus)[END_REF]. It should however be noted that in anurans, spermatogenesis lasts 5 or 6 weeks on average [START_REF] Jørgensen | Growth and Reproduction[END_REF]. With regard to the rather short duration of ALAN exposure used in our protocol (males have been exposed to ALAN for 12 days only), a marked deleterious effect on spermatogenesis would be limited. Another possibility is that the fertilisation rate decrease can likely be explained by an alteration of reproductive hormone synthesis. In vertebrates, the effects of ALAN occur at all levels of the hypothalamic-pituitary-gonadal axis, leading to variations in testosterone levels (reviewed in [START_REF] Ouyang | Hormonally mediated effects of artificial light at night on behavior and fitness: linking endocrine mechanism with function[END_REF][START_REF] Brüning | Influence of artificially induced light pollution on the hormone system of two common fish species, perch and roach, in a rural habitat[END_REF]. Testosterone would not be involved in the reduced fertilisation rate of common toads as ALAN did not modify salivary testosterone levels. Present observation in toads therefore contrasts with results in birds in which ALAN modified the testosterone secretion pattern during a breeding period [START_REF] Dominoni | Artificial light at night advances avian reproductive physiology[END_REF]) and increased corticosterone levels [START_REF] Ouyang | Stressfull colours: corticosterone concentrations in a free-living songbird vary with the spectral composition of experimental illumination[END_REF]. The quality of females paired with males cannot be an explanation for our results since females were not subjected to ALAN and we have controlled that female body mass and the ratio between male and female size did not statistically differ among light treatments, thus excluding a female effect. Finally, an alteration of the synchronization of gametes released and/or a reduction in the amount of sperm released by the male can likely explain the fertilisation rate reduction. Indeed, as in all external fertilisation situations, the male must synchronize its sperm release so that it coincides with egg release [START_REF] Davies | Optimal mate selection in the toad Bufo bufo[END_REF]. Since males exposed to ALAN show increased basal metabolic rate but reduced activity-related energy expenditure [START_REF] Touzot | Artificial light at night disturbs the activity and energy allocation of the common toad during the breeding period[END_REF], sperm emission that requires smooth muscle contractions might also be impaired.

Conclusion

Although some studies have investigated the effects of ALAN on the mortality component of fitness [START_REF] Rodríguez | Fatal attraction of Short-Tailed Shearwaters to Artificial lights[END_REF][START_REF] Van Doren | Highintensity urban light installation dramatically alters nocturnal bird migration[END_REF][START_REF] Willmott | Artificial light at night alters life history in a nocturnal orb-web spider[END_REF], reports of effects on the breeding component are still scarce, especially in amphibian species. This study showed that relevant nocturnal artificial light intensities can have a major effect on the breeding component of fitness of animals in the wild. Even if a reduction in fertilisation rate was observed at the rather high intensity of 5 lux, the effect although weaker probably occurs at lower light intensities. The separation rate during amplexus, for instance, was affected at an intensity as low as 0.1 lux, which is equivalent to a full moon. If extrapolated to habitats exposed to human activity-related ALAN, present experimental findings suggest that many toad populations in Europe are expected to incur reproductive losses in their breeding habitats. A better mechanistic understanding of the effects of ALAN, particularly on reproduction, is necessary to predict long term consequences on wildlife and propose appropriate management solutions in an increasingly lighted world.

Fig. 1 .

 1 Fig. 1. Successful amplexus latency (hour) of males previously exposed for 12 days to 0.01

Fig. 2 .

 2 Fig. 2. Fertilisation rate (%) of males previously exposed for 12 days to 0.01 (black dots and

Acknowledgements

We thank A. Clair, J. Ulmann and L. Averty for their technical assistance in the EcoAquatron; L. Guillard for his help with the setting up of LED ribbons; and Elsevier for English corrections.

Funding

This work was supported by the French Government [PhD grants 2017-2020], by the 'LABEX IMU Laboratoire d'excellence Intelligence des Mondes Urbains', by IDEX Initiative d'excellence Université Lyon. The authors declare no competing or financial interests.