R.-C Zhuang

T Burghardt

E Mäder

Study on Interfacial Adhesion Strength of Single Glass Fibre/Polypropylene Model Composites by Altering the Nature of the Surface of Sized Glass Fibres

Keywords: A. Glass fibres, A. Coupling agents, A. Polymer-matrix composites (PMCs), B. Interfacial strength, B. Surface treatments

Polypropylene (PP) compatibly sized glass fibres (GFs) were treated with boiling water and toluene, respectively, to reveal the interactions of water and toluene with different components in the sizing of sized GF and their influences on the interfacial adhesion strength of GF/PP model composites.

Compared to control GF/PP model composites, about 30% increase of interfacial adhesion strength was achieved for composites with water-treated GF, whereas a small decrease of interfacial adhesion strength was revealed for composites with toluene-treated GF. X-ray photoelectron spectroscopy, Fourier transform infrared spectroscopy, Zeta-potential measurement, and water contact angle measurement demonstrated that the boiling water treated GFs posses a more polar and hydrophilic surface with homogeneously distributed derivatives of γ-aminopropyltriethoxysilane, which is related to a higher interfacial adhesion strength for water treated GF/PP model composites.

In contrast, hot toluene treated GFs led to a more hydrophobic surface with low molar mass PP and surfactants enriching on the outermost surface.

Introduction

The interphase between reinforcement GF and matrix in GF/polymer composites is of vital importance since the mechanical properties of composite materials are mainly determined by whether the mechanical stresses can be efficiently transferred from matrix to the reinforcing fibres [START_REF] Kim | High strength, high fracture toughness fibre composites with interface control-A review[END_REF][START_REF] Pukánszky | Interfaces and interphases in multicomponent materials: past, present, future[END_REF][START_REF] Ishida | A review of recent progress in the studies of molecular and microstructure of coupling agents and their functions in composites, coatings and adhesive joints[END_REF][START_REF] Mäder | Interfacial design in fiber reinforced polymers[END_REF]. It is established that the interfacial properties of composites are mainly determined by the reactivity and compatibility between the matrix composition and the surface properties of used GFs. Tailoring the nature of the GF surface, e.g. chemical composition or geometry, has long been used to enhance GF-matrix adhesion by increasing the covalent bond density, mechanical interlock, etc [START_REF] Jones | Interphase formation and control in fibre composite materials[END_REF]. From the industrial production point of view, on-line coating as-spun GF with an appropriate sizing is the most straightforward means to meet this objective [START_REF] Thomason | Sizing up the interphase: an insider's guide to the science of sizing[END_REF].Usually, a sizing is an aqueous mixture of polymeric film former, coupling agent, processing aids (such as lubricant) and/or other additives [START_REF] Frenzel | Influence of different glass fiber sizings on selected mechanical properties of PET/glass composites[END_REF][START_REF] Frenzel | Influence of different interphases on the mechanical properties of fiberreinforced polymers[END_REF]. In general, the good combination of sizing components plays an important role in determining the GF-matrix adhesion [START_REF] Zinck | Role of silane coupling agent and polymeric film former for tailoring glass fiber sizings from tensile strength measurements[END_REF].

It is noted that under aqueous condition the functional groups, mostly amino group, of (hydrolyzed and condensed) silanes and the polar groups of surfactants and additives are able to interact with the surface silanol group of the GF via H-bonding, electrostatic interaction or dipole-dipole interaction [START_REF] Ofir | Controlled amine functionality in self-assembled monolayers via the hidden amine route: chemical and electronic tunability[END_REF][START_REF] Lee | Wettability and conformation of reactive polysiloxanes[END_REF][START_REF] Bascom | Structure of silane adhesion promoter films on glass and metal surfaces[END_REF][START_REF] Ishida | Fourier transform infrared spectroscopic study of the structure of silane coupling agent on E-glass fiber[END_REF][START_REF] Naviroj | Structure and adsorption characteristics of silane coupling agents on silica and E-glass fiber; dependence on pH[END_REF]. Meanwhile, the physically adsorbed polysiloxanes may degrade the GF-matrix adhesion [START_REF] Wu | Polymer interface and adhesion[END_REF]. As a result, the fracture of GF reinforced composites usually initiates at weak areas in the interphase, e.g. with low coupling agent density, and then progresses through the matrix. Great efforts have been conducted to understand the influence of these weak aspects [START_REF] Ishida | A review of recent progress in the studies of molecular and microstructure of coupling agents and their functions in composites, coatings and adhesive joints[END_REF][START_REF] Jones | Interphase formation and control in fibre composite materials[END_REF][START_REF] Plonka | Adhesion of epoxy/glass fibre composites influenced by aging effects on sizings[END_REF][START_REF] Wu | Effects of silane coupling agents on the interphase and performance of glass-fiber-reinforced polymer composites[END_REF][START_REF] Gorowara | Molecular characterization of glass fiber surface coatings for thermosetting polymer matrix/glass fiber composites[END_REF][START_REF] Cheng | Effect of fibre conditioning on the interfacial shear strength of glass-fibre composites[END_REF]. Among them, treating sized GFs with solvents, usually acetone or water, to partly wash out unwanted components is a simple way for fundamental research. Prior works in this field mainly focused on GFs sized with pure silane coupling agents [START_REF] Ishida | A review of recent progress in the studies of molecular and microstructure of coupling agents and their functions in composites, coatings and adhesive joints[END_REF], only a few studies were performed on GFs sized with the complete sizing package and most of them were conducted on thermosetting polymer compatible GFs [START_REF] Gorowara | Molecular characterization of glass fiber surface coatings for thermosetting polymer matrix/glass fiber composites[END_REF][START_REF] Cheng | Effect of fibre conditioning on the interfacial shear strength of glass-fibre composites[END_REF][START_REF] Thomason | The interface region in glass fibre-reinforced epoxy resin composites: 3. Characterization of fibre surface coatings and the interphase[END_REF][START_REF] Liu | XPS and AFM study of interaction of organosilane and sizing with E-glass fibre surface[END_REF][START_REF] Mallarino | Glass fibre sizing effect on dynamic mechanical properties of cyanate ester composites I. Single frequency investigations[END_REF]. There are limited studies on PP compatibly sized GFs. In this study, water and toluene were used to selectively extract a PP compatible GF.

Extracting GF with hot water is similar to aging. The aging of sizings was already investigated by Ishida and Koenig in 1979 [START_REF] Ishida | An investigation of the coupling agent/matrix interface of fiberglass reinforced plastics by fourier transform infrared spectroscopy[END_REF], the significant changes on silane treated E-glass surface after storage in water at 80 o C were observed. However, they did not correlate the surface changes with the adhesion strength changes in composites. Jones et al. extracted 1% 3aminopropyltriethoxysilane (γ-APS) sized GF with 40 o C warm water and 100 o C hot water sequentially and confirmed that the sizing layer structure is the same as the reported one [START_REF] Liu | XPS and AFM study of interaction of organosilane and sizing with E-glass fibre surface[END_REF][START_REF] Wang | Surface analytical study of the interaction between -amino propyl triethoxysilane and E-glass surface Part II: X-ray photoelectron spectroscopy[END_REF][START_REF] Wang | Surface analytical study of the interaction between -amino propyl triethoxysilane and E-glass surface Part I: Time-of-flight secondary ion mass spectrometry[END_REF]. Moreover, it was found that adsorbed water molecules could migrate towards the sizing/GF interface through the thin sizing, resulting in the dissolution/decomposition of polysiloxane [START_REF] Wu | Polymer interface and adhesion[END_REF][START_REF] Roy | Effects of moisture on the mechanical properties of glass fibre reinforced vinylester resin composites[END_REF] or the growth of a silica gel layer [START_REF] Axelos | Small-angle X-ray scattering investigation of the silica/water interface: evolution of the structure with pH[END_REF]. On the other hand, it is known that surfactant adsorbed on glass plates can be replaced with polymer solution or washed away by solvents, even though Jones et al. found that solvent extraction could not remove sizing resins efficiently [START_REF] Yumitori | The role of sizing resins in carbon fibre-reinforced polyethersulfone (PES)[END_REF][START_REF] Cheng | Sizing resin structure and interphase formation in carbon fibre composites[END_REF]. Recently, it was revealed by Mäder et al. that extracting only γ-APS sized GFs with hot water led to a great increase in the local interfacial shear strength and the energy release rate of epoxy/GF single fibre model composite since water washed off excessive physically adsorbed γ-APS [START_REF] Plonka | Adhesion of epoxy/glass fibre composites influenced by aging effects on sizings[END_REF]. The same effect was also observed by Jones et al. in their studies on AR glass and E-glass reinforced vinyl ester composites [START_REF] Cheng | Effect of fibre conditioning on the interfacial shear strength of glass-fibre composites[END_REF]. Hence, it is worthwhile to know the interaction of water molecules with PP compatibly sized GF and the influence of the surface change on the properties of the resulting model composite.

At elevated temperature, toluene is a good solvent for PP, polymeric film former of PP compatible sizings, and the physically adsorbed polysiloxane [START_REF] Basarir | Preparation of γ-APS monolayer with complete coverage via contact printing[END_REF]. Therefore, extracting sized GF with hot toluene can remove the physically adsorbed polysiloxane, the low molecular weight (MW) fraction of PP [START_REF] Mäder | Characterization and design of interphases in glass fiber reinforced polyproplyene[END_REF], and surfactants. On the other hand, long time exposure to hot toluene can also lead to the enrichment of hydrophobic PP, especially the small MW fraction of PP [START_REF] Demarquette | Influence of temperature, molecular weight, and molecular weight dispersity on the surface tension of polystyrene, polypropylene, and polyethylene. II. Theoretical[END_REF], on the outermost surface of sizing layer due to solvent annealing effect, this might lead to a more ductile or a weaker interphase.

In this work, surface sensitive analysis methods (dynamic contact angle measurement, atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS), and Zeta-potential measurement) were used to probe the changes of surface properties of as-spun and differently treated GFs. The influence of hot water and toluene treatments on the interfacial adhesion strength of single fibre/PP model composite was studied using single fibre pull-out test.

Experimental Details

Materials. Toluene (Acros) and γ-APS (Degussa) were used as received. Water was deionized to a resistivity of 15 M •cm using a Millipore Elix 5 water purification system. E-glass fibres sized by -APS in conjunction with PP film former (about 1:2.6 in dry weight ratio) were spun at the Leibniz Institute of Polymer Research Dresden using a continuous spinning device comparable to industrial ones. The average diameter of used sized GF is 15 m. The as-prepared sized GF is named M1. M1 was extracted with boiling water and toluene for 8 h using a Soxhlet extractor with the aim to selectively extract sizing layer, the resulting GFs are referred as M1-W and M1-T (shown in Tab. 1),

respectively. An isotactic polypropylene (weight average molecular weight (M w ) = 23.8 × 10 4 g/mol) compounded with 2 wt.% MAH-g-PP (Polybond 3200) was used as matrix.

Methods.

High temperature gel permeation chromatography (HT-GPC) was performed at 150 o C using trichlorobenzene as solvent and eluent on a PL GPC220 (Polymer Laboratories) equipped with 2 PL Mixed B LS separating columns using triple detection (refractive index, light scattering, and viscosity). Room temperature GPC was carried out on a PL GPC220 (Polymer Laboratories) equipped with a PL Mixed-C column using tetrahydrofurane as solvent and eluent. XPS investigations were performed on a Kratos AXIS Ultra X-ray photoelectron spectrometer.

Areas of approximately 300 x 700 microns were analyzed with a monochromatic Al Kα X-ray source. The samples were analyzed via angle-resolved XPS (AR-XPS) at normal (90º) and glancing (60º, 30º, and 15º) take-off angles (TOAs) in order to investigate the sizing surface chemistry as a function of analysis depth. The TOA is the angle between the analyzer and sample surface. At a 90º TOA, the XPS is capable of analyzing the top 8 nm of the sample surface, while at a 15º TOA only the top 2-2.5 nm of the sample surface is investigated. Due to the insulating nature of the samples, charge neutralization of the sample surface was required. Survey spectra were collected from six different areas of each sample at 90º TOA and 2 areas of each sample at 60º, 30º, and 15º TOAs.

The survey spectra were collected over a wide binding energy range (0-1300 eV) and were used to evaluate all of the elements present (except H and He) within the sample surface. The survey spectra were acquired at a pass energy of 160 eV and a step size of 1 eV. The high-resolution spectra were dissected by means of the spectra deconvolution software. The parameters of the component peaks were their binding energy, height, full width at half maximum, and the Gaussian-Lorentzian ratio.

Tapping mode AFM measurements were carried out using Dimension 3100 (Digital Instruments, Santa Barbara) at room temperature. The values of root-mean-square roughness (R q ) and maximum height roughness (R max ) were calculated over the AFM images (3 µm x 3 µm).

Electrokinetic properties, Zeta-potential as a function of pH, of sized GFs were obtained by electrokinetic analysis (EKA) on an electrokinetic analyzer (Fa. Anton Paar KG, Austria).

The dynamic advancing contact angle (θ a ) and receding contact angle (θ r ) measurements on single GFs were performed on a tensiometer K14 (Krüss GmbH, Hamburg, Germany).

The FTIR investigations were carried out on a Bruker IFS66 spectrometer. Transparent polymer films of PP film former and its acetone insoluble part with a thickness of 250 µm were prepared by compression molding at 190 o C. All extractions were analyzed in form of KBr pellets.

The NMR spectra were collected on a Bruker DRX 500 NMR spectrometer at 500.13 MHz for 1 H NMR spectrum and 125.75 MHz for 13 C NMR spectrum. The signal assignment was done by 1 H-1 H COSY and 1 H- 13 C HMQC 2D NMR experiments using the standard pulse sequences provided by Bruker. For internal calibration the solvent peaks of DMSO-d 6 were used: δ ( 13 C) = 39.60 ppm; δ ( 1 H) = 2.50 ppm. 13 C NMR spectrum was used for peak assignment and is not shown at here. Single fibre pull-out test was performed on single fibre model composites to study the interfacial adhesion strength. The fibres were end-embedded 300 m deep into matrix at 200 o C with a heating and cooling rate of 50 K/min. From each force-displacement curve, the force F d at the start of debonding and the embedded length, l e , were derived. The apparent adhesion strength τ app was determined according to eq. 1

e f d app l D F π τ = , (1)
where D f is fibre diameter.

The local adhesion strength τ d and the critical interphase release rate G ic were calculated

according to ] ) ( ( [ ) 2 / ( 2 / 1 2 2 ic f d G q p p D - + - = π τ (2)
where p and q(G ic ) are terms depending on fibre and matrix properties and specimen geometry [START_REF] Zhandarov | Investigation of load transfer between the fiber and the matrix in pull-out tests with fibers having different diameters[END_REF].

Results and discussion

Remarks on M1 fibre and used PP film former

The organic content of M1 fibre is 1.1 wt.% determined by pyrolysis following DIN EN ISO 1172.

The average sizing thickness is about 36 nm assuming a homogenous sizing was formed and the density of sizing is 1 g/cm 3 . The commercially available PP film former is in the form of aqueous dispersion with a solid content of 35 wt.%, which was added drop-wise to acetone to get an acetone soluble part (Part-I) and an acetone insoluble part (Part-II), the weight ratio of Part-II to Part-I is about 4:1. Part-I was subjected to NMR, FTIR, and GPC analyses while Part-II was analyzed using FTIR and HT-GPC. The results are shown in Fig. 1 and Fig. 2 for 1 H NMR spectrum and FTIR spectra, respectively. The NMR spectrum of Part-I (Fig. 1) revealed that the main fraction of Part-I is alkyl polyethylene glycol ether. The peak assignment is detailed in following. The triplet peaks centered at 0.85 ppm are assigned to the CH 3 -of alkyl group. The intensive peak at 1.24 ppm arises from the -CH 2 -of alkyl group. A group of multiplet peaks between 3.3~3.6 ppm, except the resonance peak of H 2 O at 3.51 ppm, are due to the resonance of -CH 2 O-. Additionally, Part-I has a small amount of low molecular amines, most likely N-methyl ethylamine and/or its derivatives. The singlet peak at 2.37 mainly arises from the CH 3 N-of N-methyl ethylamine, while the quadruplet peaks between 2.76~2.80 are attributed to the resonance peak of -CH 2 N-of N-methyl ethylamine next to methyl group, which gives raise to the triplet peaks around 1.06 ppm. On the other hand, the FTIR spectrum of Part-I (Fig. 2d) shows only the typical absorbance bands of alkyl polyethylene glycol ether. In detail, the strong absorbance bands between 1080~1150 cm -1 are attributed to the stretching vibration of C-O, the intensive band at 1464 cm -1 arises from the bending vibration of CH 2 , while the broad band between 3200~3600 is assigned to the stretching vibration of OH and/or NH. GPC analysis revealed that the number average molecular weight (Mn) of Part-I is 920 g/mol with a narrow polydispersity index of 1.25. The main component of Part-II is MAH-g-PP, as revealed by FTIR in Fig. 2e. Except the typical absorbance bands of isotactic-PP, additional bands at 1775 and 1735 cm -1 are attributed to the stretching vibration of anhydride and other carbonyl groups. HT-GPC showed that Part-II has Mn of 75400 g/mol, which is quite close to that of PP dispersion (80600 g/mol). Moreover, Part-II has also a tiny amount of surfactant or other low MW components, as a weak peak at low molar mass region was present in the HT-GPC curve.

Analyses on extractions

Extracting M1 fibre in hot water and toluene, respectively, both led to a weight loss, the weight loss of M1 fibre is slightly higher in the case of hot water treatment (Tab. 1). It is noted that both hot water and toluene extracted mainly one component from the sizing layer, basing on the following observations. The extract in hot water was subjected to FTIR analysis and the result is shown in Fig. 2a. For a better comparison, Fig. 2c and Fig. 2b show the FTIR spectra of acetone soluble part of PP dispersion and the extract of only γ-APS sized GF using hot water, respectively. For the extract of only γ-APS sized GF (Fig. 2b), the absorbance bands at 2923 and 2852 cm -1 are assigned to asymmetric and symmetric stretching vibration of >CH 2 , the strong and broad peak at 1029 cm -1 is the typical stretching absorbance band of Si-O-Si. The absorbance bands between 1650~1130 cm -1 are attributed to the stretching vibration of carbonyl group and bicarbonate salts due to the interaction between the amino group of γ-APS based components and environmental CO 2 and water [START_REF] Bayer | FTIR spectroscopic studies of interfacial reactions between amino functionalized silicon surfaces and molten maleic anhydride copolymers[END_REF][START_REF] Ishida | The structure of an aminosilane coupling agent in aqueous solutions and partially cured solids[END_REF]. From the similarity between Fig. 2a and Fig. 2b one can conclude that the wash-out of M1 fibre in hot water mainly is γ-APS based components. Since the intensity of the peaks at 2923 and 2852 cm -1 is stronger in Fig. 2a, alkyl polyethylene glycol ether could be also included. However, the wash-out might also comprise small molecular amines and small amounts of PP, which bond or interact with physically adsorbed γ-APS based components and were extracted into water with physically adsorbed γ-APS based components.

Upon extraction in hot toluene, mainly alkyl polyethylene glycol ether was washed out as revealed in the FTIR spectrum (Fig. 2d). However, components with Mn of 23800 g/mol were detected by HT-GPC, this fraction can be assigned to PP or the complex of PP with γ-APS, since it is known that the physically adsorbed γ-APS based components do not posses such high MW [START_REF] Wang | Surface analytical study of the interaction between -amino propyl triethoxysilane and E-glass surface Part I: Time-of-flight secondary ion mass spectrometry[END_REF].

Nevertheless, one can not exclude the presence of this part, its concentration might be just beyond the sensitivity of methods used.

Surface properties of fibres

The impact of solvent treatment on the surface properties of GFs was firstly studied by mapping the surface topography using tapping mode AFM. Fig. 3 presents AFM height images of M1-T and M1-W fibres as well as M1 fibre, the surface roughness data of GFs are summarized in Tab. 1. Fig. 3a shows that the surface of M1 fibre looks like pine tree bark characterized by the irregular islands in a sea. Upon extraction with hot toluene, the surface of M1-T fibre turns rougher (Fig. 3b), which probably is due to the swelling of toluene in sizing during extraction, which collapsed or shrank in the course of drying at 80 o C under vacuum. Correspondingly, the R q and R max values increase from 3.5 and 41.5 nm to 9.6 and 88.6 nm, respectively. On the other hand, extracting M1 fibre in hot water did not change the surface topography of the resulting fibres as much as in hot toluene. There is only a small increase in both R q and R max values.

Water contact angle measurements were performed to obtain information concerning the hydrophobicity of the sizing of single fibres. Tab. 1 summarizes dynamic advancing contact angles (θ a ) and receding contact angles (θ r ) as well as the hysteresis value (θ a -θ r ). The control M1 fibre has θ a of 94.3 o and θ r of 68.5 o . After toluene extraction, θ a and θ r of M1-T fibre increase a little bit to 99.2 o and 72 o , respectively. As FTIR and HT-GPC analyses revealed that surfactants and PP with low MW were extracted into hot toluene, the higher water contact angle is related to the enrichment of low MW PP on the topmost surface since PP with lower MW has lower surface tension compared to the high MW analogues [START_REF] Demarquette | Influence of temperature, molecular weight, and molecular weight dispersity on the surface tension of polystyrene, polypropylene, and polyethylene. II. Theoretical[END_REF]. Extracting M1 fibre with boiling water altered the surface of sized GF from hydrophobic to hydrophilic, indicated by the significant decrease of both θ a and θ r . This could be due to two reasons. One is that amide groups, imide groups and/or ester groups converted into more hydrophilic carboxyl groups via hydrolysis reaction, meanwhile the polysiloxane also underwent hydrolysis resulting in more polar silanol groups. This is confirmed by XPS analysis demonstrated in next paragraphs. The other reason is the annealing affect, the high temperature and highly humid atmosphere drove polar groups to migrate towards the sizing/air interface.

AR-XPS was used to probe the radial distribution of sizing components within the sizing, the average surface compositions in atomic percentage are presented in Tab. 2. The C 1s high resolution spectra were curve-fitted with four peaks. The corresponding peak assignments are listed in Tab. 3.

The peaks of interest in the C 1s spectra are C-1 and C-2, which correspond to C-C and C-N/C-O chemical states, respectively. C-3 and C-4 correspond to different types of surface oxidation of the carbon. In addition, the N 1s high resolution spectra were curve-fitted with two peaks, which correspond to amine and protonated amine chemical states. Here, only the degrees of nitrogen protonation are reported in Tab. 3.

For the M1 fibre, a 20% increase in C and 40% decrease in N were found at 15 o TOA versus at 90 o TOA, while the percentage of C-C bonds remained constant and there was a 10% decrease in C-N/C-O bonds. This agrees with the results of contact angle measurements indicating the non-polar components of polymeric film former enrich on the outermost surface. In addition, it was determined that at 90 o TOA 10% of the amine groups are protonated while no protonation was observed at the air/sizing interface. Usually it is assumed that the protonation of the amino group is mostly induced by the interaction of amino groups with silanol groups either via intra-molecule or inter-molecule H-bondings [START_REF] Ofir | Controlled amine functionality in self-assembled monolayers via the hidden amine route: chemical and electronic tunability[END_REF][START_REF] Lee | Wettability and conformation of reactive polysiloxanes[END_REF][START_REF] Bascom | Structure of silane adhesion promoter films on glass and metal surfaces[END_REF][START_REF] Ishida | Fourier transform infrared spectroscopic study of the structure of silane coupling agent on E-glass fiber[END_REF][START_REF] Naviroj | Structure and adsorption characteristics of silane coupling agents on silica and E-glass fiber; dependence on pH[END_REF]. Thus a higher degree of protonation serves as indirect evidence supporting the above observation that the aminosilane is enriched at glass/sizing interface.

For the toluene washed fibre (M1-T), at TOA of 90 o , a 5% decrease in C was detected while the N percentage was similar to the M1 fibre. This small drop in C correlates to the FTIR and GPC results, which revealed surfactants and PP in the toluene extract. In addition, a 30% increment in C and 35% decrease in N were observed at 15 o TOA in comparison with at 90 o TOA. These results are similar to those found for the M1 fibre prior to extraction. Moreover, C-C bonds show about a 10% increase while about 40% reduction in C-N/C-O bonds was found compared to the M1 sample, which is in a good agreement with the FTIR analysis of the wash-off of M1 fibre in hot toluene. On the other hand, a 5% drop in C-C bonds and about 30% increment in C-N/C-O bonds were observed as the TOA was decreased. The large increase in the C-2 peak is probably due to an enrichment of the C-O bonds from either polyethylene glycol or oxidation of the outermost surface of the fibre or more amino groups are present in the outermost surface upon extraction as observed by Zetapotential measurement. Moreover, it was found that 20% of the amine groups at TOA of 90 o are protonated while no protonation was found at the outermost surface of the fibre.

For the water washed fibre (M1-W), at TOA of 90 o , a 15% drop in C and 40% decrease in N were observed compared with the M1 sample. A 10% increase in C-C bonds and 30% decrease in C-N/C-O bonds were observed compared to the M1 sample. This suggests that the boiling water extraction removed physically adsorbed and/or chemisorbed coupling agent and surfactants from sizing and consists with the FTIR analysis on the wash-off of M1 fibre using hot water. Furthermore, a 12% reduction in C was detected at TOA of 15 o versus at TOA of 90 o , while the N percentage remained constant as a function of probing depth. In addition, it was observed that almost 50% of the amine groups at TOA of 90 o have been protonated, while 35% are protonated at the outermost surface. This agrees with the contact angle observation that the polar groups are present on the outermost surface, thus creating a hydrophilic surface. On the other hand, the C-1 and C-2 percentages remained constant as a function of depth probed into the surface. This suggests that the concentration of aminosilane does not change greatly within the detected information depth.

To better understand the influence of solvent treatments on the surface properties of GFs, Zetapotential of GF was determined using EKA to derive information about the surface functional groups. The Zeta-potential ~ pH curves are shown in Fig. 4. Compared with M1 fibre, a significant shift in iso-electric point (IEP) to the basic region after toluene treatment or to the acidic region after water treatment was found. After toluene treatment a significant amount of surfactant and a part of the PP film former were removed. As a consequence, the basic NH 2 -groups of the aminosilane are more accessible, correspondingly the Zeta-potential at the plateau shifted to higher positive values, meanwhile the IEP also moved from 7.0 to more basic value. On the contrary, after water treatment the Zeta-potential of M1-W fibre at native pH shifted from positive to negative and preferably acidic groups were detected at the surface, moreover the IEP was shifted to about pH 4.

These results also suggest the hydrolysis of siloxane groups and migration of aminosilane from sizing/fibre interface to air/sizing interface.

From above analyses the radial chemical compositions of GF sizing layer become clear and are presented in following. After sizing application and drying, the sizing of control fibre (M1) shows enrichment of aminosilane in the sizing/GF interface while PP film former predominating at the air/sizing interface. The driving force for this is related to the low surface free energy of PP and the good affinity between aminosilane and GF surface. Upon extraction in hot toluene, the low MW fraction of PP film former and surfactants are partly washed off, making the amino groups of aminosilane more accessible; however, the low MW PP and surfactants enrich on the outermost surface. Extracting sized GF in hot water can wash off physically adsorbed and chemisorbed aminosilane and surfactants. Under high humidity and high temperature, polysiloxane hydrolyzes and distributes evenly throughout the detected information depth. Moreover, hot water extraction led to GF with a more hydrophilic surface.

Single fibre pull-out test

Interfacial shear strength is an evaluation of the efficiency of the interface to transfer the applied stress from the matrix to the fibre before interfacial debonding occurs. In-house single fibre pull-out test device was used to determine the interfacial adhesion strength of differently treated GF/PP model composites. Tab. 1 shows a modest decrease of the local adhesion strength τ τ τ τ d and G ic when M1 fibre was extracted in toluene, the τ τ τ τ d and G ic slightly drop from 13.1 MPa and 5.5 J•m -2 to 10.6

MPa and 3.7 J•m -2 , respectively. Based on the results of Zeta-potential measurement this was not expected, since it revealed a better accessibility of the amino groups of aminosilane, which can prompt the formation of the covalent bond between amino group and anhydride group. However, if the enrichment of low MW PP and surfactants at the outermost surface is taken into consideration, the modest drop in interfacial adhesion strength is reasonable and could relate to the presence of weak points in the interphase. This is consistent with our previous observation that the bond strength was especially low when low MW PP was applied as film former [START_REF] Mäder | Characterization and design of interphases in glass fiber reinforced polyproplyene[END_REF]. Further works will be dedicated to Nano-indentation and Nano-TA studies on differently treated GFs and corresponding composites to evaluate the mechanical property and chemical composition gradient within sizing/interphase thickness with the aim to deepen the understanding on the correlation between the surface/interphase properties and composite material mechanical properties.

  On the other hand, water extraction removed a part of the silane coupling agent, which is physisorbed on the GF surface, and small molecule surfactants. Additionally, the interpenetrating network of polymeric film former and aminosilane caused by the hot water extraction enhances the inter-diffusion of sizing and matrix promoting the formation of covalent bonds between amino groups and anhydride groups. In this case, the local adhesion strength τ τ τ τ d and the critical interface energy release rate G ic are significantly increased up to 21.0 MPa and 14.7 J•m -2 , respectively. Conclusion PP compatibly sized glass fibres (GFs) were subjected to extraction either in hot water or in toluene to simulate a different attack of sizing depending on the media and in depth altering of the sizing, respectively, and to obtain indirect information about the gradient within the interphase ranging from the air/sizing interface to the sizing/glass interface. FTIR and GPC anaylses on the wash-offs, surface properties of GFs and the interfacial adhesion strength of single fibre/PP model composites upon extraction in different liquids proved to be surface sensitive methods, revealing the chemical composition within the sizing. Extracting control GF in hot toluene, surfactants and the low molecular weight fraction of PP film former are partly washed off, making the amino groups of aminosilane more accessible; however, the enrichment of low molecular weight PP and surfactants on the outermost surface creates weak points in the interphase. The latter one overwhelms the former one leading to slight decrease in interfacial adhesion strength. On the other hand, extracting control GF in hot water could wash off physically adsorbed and chemisorbed aminosilane and surfactant. Meanwhile polysiloxane hydrolyzes and distributes evenly throughout the sizing thickness, as a result of this the observed interfacial adhesion strength value shows a 30% increase.

able 3 :

 3 Average C surface compositions and the degree of nitrogen protonation obtained from M1 fibres igure captions ig. 1. 1 H NMR spectrum of acetone soluble part of PP dispersion and the peak assignment ig. 2. FTIR spectrum comparison of (a) wash-off of M1 fibre in water, (b) extraction of only γ-APS sized glass fibre in water, (c) acetone soluble part of PP dispersion, (d) wash-off of M1 fibre in toluene, (e) acetone insoluble part of PP dispersion, and (f) PP dispersion ig. 3: Tapping mode AFM surface topography images of glass fibres (a) M1 fibre, (b) M1-T fibre, and (c) M1-W fibre, the Z scale is 200 nm ig. 4. Zeta-potential as a function of pH for M1, M1-W, and M1-

FiguresFig. 1

 1 Figures

. 1 HFig. 2 .

 12 Fig. 1. 1 H NMR spectrum of acetone soluble part of PP dispersion and the peak assignment

Fig. 3 :Fig. 4 .

 34 Fig. 3: Tapping mode AFM surface topography images of glass fibres (a) M1 fibre, (b) M1-T fibre, and (c) M1-W fibre, the Z scale is 200 nm

Table captions able 1 .

 captions1 Surface properties and pull-out test results of fibre M1 as reference compared with them

	extracted either by toluene (T) or water (W)
	able 2: Absolute average surface compositions obtained from XPS investigation on M1 fibres

Table 1 .

 1 Surface properties and pull-out test results of fibre M1 as reference compared with them extracted either by toluene (T) or water (W) wt. loss: weight loss, 2 θ a : advancing contact angle, 3 θ r : receding contact angle, 4 R q : root-mean-square roughness, 5 R max : maximum height roughness, 6 τ d : local adhesion strength,7 G ic : critical interphase release rate, 8 τ app :

	Fibre	wt. loss 1 (wt.%)	θ a (deg) 2	θ r (deg) 3	θ a -θ r (deg)	R q (nm) 4	R max 5 (nm)	τ d (MPa) 6	G ic (J m -2 ) 7	8 (MPa) τ app
	M1	/	94.3	68.5	25.8	3.5	41.5	13.1	5.5	8.2
	M1-T	0.35	99.2	72.0	27.2	9.6	88.6	10.6	3.7	6.4
	M1-W	0.48	74.4	39.6	34.8	4.6	59.2	21.0	14.6	9.4
	apparent adhesion strength

1

Table 2 :

 2 Absolute average surface compositions obtained from XPS investigation on M1 fibres

	Fibre	TOA (deg)	Information depth (nm)	O	Average surface composition (in at. %) N Ca C Si	Al
	M1	90	8	18.1	1.5	1.3	71.2	5.9	2.0
		60		14.9	1.2	0.9	74.3	4.7	4.0
		30		10.7	1.1	0.6	80.7	3.7	3.2
		15	2-2.5	8.9	0.9	0.6	86.3	2.6	0.8
	M1-T	90	8	20.6	1.5	1.9	66.6	7.5	1.8
		60		17.7	1.4	1.5	72.2	6.1	1.3
		30		11.9	1.4	1.0	79.6	5.1	1.0
		15	2-2.5	8.8	1.0	0.7	85.6	3.8	0.3
	M1-W	90	8	24.8	0.9	1.4	60.0	11.1	1.9
		60		24.2	0.8	1.2	61.5	10.3	2.0
		30		21.2	0.9	0.8	64.6	10.6	1.9
		15	2-2.5	18.7	1.1	0.8	67.8	10.6	0.9

Table 3 :

 3 Average C surface compositions and the degree of nitrogen protonation obtained from M1 fibres

	Fibre	TOA (deg)	C-1	C surface composition (in at.%) C-2 C-3	C-4	N + (in at.%)
	M1	90	73.2	20.7	4.3		1.8	10.5
		60	73.3	20.4	4.5		1.8	0
		30	74.9	18.5	4.9		1.7	0
	M1-T	90	81.9	11.8	4.0		2.3	20.2
		60	79.2	14.1	4.2		2.6	25.2
		30	77.5	15.9	4.2		2.5	0
	M1-W	90	79.6	14.4	3.8		2.2	48.4
		60	78.9	14.1	4.7		2.4	42.2
		30	79.1	14.3	4.3		2.3	35.1
	Binding energy (eV)	285.0	286.4-286.5	287.4-287.5	288.7-289.0
	Chemical state	C-C	C-N/C-O	C=O/O-C-O C-O-(C=O)/CO 3

Acknowledgements

RCZ would like to thank Johns Manville and Leibniz Institute of Polymer Research Dresden for funding the stay as postdoc. The authors are indebted to Mrs. G. Adam, Dr. C. Bellmann, Dr. H. Brodowsky, Mrs. R. Plonka, Mr. J. Rausch, Mrs. A. Rothe, Mrs. P. Treppe (IPF Dresden e.V), Dr. J. Asrar, Dr. D. Blasini, Dr. A. Ferryman, and Dr. K. Gleich (JMTC) for their experimental assistance and helpful interpretations.


Emilie Planes

Laurent Chazeau
email: laurent.chazeau@insa-lyon.fr

Gérard Vigier

Thomas Stuhldreier

Influence of silica fillers on the ageing by gamma radiation of EDPM nanocomposites

Keywords: Nanocomposites, mechanical properties, environmental degradation

This study evidences that the presence of silica fillers as well as their surface treatment influences the impact of gamma irradiation on the mechanical properties of the filled materials. It influences it both chemically, by a modification of the kinetics of the degradation reactions, and physically, through the complex modification of the filler-filler and fillermatrix interactions involved in the mechanical properties of the filler network.

Introduction

Some applications of filled elastomer can involve ageing by gamma irradiation. For instance, the elastomers used as cable insulation in nuclear power plants are submitted to irradiation during their lifetime. Several studies have been devoted to the understanding of the consequences of ageing by gamma radiation on the mechanical properties of unfilled elastomers such as Ethylene-Propylene Diene Monomer (EPDM) or Poly DiMethyl Siloxane (PDMS) [START_REF] Planes | Evolution of EPDM networks aged by gamma irradiation -Consequences on the mechanical properties[END_REF][START_REF] Planes | Crystalline microstructure and mechanical properties of crosslinked EPDM aged under gamma irradiation[END_REF][START_REF] Rivaton | Radiochemical ageing of EPDM elastomers. 2. Identification and quantification of chemical changes in EPDM and EPR films gamma-irradiated under oxygen atmosphere[END_REF][START_REF] Rivaton | Radiochemical ageing of EPDM elastomers. 3. Mechanism of radiooxidation[END_REF]5,6]. Thus, a scheme of the chemical reactions involved in the degradation process of EPDM under gamma radiation has been established from a fine analysis of the degradation products [START_REF] Rivaton | Radiochemical ageing of EPDM elastomers. 2. Identification and quantification of chemical changes in EPDM and EPR films gamma-irradiated under oxygen atmosphere[END_REF][START_REF] Rivaton | Radiochemical ageing of EPDM elastomers. 3. Mechanism of radiooxidation[END_REF]. From a microstructural point of view, these reactions can be, within more or less important simplification, related to scissions and/or crosslinkings of the polymer chains [5,6] However in most applications, elastomers are formulated with fillers, which are in many cases nanoscopic. For instance, incorporation of nanoscopic silica fillers in elastomers is well known to improve their mechanical properties [START_REF] Chazeau | Handbook of "organic-inorganic hybrid materials and nanocomposites[END_REF]. However, the use of such fillers, with a very large interface area, can also influence the degradation mechanisms. They can modify the polymer degradation by trapping radicals or degradation by-products which can interact with intermediate chemical species involved in the matrix degradation. They can also be degraded and lead to the formation of supplementary degradation by-products, which can interact with the matrix degradation processes. Moreover, whether fillers are still inert or not during the matrix degradation, this matrix degradation can induce a modification of the fillermatrix interaction: this may have consequences in the reinforcement efficiency of the filler, and therefore be involved in the consequences of the degradation in the overall properties of the composite.

Very few studies were devoted to the irradiation effect on silica filled rubbers, and they mainly focused on PDMS filled silica systems [START_REF] Stevenson | Influence of SiO 2 fillers on the irradiation ageing of silicone rubbers[END_REF][START_REF] Chien | Characterization of radiationinduced aging in silica-reinforced polysiloxane composites[END_REF][START_REF] Patel | Gamma radiation induced effects on Silica and on silica-polymer interfacial interactions in filled polysiloxane Rubber[END_REF][START_REF] Maxwell | Residual dipolar coupling for the assessment of cross-link density changes in -irradiated silica-PDMS composite materials[END_REF][START_REF] Maxwell | The Effects of gammaradiation on the thermal, mechanical, and segmental dynamics of a silica filled, room temperature vulcanized polysiloxane rubber[END_REF]. To our knowledge no study concerns the ageing of nanoscopic silica filled (EPDM). However, EPDM is often preferred to silicone because of its excellent capability in electric insulation. When introducing silica in EPDM, the created filler-matrix interactions are very different from those found in silica filled PDMS: there are not strong hydrogen bonds between silica and the matrix. Moreover, in silica/PDMS system, it was shown that covalent bonds are formed at the interface during irradiation [START_REF] Stevenson | Influence of SiO 2 fillers on the irradiation ageing of silicone rubbers[END_REF]. Therefore, the chemistry of the system as well as the initial polymer-filler interaction (van der Waals interaction) being very different in silica-filled EPDM, the consequences of ageing under irradiation could be very different from those found with silica filled PDMS systems. This motivates the present study. In order to understand the role of the semi-crystalline phase in the polymer matrix, the materials after irradiation at room temperature are studied both in the rubbery state (i.e. at 80°C) and in the semi-crystalline state (at room temperature). In most applications, these materials are formulated with a specific coupling agent, such a as bifunctionnal organosilane, which promotes the formation of covalent bonds at the filler matrix interface. For this reason, we also present results obtained with a formulation containing this treatment.

Experimental

Materials

EPDM elastomer (Nordel IP NDR 3722 P from Dow Chemical Company) is made of 70% ethylene, 29.5% propylene and 0.5% ethylene norbornene (ENB) and the crosslinking agent is dicumyl peroxide (Perkadox BC-FF from Akzo Nobel). EPDM is reinforced with silica ULTRASIL VN3 supplied by Degussa. The filler is highly dispersible silica with a specific surface of 175 m 2 /g, the estimated size of a primary particle is c.a. 15 nm. The filler ratio in

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the studied materials is constant and equal to 50 phr (50 weight ratios for 100 weight ratios of EPDM, this corresponds to a weight fraction of 33%, and a volume fraction of 17%). The surface treatment chosen to improve the filler-matrix interactions is a vinyltrimethoxysilane Z6300 supplied by Dow Corning. It is made of a reactive foot with three ethoxy groups, which react with the silanol of the silica surface, and an alkyl chains with a vinyl function, which can react with the matrix during crosslinking [START_REF] Pal | Studies on peroxide vulcanization of silica filled EPDM rubber in presence of vinyl silane coupling agent[END_REF]. Following the procedure of Ramier et al [START_REF] Ramier | Grafting of silica during the processing of silica-filled SBR : comparison between length and content of the silane[END_REF], the optimal quantity of surface treatment, to effectively cover this type of silica surface is estimated to 3 phr.. The so called E-SiU material contains untreated fillers, conversely to the E-SiT material, which contains treated fillers. The samples are processed as follows [START_REF] Planes | Evolution of EPDM networks aged by gamma irradiation -Consequences on the mechanical properties[END_REF][START_REF] Planes | Influence of fillers on mechanical properties of ATH filled EPDM during ageing by gamma irradiation[END_REF]: (i) mixing 2 min of the matrix in an internal mixer, (ii) introduction of the fillers (for treated fillers, the organosilane molecules are introduced with 2/3 of the silica, and then the rest of filler is introduced) and mixing for 5 min, (iii) introduction of 3 phr of peroxide and mixing for 10 min, at 80°C, (iv) mixing in an external mixer (cylinders) for 10 min at 80°C, (v) pressing the compound as 1-mm-thick films at 170 °C (crosslinking step).We used the common industrial method which consists in measuring with a MONSANTO rheometer the curing time needed to obtain 98% of the maximum torque extrapolated at infinite cure time (t 98 at 170°C). Thus the vulcanisation at 170°C is stopped after 10 minutes for the unfilled EPDM and after 5 minutes for silica filled EPDM. It was checked by Transmission (JEOL 200CX, on samples obtained from cryo-ultra microtomy) and by Scanning Electron Microscopy performed with an ESEM equipped with a Field Ion Beam (SEM-FIB Nvision40 Zeiss) the good silica dispersion within the polymer matrix (expected for such classical material processing and silica fillers), whether the silica fillers were treated or not. As an example, figure 1 presents a nice SEM-FIB image of E-SiU sample: prior to observation, the sample was gold coated. Then an excavation was performed followed by the

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creation by the FIB of a perfectly flat surface in the sample thickness direction. This surface was then observed in SE mode using an 750V accelerating voltage, and an InLens detector.

Figure 1: SEM image of a FIB prepared surface of E-SiU sample

The crystallization temperature domain of our EPDM is large [START_REF] Planes | Crystalline microstructure and mechanical properties of crosslinked EPDM aged under gamma irradiation[END_REF]: from about -50°C up to 60°C. A thermal treatment was applied to all samples to obtain a stable crystallinity ratio around 22°C, in particular during the ageing in the pool at 18°C, and the post-irradiation at 22°C: (i) A heating ramp up to 80°C to erase the previous thermal history, (ii) then a rapid cooling down to the optimal crystallization temperature 22°C and storage during 12h at this temperature (iii) and then a heating ramp of 1°C/min up to 38°C, the temperature at which the samples are maintained during 6h. Then the samples are cooled down to 22°C and stored at this same temperature. This treatment enables to get a unique well defined melting peak for unfilled EPDM materials and a double peak for silica filled EPDM. Thus these materials have one or two crystallite populations with melting temperatures above 22°C.

Ageing conditions

E-CR, E-SiU and E-SiT samples (for unfilled EPDM, and EPDM filled with untreated and treated silica respectively) are exposed to -radiation of a 60 CoO source at a dose rate of 1 kGy/h in an oxygen atmosphere for doses 50, 165, 300, 510 kGy (Arc Nucleart -Grenoble France): the water temperature of the pool, where exposures are performed, is about 18°C.

Afterwards, the samples are stored under vacuum at about 22°C. The samples will be named hereafter E-CR-XXX, E-SiU-XXX and E-SiT-XXX with XXX the irradiation dose.

IR spectroscopy showed for all the irradiated samples an increase in absorbance in the hydroxyl absorption region (3800-3000 cm -1 ) and in the carbonyl region (1600-1800 cm -1 ) [START_REF] Rivaton | Radiochemical ageing of EPDM elastomers. 2. Identification and quantification of chemical changes in EPDM and EPR films gamma-irradiated under oxygen atmosphere[END_REF].

Because of the filler presence, it is not possible to quantify these products by ATR and thus to compare the oxidation level of the different elastomers. Nevertheless, oxidation (carbonyl) profiles were evaluated from IR-microspectroscopy using a Continuum microscope coupled to a Nexus Nicolet optical bench (32 scans, resolution of 4 cm -1 , analysis conducted at the CNEP -Clermont Ferrand, France). A slice of sample of about 40 µm was cut by cryomicrotom in the thickness direction. Variations in absorbance at 1713 cm -1 were measured on this slice every 50 µm. Profiles for E-CR-510 and E-SiU-510, were found nearly flat, indicating that the oxidation processes can be considered homogeneous within the thickness.

Instruments

Swelling measurements 1.3.1.1. Swelling in xylene

Samples with an initial mass M i are introduced in xylene for 8 days to achieve the swelling equilibrium; then the materials with a swelled mass M s are dried under vacuum at 60°C during 1 day and finally weighted to obtain the dried mass M d . In the case of the composites, TGA (TGA F3 apparatus from Netzch) enables to estimate the filler weight ratio i in the initial sample and the filler weight ratio d in the dried sample after swelling. Therefore, the swelling ratio Q and the polymer soluble fraction F p (%) can be evaluated from:

( ) ( ) 1 1 1 polymer s d xylene d d M M Q M ρ ρ ε - = + - ( ) ( ) ( ) ( ) 1 1 2 1 i i d d p i i M M F M ε ε ε - -- = -
At low dose, given the low sol fraction measured for E-SiU and E-SiT materials, the difference found between d and i is within the experimental uncertainties. However, for high irradiation dose, actually for total sol fractions above 20%, it is found that d is clearly lower than i , meaning that the sol fractions contain a significant amount of fillers.

Water uptake

Because of the presence of silanol on its surface and of its large specific surface, silica can absorb a significant amount of water. To measure it, the samples, cut as identical parallelepipeds to offer a constant surface to the water, are firstly dried under vacuum for 24 h at 100°C to remove the maximum amount of water on the silica surface. After weighting dried samples (M dried ), they are put in distilled water at room temperature for one month and then weighted again (M swollen ). The water uptake S w (expressed in %) is then calculated from:

( )

100 3 swollen dried w dried M M S M - = ×

Tensile tests

Experiments (on an MTS device equipped with a temperature controlled chamber) are conducted at 80°C and at 25°C with a true strain rate 0.01 s -1 . An image processing acquisition system (Apollor VideoTraction System) is used to obtain the true stress -true strain curve. The samples are dumbbell-shaped with dimensions 20 x 4 x 1 mm 3 . For one type of samples, three specimens are tested; on figures only one test is reported, but the average stresses and strains at break on the three specimens are given in a separated table 2. Note that the rupture has not been reached for the E-SiU samples. Besides, the elastic modulus E is calculated as the slope of the linear initial portion of the curve. Tensile test cycles are also performed at 80°C. Sample is stretched up to a 0.2 true strain, then relaxed during 5 minutes; then, it is stretched again up to a 0.4 true strain. This experience is repeated with a 0.2 true strain increment until the sample rupture.

Results and discussions

Preliminary characterization of the studied materials

As reported in figure 2, the swelling ratio Q of E-SiU is higher (4.2) than the matrix one (3.7).

As suggested in the experimental part, the polymer matrix of the composite may be slightly less crosslinked than the unfilled EPDM because of the choice of the curing time. This is in agreement with the sol fraction F p values (cf. Table 1): E-SiU contains a soluble fraction of about 3.2%, conversely to the unfilled sample, although this result must be considered with caution because of the experimental uncertainties. Note that this soluble fraction could also be due to occluded polymer in the complex filler structure. The elastic modulus at 80°C of E-SiU (Table 1) is about 4.5 times higher than the modulus of E-CR. This is much higher than the reinforcement predicted by the Christensen and Lo model [START_REF] Christensen | Solutions for effective shear properties in three phase sphere and cylinder models[END_REF]. This model has shown its validity for micron size filler [START_REF] Planes | Influence of fillers on mechanical properties of ATH filled EPDM during ageing by gamma irradiation[END_REF] and predicts a reinforcement of a factor 1.6 for 20%vol. of filler. This discrepancy between theory and experiments is generally explained by the formation of a strong filler network [START_REF] Chazeau | Handbook of "organic-inorganic hybrid materials and nanocomposites[END_REF]. The efficiency of the silica surface treatment in E-SiT is first evidenced by the water uptake measurements (cf. Figure 3): S w is more important for E-SiU than for E-SiT. Nevertheless, the soluble fraction of E-SiT is higher than the one found for E-SiU. Thus, the E-SiT polymer phase is certainly slightly less crosslinked than that of E-SiU. The reasonable explanation is that a part of the peroxide has been used in the filler/matrix coupling reactions. Despite this, the swelling ratio of E-SiT is smaller (2.8) than the one of E-CR. This is not surprising: indeed, in the case of nanocomposites such as the studied materials, in which nanoscopic fillers likely form a percolating reinforcing structure [START_REF] Chazeau | Handbook of "organic-inorganic hybrid materials and nanocomposites[END_REF], filler-matrix covalent bonds become important in the material response at larger strains, in particular for strain level such as that involved in the swelling measurements, or for strains at which hardening appears in stressstrain curve, that is when the filler structure is stretched (cf. Figure 4a and4b).

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physical, is not a key parameter, presumably because they are in any case strong enough to transfer the stress at the filler-matrix interface. Thus, the elastic modulus at 80°C of E-SiT is slightly smaller than the one of E-SiU (cf. Table1). This lower modulus can be attributed to the slightly less crosslinked matrix previously suggested, or to a less reinforcing filler structure [START_REF] Ramier | Payne effect in silica-filled styrene-butadiene rubber. Influence of surface treatment[END_REF]: the filler treatment may lead to a decrease in the filler-filler interactions by promoting the filler-matrix ones, leading to a less reinforcing filler.

The rupture behaviour cannot be discussed for the E-SiU samples, since they systematically break in the clamps. The authors have no explanation for this peculiar behaviour. However, it can be observed that E-SiT breaks earlier and not in the clamps. The silane treatment seemingly leads to a more brittle behaviour of this material, as already observed in the study of treated and untreated ATH filled EPDM [START_REF] Planes | Influence of fillers on mechanical properties of ATH filled EPDM during ageing by gamma irradiation[END_REF]. This might be attributed to the absence of decohesion mechanism which might delay sample rupture by decreasing the local matrix stretching.

Degradation kinetic -Consequences on mechanical properties at 80°C

E-CR material:

The consequences of irradiation on unfilled E-CR are already reported in reference 1. At the beginning of irradiation (below 50 kGy), no change of the swelling ratio (cf. Figure 2), a slight increase in the soluble fraction (cf. Table 1) and in the elastic modulus measured at 80°C, i.e. above the melting temperature, (cf. Table 1) suggests that this material is submitted to concomitant crosslinking / chain scissions processes. Note that this interpretation is also supported by the evolution of initially uncrosslinked EPDM subjected to gamma irradiation [START_REF] Planes | Evolution of EPDM networks aged by gamma irradiation -Consequences on the mechanical properties[END_REF]). Then at higher irradiation dose, the increases in the swelling ratio and in the soluble fraction indicate that chain scissions become the preponderant occurring phenomenon. The sol fraction evolution can be correctly described using the Charlesby approach which enables to calculate the chain scission kinetic. The consequences of the degradation process on the mechanical properties are recalled on figure 4 and tables 1 and 2:

the modulus at 80°C decreases as well as the stress level of the tensile curves, and the ultimate properties deteriorates.

Table 1: Soluble fraction F p (%), elastic moduli E at 80°C (MPa) of the different materials

E-SiU material:

Between 0 kGy and 50 kGy, E-SiU does not show any significant evolution of the swelling ratio (cf. Figure 2), of the sol fraction (F p ) and of the elastic modulus at 80°C (cf. Table 1). Thus, at 50 kGy, there is not a modulus maximum like in the case of the E-CR.

Given the assumed crosslink density lower in this material than in E-CR, and assuming the occurrence of a crosslinking process induced by radiation (such as the one suggested by the slight modulus and sol fraction evolutions in the case of E-CR), it can be assumed that the matrix in the filled sample has also been submitted to the same competing mechanisms of crosslinking and scissions. Within this assumption, the absence of modulus maximum at 50 kGy might be due either to its shift at a lower irradiation dose (experiments at lower radiation dose could then confirm it), or to the fact that the crosslinking process never counterbalances the chain sicssions. Figure 1), as well as the polymer soluble fraction F p (cf. Table 1). Chain scissions take place, but as indicated by the larger extracted polymer ratio F p of the filled rubber (at 165 kGy, 25% against 6% for E-CR), this phenomenon is much more important than in the case of unfilled material. The same amount of sol fraction is found in E-SiU at 165 kGy as in E-CR material at 510 kGy. This sol fraction variation is correlated with the decreases in the elastic modulus at 80°C (cf. Table 1) and in the stress levels measured in tensile test at 80°C (cf. Figure 4). In addition, a modification of the silica surface is evidenced by the water uptake measurements, which show a strong decrease of S w between 0 and 165 kGy (cf. Figure 3), indicating that the silica surface becomes less hydrophilic, i.e. that the silanol initially on its surface are degraded. These results strongly suggest that the fillers promote supplementary reactions in the filler vicinity. Indeed, it has been reported that the irradiation of silica involves the formation of numerous radicals in the bulk [START_REF] Cataldo | radiolyzed amorphous silica: A study with 29 Si CP-MAS NMR spectroscopy[END_REF][START_REF] Stevenson | Influence of SiO 2 fillers on the irradiation ageing of silicone rubbers[END_REF][START_REF] Patel | Gamma radiation induced effects on Silica and on silica-polymer interfacial interactions in filled polysiloxane Rubber[END_REF]. They may tend to recombine or diffuse to the surface, making the surface reactive, and accelerating degradation reactions, in the filler-surface vicinity.

Dose

At doses above 165 kGy, both modulus and soluble fraction tend to stabilize. Moreover, the stability of S w suggests that the silica surface hydrophilicity is not significantly modified anymore. However, the swelling ratio goes through a maximum at 300 kGy to decrease at 510 kGy. This indicates that an additional mechanism of crosslinking occurs and counterbalances the chain scission process. Such phenomenon was not observed in the case of polymer E-CR.

As shown in the material stress-strain curves of E-SiU at 510kGy (cf. figure 4), the formation of these supplementary crosslinks leads to an increase in hardening. Thus, there are two sequences in the modification of the silica surface under irradiation: first, it is made more hydrophobic, and then it reacts with the polymer matrix in its vicinity to form covalent bonds.

To confirm the strengthening of the filler-matrix interaction, non-irradiated and irradiated E-SiU samples have been subjected to cycle tensile tests at 80°C (cf. Insert in figure 5). The area delimited by two successive tests, which is significant of the energy damage, is plotted as function of the maximal stress reached for each cycle in figure 5 b). Unexpectedly, Irradiation does not strongly modify the cycle area versus stress curve. This means that the bonds formed during irradiation are not numerous enough to lead to a significant modification of the damage behaviour.

E-SiT (cf. table 2)

E-SiT material:

In this case, between 0 and 50 kGy, a slight crosslinking phenomenon is noticeable as evidenced by a modulus increase and the swelling ratio decrease. This can be due to some additional filler-matrix coupling reactions formed by some residual silane treatment, which has not been covalently bonded to the matrix. This can also be due to crosslinking reactions in the matrix provided by some residual peroxide, since it has been shown that the polymer phase of this material is slightly less crosslinked. As shown in figure 3, the consequences of this crosslinking on the stress-strain curves are a more important hardening and an earlier rupture in addition to an increase in modulus.

Above 50 kGy and up to 300 kGy, the swelling ratio (cf. Figure 2) and the soluble fraction (cf. Table 1) also increase with the radiation dose, but these increases are much less important than for E-SiU; actually they are comparable to those measured for E-CR. Thus, the polymer degradation is seemingly similar to that of E-CR. There is no indication of a more rapid scission mechanism such as that found in the case of E-SiU. However one can note between 300 kGy and 500 kGy that the swelling ratio and the sol fraction rapidly increases and reaches the values found for E-SiU. Thus, the fast scission mechanism seems to be delayed by the silane treatment and to occur at larger radiation dose. Such interpretation is supported by the evolution of the mechanical properties with the radiation dose. The stress-strain curves show a delayed decrease of the stress level at a given strain when compared with the stress-strain curves of E-SiU: the decrease becomes significantly visible from 300 kGy and it accelerates between 300Gy and 500 kGy. Eventually, after ageing at 500kGy, the materials with treated and untreated silica have very similar stress-strain curves at 80°C; moreover, given their same swelling ratio, it can be deduced that both materials have about the same number of covalent bonds at the filler-matrix interface. E-SiT samples have also been subjected to cyclic tensile tests at 80°C (cf. Figure 5). The non irradiated E-SiT shows an increase in the energy for a higher stress than E-SiU: this evidences the strongest filler-matrix interface in E-SiT, which breaks at higher stress than E-SIU. The cycle performed on E-SIT-510 leads to an energy curve similar to the one of E-SIU510, with an increase in energy which begins at the same stress as E-SiU-510. This confirms that a large number of initially mechanically active In addition, the modulus value lower for E-SiT than for E-SiU might suggest that, especially at 510kGy, all things equal, i.e. the matrix crosslink density, and the filler-matrix interaction, the filler structure in E-SiT is actually less reinforcing than the one in E-SIU; this would be not surprising since the use of a filler treatment is known to impact the filler network by reducing the filler-filler interaction [START_REF] Pal | Studies on peroxide vulcanization of silica filled EPDM rubber in presence of vinyl silane coupling agent[END_REF].

Consequences on mechanical properties at 25°C

As reported in [START_REF] Planes | Crystalline microstructure and mechanical properties of crosslinked EPDM aged under gamma irradiation[END_REF] E-CR presents a lamellar microstructure in the undeformed state which is not strongly modified by irradiation (only a very limited chemi-cristallisation process has been evidenced). The cristallinity ratios of E-SiU, and E-SiT deduced from DSC measurements (on a Perkin Elmer Pyris Diamond) prior and after irradiation, as well as their melting temperature are not significantly different from those found for E-CR. This indicates that, irradiation has the same negligible impact on the polymer crystalline microstructure, with or without the filler presence. This was expected since the introduction of silica does not either significantly modify the polymer cristallinity in the non irradiated E-SiU and E-SiT. are very different because of the presence of crystallites at room temperature. E-Si and E-SiT have the same modulus which is, like at 80°C, much higher than that of the unfilled sample:

the slightly higher ratio of the filled sample modulus by the modulus of the unfilled sample (around 6 at 25°C against 4.5 at 80°C) suggests that the reinforcement is due to a mixed crystallite silica network. This idea is supported by the size of silica fillers (around 15 nm) which is in the same range as the crystallite size. The modulus slightly increases in the beginning of irradiation, due to the small chemicrystallisation phenomenon. Thus, like observed in the study of the effect of irradiation on E-CR [START_REF] Planes | Crystalline microstructure and mechanical properties of crosslinked EPDM aged under gamma irradiation[END_REF], the crystallite presence strongly decreases the impact of the degradation on the mechanical properties in the semi-crystalline state. Such phenomenon has also been highlighted for others semi-crystalline polymers, such as, for instance, polyethylene [START_REF] Aslanian | Effect of radiation on the crystallinity of low-density polyethylene[END_REF]. This holds true for strain below 0.4 since the tensile curves obtained at the different irradiation doses for E-SiU of for E-SiT are nearly superimposed (Figure 5). At larger strain, the degradation of the amorphous phase, already evidenced at 80°C, obviously decreases the stress transfer between crystallites during the matrix deformation, leading to a decrease in the hardening phenomenon.

The creation of filler-matrix bonds in the E-SiU samples by high irradiation dose has a limited impact on the hardening process of the corresponding tensile curve at 25°C. As assumed above, this means that these bonds are in small numbers. Besides, even if E-SiU and E-SiT have similar stress-strain curves at 80°C, all the E-SiT curves obtained at 25°C show a much more important hardening than the tensile curves obtained with the E-SiU samples. Whatever the irradiation dose, the stress level measured for E-SiT is much higher than the one measured with E-SiU (a ratio of about 2 is observed for a true strain equal to 1). Thus, the crystallites presence partly heals the desactivation of the filler-matrix covalent bonds.

Conclusions

The incorporation of nanoscopic fillers in EPDM induces the formation of a filler network with a larger reinforcement efficiency than when the fillers are micron size. The materials with treated fillers have an elastic modulus close to the one of EPDM filled with untreated fillers. But in the amorphous state like in the semi-crystalline state, they show more important hardening with a faster rupture. At 25°C, the reinforcement measured for both materials is higher than the one found at 80°C, which indicates the formation of a mixed crystallite silica network.

These materials have been irradiated at room temperature:

• Without treatment, the polymer phase degradation is accelerated. Our explanations is the formation of supplementary radicals induced by the silica irradiation. At the highest irradiation dose studied, a particular mechanism can be observed: due to the modification of the silica surface, stronger interactions between filler and matrix are formed.

• The silica treatment enables to delay the degradation acceleration to the highest radiation dose studied. In addition, irradiation, leads to a mechanical deactivation of the initially present covalent bonds at the interface, either by their direct degradation, or by the degradation of the polymer in their vicinity.

• As a consequence, both filled rubbers (with and without silane treatment) have similar mechanical behavior at 80°C at 510 kGy. At room temperature, i.e. in the semi crystalline state, their properties are mainly controlled by the crystalline microstructure which is not significantly modified by radiation. In addition, at room temperature, conversely to the filler-matrix covalent bonds created by the silane treatment, those created by gamma irradiation in the composite with untreated silica have a much lower impact on the mechanical properties.

To conclude, the different experimental results clearly show that the presence of silica fillers as well as their surface treatment influences the impact of irradiation on the mechanical properties of the filled materials. They influence it both chemically, by a modification of the kinetics of the degradation reactions, and physically, through the complex modification by irradiation of the filler-filler and filler-matrix interaction involved in the mechanical properties of the filler network.

Figure 2 :

 2 Figure 2: Swelling ratio Q, as a function of radiation dose, dashed lines are guide for the

Figure 3 :

 3 Figure 3: Water uptake S w as a function of radiation dose

Figure 4 :

 4 Figure 4: Large deformation behavior at 80°C of a) E-SiU (no rupture is observed) and of b)

Figure 5 :

 5 Figure 5: Evolution of the cycle area versus true stress for E-SiU and E-SiT at 0 kGy and at

Figure 6 :

 6 Figure 6: Large deformation behavior at 25°C of a) E-SiU (no rupture is observed) and of b)

Table 2 :

 2 Average stresses and strains at break for E-SiT and E-CR at 80°C and at 25°C

	Samples	At 80°C	At 25°C
		Strain at break	Stress at break	Strain at break	Stress at break
			(MPa)		(MPa)
	E-CR-0	0.8 ± 0.1	2.8 ± 0.3	1.6 ± 0.2	16.8 ± 4
	E-CR-510	0.5 ± 0.1	0.7 ± 0.1	1.2 ± 0.1	7.2 ± 1.3
	E-SiT-0	1 ± 0.1	16 ± 1.6	1.4 ± 0.1	60.4 ± 18
	E-SiT-50	0.6 ± 0.1	6.2 ± 1	1.3 ± 0.1	47 ± 12
	E-SiT-165	0.8 ± 0.1	10.6 ± 2	1.4 ± 0.2	47 ± 15
	E-SiT-300	0.6 ± 0.1	4.6 ± 0.5	1.2 ± 0.2	32 ± 11
	E-SiT-510	0.6 ± 0.1	2.5 ± 0.35	1.1 ± 0.2	21 ± 6
	Figures 6 a), 6 b) and table 2 and 3 present tensile test results at 25°C of E-SiU and E-SiT, E-
	CR and E-CR-510. As shown on these figures, the mechanical properties at 80°C and at 25°C

Table 3 :

 3 Elastic moduli E at 25°C (MPa) for different materials

	ACCEPTED MANUSCRIPT
	Dose	E-CR	E-SiU	E-SiT
	(kGy)
	0	5.8 ± 0.6	33.8 ± 3.4	34.4 ± 3.4
	50	5.8 ± 0.6	33.8 ± 3.4	39 ± 3.9
	165	5.8 ± 0.6	40.5 ± 4.1	39.4 ± 3.9
	300	6 ± 0.6	44.7 ± 4.5	43 ± 4.3
	510	5.9 ± 0.6	41.8 ± 4.2	39.1 ± 3.9

Acknowledgments

It is a pleasure to acknowledge the joint research program "COPOLA" between EDF, NEXANS France, LABORELEC, CEA, INRA and CNRS. We also greatly acknowledge B.

Van De Moortele for the SEM-FIB images.


Stefano Pongolini
email: stefano.pongolini@bs.izs.it

Federica Bergamini

Alessandra Iori

Sergio Migliore

Attilio Corradi

Stefano Bassi

Prion protein genotypes of Italian sheep breeds with lysine-171 and phenylalanine-141 detection

A c c e p t e d M a n u s c r i p t 2 Abstract Amino acid polymorphisms of the prion protein gene influence sheep susceptibility to classical and atypical scrapie. Substitutions at codons 136, 154 and 171 play an important role in classical scrapie. Codon 141 leucine to phenylalanine mutation (AFRQ) has been recognized as an increased risk factor for atypical scrapie. In addition a rare allele with lysine at codon 171 (ARK) has been detected in Mediterranean sheep breeds. The presence of ARK poses two problems: the determination of its frequency and its possible interference with genotyping output of routine methods lacking specific detection capacity for ARK. The aim of our work was the development of a routine genotyping method with the capacity to identify ARK and AFRQ in addition to the normally detected alleles and to determine the frequencies of all these alleles in 5 main Italian breeds: Sarda (n=2494), Bergamasca (n=2686), Appenninica (n=297), Comisana (n=361) and Massese (n=402). A multiplex primer extension assay targeting the 6 single nucleotide polymorphisms of interest was developed. Allele frequencies revealed a very low level of ARR in Bergamasca (6.91%) as opposed to the other breeds, very diverse levels of AFRQ ranging from absence in Comisana to (10.70%) in Massese and a restricted presence of ARK. This allele has only been detected in Bargamasca with a significant 3.67% and marginally in Appenninica (0.34%). These results underline the need for adequate routine methods for genotyping of breeds with alleles that can interfere with typing of important codons such as the case of ARK for codon 171. Keywords: genotyping, PrP polymorphism, scrapie, sheep, 141-phenylalanine, 171-lysine A c c e p t e d M a n u s c r i p t

Introduction

Scrapie is a fatal neurodegenerative pathology of sheep and goat belonging to the group of prion diseases of animals and humans. Likewise the other prion diseases it is characterized by the accumulation of an abnormal isoform (PrPsc) of the host encoded cellular prion protein (PrPc). Symptoms of classical scrapie include various neurological disorders followed by progressive wasting and death, the onset of the disease usually occurs not earlier than 18 months of age and susceptibility varies depending on the strain of the agent, the breed and the genotype of the animal (Goldman et al., 1994;[START_REF] O'rourke | PrP genotypes and experimental scrapie in orally inoculated Suffolk sheep in the United States[END_REF].

With regard to the genotype effects on scrapie, amino acid polymorphisms in different positions of the ovine prion protein are known which condition the susceptibility of sheep to the disease. In particular amino acid positions 136, 154 and 171 play a fundamental role and several alleles have been identified with reference to these positions [START_REF] Hunter | PrP genetics in sheep and the applications for scrapie and BSE[END_REF].

These alleles are commonly reported using a three letters code (e.g. ARQ for alanine136/arginine154/glutamine171). Some of them are more commonly encountered and widespread among breeds of diverse origin, specifically alleles ARQ, ARR, AHQ, ARH and VRQ (Baylis and Goldman, 2004) , however more rarely encountered polymorphisms have been described in these positions: ARK [START_REF] Guo | Identifications of a novel lysine-171 allele in the ovine prion protein (PRNP) gene[END_REF] , TRQ (Billinis, et al., 2004), and ALQ [START_REF] Alvarez | Identification of a new leucine haplotype (ALQ) at codon 154 in the ovine prion protein gene in Spanish sheep[END_REF]. Evidence have accumulated indicating that the ARR allele is associated with minimum susceptibility particularly in case of the homozygous genotype ARR/ARR while ARQ and VRQ confer the highest susceptibility, an intermediate effect is attributed to the other common alleles [START_REF] Belt | Identification of five allelic variants of the sheep PrP gene and their association with natural scrapie[END_REF][START_REF] Clouscard | Different allelic effects of the codons 136 and 171 of the prion protein gene in sheep with natural scrapie[END_REF][START_REF] Hunter | Natural scrapie in a closed flock of Cheviot sheep occurs only in specific PrP genotypes[END_REF][START_REF] Hunter | Natural scrapie and PrP genotype: case-control studies in British sheep[END_REF]. However, recently 2 cases of natural scrapie were reported by [START_REF] Groschup | Classic scrapie in sheep with the ARR/ARR prion genotype in Germany and France[END_REF] in sheep with ARR/ARR genotype. The biochemical and transmission characteristics of these 2 cases made them referable to classical scrapie albeit symptoms were not well defined, they were single cases in their flocks of origin and showed reduced proteinase-K resistance of PrPsc. This has led A c c e p t e d M a n u s c r i p t 4 these authors to conclude that the agent they described could be a particular scrapie strain responsible for extremely rare infections.

Alongside classical scrapie, atypical forms of the disease have been identified which show different epidemiology, clinical signs and distinct biochemical characters [START_REF] Benestad | Cases of scrapie with unusual features in Norway and designation of a new type[END_REF]. Furthermore prion protein genotypes condition atypical scrapie in a very different way compared to classical scrapie. Specifically, alleles AHQ and AF141RQ (hereafter AFRQ), an ARQ variant with L to F mutation at codon 141, have been associated with increased risk while the ARR allele gives no protection [START_REF] Moum | Polymorphisms at codons 141 and 154 in the ovine prion protein gene are associated with scrapie Nor98 cases[END_REF][START_REF] Arsac | Similar biochemical signatures and prion protein genotypes in atypical scrapie and Nor98 cases, France and Norway[END_REF][START_REF] Lühken | Epidemiological and genetical differences between classical and atypical scrapie cases[END_REF][START_REF] Benestad | Atypical/Nor98 scrapie: properties of the agent, genetics, and epidemiology[END_REF].

In this context over the past decade selection programs have been implemented in European Union countries and elsewhere with the goal of increasing ARR allele frequency in order to protect sheep populations from classical scrapie, but we still have limited knowledge about the frequency of alleles that are not detected in routine genotyping for selection programs of ovine breeds. Among these are AFRQ and alleles confined to particular breeds such as ARK in some Italian breeds. The precise knowledge of these frequencies may contribute to the revaluation of selection programs for classical scrapie and give insights into susceptibility of the different breeds to atypical scrapie.

The aims of our work were two. Firstly, the development of a robust and accurate routine PrP genotyping assay for codons 136, 141, 154 and 171 with added capacity to identify 171-lysine (ARK), already demonstrated by Acutis et al. (2004) Resampling occurred for some flocks over this period. In case of resampling in a flock, only animals fulfilling the following criteria were included in the study: a) no resampling of the same animal occurred; b) animals were not born after that males of known genotype had become available to the breeder for genotype-directed mating; c) animals born after that females of the same flock had been genotyped were included in the study provided that no mating groups of selected females were created in the flock. Genomic DNA was extracted from thawed whole blood using NucleoSpin 96 Blood (Macherey-Nagel) on a Hamilton StarLet robotic workstation (Hamilton Robotics); the extraction protocol of the manufacturer was modified as follows: 20 l of blood were added to 180 l of phosphate buffered saline prior to proteinase-K digestion.

Samples collection and DNA extraction

Genotyping

The segment of ovine PrP coding region from nucleotide 306 to 650 was amplified by PCR from genomic DNA with the following primers: 5'-GAACAGCCCAGTAAGCCA-3' (forward), 5'-CACATTTGCTCCACCACTC-3' (reverse). The reaction volume of 10 l contained 2 mM MgCl2, 100 mM dNTPs, 0,5 U FastStart Taq DNA Polymerase (Roche), 0,5 mM of each primer and 2 l of genomic DNA (corresponding to 10-20 ng). The amplification was carried out in a Mastercycler gradient 5330 (Eppendorf) with initial denaturation at 95 °C for 6 min followed by 35 cycles of 30 s at 95 °C, 30 s at 57 °C, 30 s at 72 °C and a final step at 72 °C for 7 min. Post-PCR clean-up was performed by adding 3 U of shrimp alkaline phosphatase (SAP) and 1.5 U of exonuclease I (EXO) (both from GE Healthcare) to the PCR tubes and incubating them for 60 min at 37 °C followed by 15 min at 75 °C for enzyme inactivation. All steps from PCR to electrophoresis were carried out with Hamilton automation.

Primer extension cross-validation

Direct sequencing was used as the gold standard to validate the newly developed primer extension assay. Ninety-seven samples were sequenced after they had been genotyped by primer extension. They represented 20 genotypes and all the alleles reported in this study.

Sequencing and primer extension were carried out starting from the same purified PCR product in order to keep to a minimum the sources of variation between the 2 protocols.

Sequencing of both strands of the amplicons from nucleotide 306 to nucleotide 613 of the coding region (encompassing the SNPs to validate), was done on a Beckman Coulter CEQ 8000 automated sequencer with DTCS Quick start kit (Beckman Coulter) according to manufacturer's instructions, with primer sequence 5'-GAACAGCCCAGTAAGCC-3' for the forward strand, and 5'-CAGTTTCGGTGAAGTTCTCC-3' for the reverse strand.

Field trial

The primer extension assay presented in this study was used in routine conditions for the genotyping of over 7000 samples in 2 years.

Genotyping assay

The developed primer extension reaction produced clearly interpretable electropherograms with balanced peak heights. This allowed for reliable identification of the PrP genotypes corresponding to the 6 single nucleotide polymorphisms (SNPs) interrogated by the corresponding primers. All known alleles corresponding to the interrogated SNPs have been detected in this study with the exception of ALQ which was reported by [START_REF] Alvarez | Identification of a new leucine haplotype (ALQ) at codon 154 in the ovine prion protein gene in Spanish sheep[END_REF]

Genotype and allele frequencies

Genotype and allele frequencies are shown in table 2 and 3 respectively. Concerning the allele frequencies reported for Sarda breed, we evidence that the contribution of 1205 samples out of 2494 by only two flocks (outbreaks) does not bias the overall figures for this breed. In fact the frequencies calculated for those two flocks only are similar to those calculated for the whole set of Sarda samples, reported in table 3. The frequencies in the two flocks are: 55.31% (ARQ), 40.08% (ARR), 2.07% (AHQ), 0.17% (ARH) and 2.37% (AFRQ). The resulting picture shows significant differences among the investigated breeds, in particular with regard to ARR, VRQ, AFRQ and ARK frequencies. Bergamasca is routine genotyping. This is particularly significant in breeds where ARK is present, such as Bergamasca. In fact, in these breeds two problems would arise without ARK detection capacity: a) no estimate of ARK frequency would be possible and b) a wrong estimate of ARQ frequency would be produced in primer extension since, without a specific primer for the first base of codon 171, ARK would be misclassified as ARQ. The outcome would be even worse using methods based on allele-specific probes. In this case, without ARKspecific probe, heterozygous animals with ARK/XXX genotypes would be misclassified as homozygous XXX/XXX with detrimental consequences (e.g. in case of ARK/ARR wrongly typed as ARR/ARR).

Primer extension for ovine PrP genotyping has already been used by others (Zsonlai et al., 2003;[START_REF] Vaccari | Primer extension assay for prion protein genotype determination in sheep[END_REF][START_REF] Alvarez | Identification of a new leucine haplotype (ALQ) at codon 154 in the ovine prion protein gene in Spanish sheep[END_REF][START_REF] Benkel | Simultaneous detection of eight single nucleotide polymorphisms in the ovine prion protein gene[END_REF] who demonstrated its reliability. Our validation experiment confirmed the accuracy of the technique. In addition, this study is the first example of use of primer extension for the detection of three consecutive SNPs in the ovine PrP gene, the three polymorphic bases of codon 171. This is A c c e p t e d M a n u s c r i p t 10 made possible by use of two overlapping forward primers for the first two bases of the codon. No interference occurred between the two primers. Of course, when the first base of the codon was mutated (ARK) the second base of the same allele was not detectable by the primer directed to the second base, because of the mismatch corresponding to the 3' end of the primer. Anyway the situation was easily interpretable given the contemporary detection of the first base mutation. The third base of codon 171 was interrogated by a reverse primer that did not interfere with the two forward primers.

The results of our survey indicate that considerable genetic difference exists among the investigated breeds. ARR frequency appears significantly low in Bergamasca (6.91%) compared to the other breeds where it ranges from 37.81% of Comisana to 46.97% of Appenninica. On the contrary VRQ shows higher frequency in Bergamasca (4.11%) than the others. In particular its presence can be considered negligible in Sarda where only 1 animal out of 2494 resulted VRQ carrier. AFRQ is remarkably variable: it has been detected in all breeds except Comisana, it is scarcely present in Appenninica (0.34%), moderately represented in Sarda (2.53%) and reaches 5.01% and 10.70% in Bergamasca and Massese respectively. These two breeds also show the highest AHQ frequencies. High AFRQ and AHQ levels render Massese and Bergamasca more genetically prone to atypical scrapie. ARK has been detected only in Bergamasca and Appenninica. Furthermore Appenninica possesses this allele with a very low frequency (0.34%). Consequently Bergamasca is the only breed of this study with high ARK level (3.67%), indeed the highest reported so far. In fact, ARK has been detected with very low frequencies in other breeds from different countries, specifically in Mongolian Sheep [START_REF] Gombojav | Amino acid polymorphisms of PrP gene in Mongolian sheep[END_REF]), in Oklahoma sheep (DeSilva et al., 2003) in Greek breeds (Billinis et al., 2004). Acutis et al. (2004) reported 2.5% of ARK in Italian Biellese breed. The same authors speculated that this allele could be present in other breeds but the use of common methods prevented its identification during routine genotyping. The results of our study confirm the need for 2006) rules out a strong protective effect of this allele but the same authors reported that the positive animal they found had no symptoms and showed only weak positivity in the obex without involvement of any other nervous or linforeticular tissue. This pathologic behaviour does not allow ultimate conclusions on the effects of ARK on susceptibility to the disease. Experiments addressing this issue are underway.

Conclusion

The genotyping method developed accurately identifies AFRQ and ARK in addition to common alleles. Our survey indicates a good level of genetic resistance to classical scrapie due to high ARR frequencies in all investigated breeds with the exception of Bergamasca.

AFRQ is very differently distributed ranging from absence to over 10%. Massese and Bergamasca appear the most susceptible to atypical scrapie based on their higher AFRQ and AHQ frequencies. ARK is restricted to two breeds: Bergamasca, where it reaches the highest frequency ever reported and Appenninica with a very low level. The exact role of ARK in scrapie susceptibility needs to be investigated.

  in the Italian breed Biellese. Secondly, the determination of allele frequencies of AFRQ and ARK alongside the common alleles, in 5 main Italian breeds.

  included in the study, they belonged to 5 Italian breeds: Sarda (n=2494, from 50 flocks), Bergamasca (n=2686, from 13 flocks), Appenninica (n=297, from 12 flocks), Comisana (n=361, from 5 flocks) and Massese (n=402, from 13 flocks). The animals were sampled within the genotyping program for scrapie resistance of Emilia-Romagna Region and the selective culling of scrapie outbreaks from Emilia-Romagna and Lombardy Regions. In outbreaks, all animals were sampled for the study and this accounted for 1205 samples from two outbreaks for Sarda and for 2479 samples from seven outbreaks for Bergamasca. No outbreaks involved Appenninica, Comisana and Massese. Concerning the genotyping program of Emilia-Romagna, all animals submitted to the program by individual breeders were included in the study. These subjects were selected on the basis of their productive performance and for potential use in reproduction within each flock. This preliminary selection process was not linked to scrapie resistance; therefore it was not prone to bias PrP allele frequencies in the study population. The differences in the number of samples and flocks per breed reflect the ovine population structure in Emilia-Romagna with the exception of Bergamasca; most samples of this breed came from Lombardy, its Region of origin. All samples were collected from March 2006 to May 2008.

  A c c e p t e d M a n u s c r i p t stored frozen until DNA extraction.

A

  multiplex primer extension reaction was performed in 10 l volume containing 2 l of purified PCR product, 4 l of SNPStarter Kit (Beckman Coulter) and 4 l of interrogation primers mix. The primers used were designed to be compatible in a multiplex reaction, 5'-polyT tails were added to obtain adequate length spacing of primers in order not to overlap in the subsequent electropherogram. The concentrations of primers were optimized to produce equilibrated signals. Sequence, position and final concentration of each primer areA c c e p t e d M a n u s c r i p t 7 reported in table 1. The reaction was performed in a Mastercycler gradient 5330 (Eppendorf) with the following thermal protocol: 25 cycles of 30 s at 95 °C, 30 s at 57 °C, 30 s at 72 °C. Unincorporated dyes terminators were removed incubating the reaction tubes with 1 U of SAP (GE Healthcare) for 30 min at 37 °C followed by 15 min at 75 °C for enzyme inactivation. Two l of SAP treated reaction product were mixed with 20 l of sample loading solution and 0.3 l of Size Standard 80 (Beckman Coulter) and subjected to capillary electrophoresis on a Beckman Coulter CEQ 8000 automated sequencer. Raw traces were analyzed and peaks identified using the "Fragments" package of CEQ 8000 software.

  in Spanish Churra breed. The Cross-validation experiment gave concordant results for all 97 sequenced samples. During the field trial approximately 1% of the samples needed retesting. The main reasons for retesting were two: a) poor genomic DNA quality or quantity, resulting in insufficient or null amplification, b) incomplete enzymatic clean-up of PCR with consequent confusing extra peaks in electropherograms. The first case was easily identified by lack of sample peaks in presence of size standard peaks; re-extraction of DNA was the routine remedy. The second case appeared as a sequence-like trace and repetition of PCR clean-up systematically removed the problem. Centrifugation of PCR plates before adding EXO-SAP prevented the problem.

  lowest ARR frequency accompanied by the highest VRQ and ARK frequency. In addition this breed shows the highest PrP genetic variability with 7 alleles detected, distributed in 25 genotypes. On the opposite side Comisana possesses only 3 alleles and 5 genotypes. ARK is almost exclusively restricted to Bergamasca with a limited frequency in Appenninica (0.34%). AFRQ is present in all breeds except Comisana. 4. Discussion In 2003 the genetic strategy for scrapie control was established in the European Union (European Commission Decision 2003/100/EC). However, technical or practical limitations of commonly used genotyping methods have prevented extended screening for both AFRQ and unusual alleles. The primer extension method that we developed has made it possible to detect AFRQ and ARK alleles with no extra costs and extra time during

  detection capacity when working with ARK positive breeds such as Bergamasca. On the other hand our results do not evidence a widespread diffusion of ARK among different breeds. The role of this allele in scrapie susceptibility is still scarcely understood. The finding of a scrapie positive sheep with ARK/ARH genotype by Acutis et al. (

Table 1

 1 Interrogation primers used for primer extension reaction at reported codons

	Codon	SNP a Amino acid Interrogation primer (5'-3') b	Concentration (M)
	136	GCC GTC	A V	(15T)GGTGGCTACATGCTGGGAAGTG	0.040
	141	CTT TTT	L F	(32T)GGAAGTGCCATGAGCAGGCCT	0.040
	154	CGT CAT	R H	(9T)GGGGTAACGGTACATGTTTTA c	0.020
	171-I	CAG AAG	Q K	(38T)CCAAGTGTACTACAGACCAGTGGAT	0.016
	171-II	CAG CGG	Q R	CAA GTGTACTACAGACCAGTGGATC	0.040
	171-III	CAG CAT	Q H	(19T)GCACAAAGTTGTTCTGGTTACTATA c	0.012

a Single nucleotide polymorphism; wild type sequence in first line, mutant in second line

b

The number of T bases added to 5'-end of primers is reported in parenthesis c Primer complementary to the reverse strand

Table 2

 2 PrP genotypes and their frequencies (%)

					Breeds (no. of flocks)
		Sarda (50)		Bergamasca (13) Appenninica (12) Massese (13)	Comisana (5)
	Genotype	n	%	n	%	n	%	n	%	n	%
	ARQ/ARQ	688 27.59	1550 57.71	52 17.51	45 11.19	126 34.90
	ARQ/ARR	1089 43.66	275 10.24	123 41.41	133 33.09	189 52.36
	ARR/ARR	451 18.08	14	0.52	64 21.55	62 15.42	40 11.08
	ARQ/AHQ	63	2.53	141	5.25	3	1.01	19	4.73
	ARR/AHQ	64	2.57	10	0.37	5	1.68	29	7.21
	AHQ/AHQ	3	0.12					2	0.50
	ARQ/ARH	6	0.24	45	1.68	19	6.40	1	0.25
	ARR/ARH	8	0.32			14	4.71	6	1.49
	AHQ/ARH			1	0.04	3	1.01	3	0.75
	ARH/ARH					2	0.67
	VRQ/ARQ			174	6.48			16	3.98	2	0.55
	VRQ/ARR			18	0.67	7	2.35	6	1.49	4	1.11
	VRQ/AHQ			3	0.11
	VRQ/ARH			2	0.08	1	0.34
	VRQ/VRQ			4	0.16
	ARQ/ARK			152	5.66	1	0.34
	ARR/ARK			16	0.59	1	0.34
	AHQ/ARK			3	0.11
	ARH/ARK			1	0.04
	VRQ/ARK			5	0.19
	ARK/ARK			6	0.22
	ARK/ARQ			8	0.29
	ARQ/AFRQ	73	2.93	198	7.37	1	0.34	30	7.46
	ARR/AFRQ	37	1.48	24	0.89	1	0.34	36	8.96
	AHQ/AFRQ	7	0.28	12	0.44			5	1.24
	ARH/AFRQ			10	0.37			3	0.75
	VRQ/AFRQ	1	0.04	11	0.41
	AFRQ/AFRQ	4	0.16	3	0.11			6	1.49
	Total	2494 100.00	2686 100.00	297 100.00	402 100.00	361 100.00

Table 3

 3 PrP alleles and their frequencies (%)

				Breeds
		Sarda	Bergamasca	Appenninica	Massese	Comisana
	Allele	%	%	%	%	%
	ARQ	52.26	76.04	42.25	35.94	61.36
	ARR	42.10	6.91	46.97	41.54	37.81
	AHQ	2.81	3.16	1.85	7.46
	ARH	0.28	1.10	6.90	1.62
	VRQ	0.02	4.11	1.35	2.74	0.83
	AFRQ	2.53	5.01	0.34	10.70
	ARK		3.67	0.34
	Total	100.00	100.00	100.00	100.00	100.00

Acknowledgments

The study was supported by Italian Ministry of Health grant IZSLE 011/03 RC (Caratterizzazione Genetica degli ovini dell'Emilia-Romagna per la resistenza alla scrapie) References