Monsieur Rivière Gilles

Monsieur Rose Jérôme

Anaïs Lajarrige

Nathalie Gontard

Sébastien Gaucel

H Besrour

Valérie Guillard

Elisa Goure

Fabrice Gaubiac

Oulaft Amin

Catherine Santaella

Emmanuelle Gastaldi

Marie-Françoise Samson

Stéphane Peyron

to the packaging of foods in bio-nanocomposite materials. En préparation de soumission dans Food Additives & Contaminants: Parts A

Keywords: nanoclays, biopolymer, apparent diffusion coefficient (Dapp), additives, food packaging nanoclays, biodegradable polymer, accelerated aging, migration, apparent diffusion coefficient (Dapp) bio-nanocomposite, food packaging, migration, additives, microbial food simulant

Cette thèse n'aurait pas été possible sans l'aide et le soutien de nombreuses personnes que j'aimerai remercier.

Je souhaite tout d'abord remercier le Labex SERENADE qui a financé cette thèse et qui m'a permis de la réaliser dans des conditions idéales. J'ai été ravie de travailler dans cet environnement enrichissant et dynamique autour des nanos, de participer aux séminaires et d'échanger avec ses membres.

Je voudrai également remercier Nathalie Gontard, ma directrice de thèse, pour m'avoir guidée dans les moments clés de ma thèse. Merci pour ses conseils avisés, ses compétences scientifiques et son aide au moment de la rédaction de mon 1 er article.

Je remercie Stéphane Peyron, mon co-directeur de thèse, qui m'a donné l'opportunité de réaliser ma thèse au sein de l'équipe ePOP et sans qui je ne me serai sûrement pas « lancée » dans la réalisation d'une thèse. Merci pour ses conseils, ses encouragements et le temps qu'il m'a accordé tout au long de ces trois années de thèse et notamment lors de la rédaction de ce manuscrit. J'ai beaucoup appris à ses côtés grâce à ses connaissances en sécurité des matériaux au contact et propriétés de transfert des matériaux. J'exprime mes remerciements à l'ensemble des membres d'avoir accepté de participer à mon jury de thèse : Madame Espuche Eliane,

General introduction

Ce travail de thèse a pour but de poser les bases et les limites de l'élaboration de nouveaux emballages alimentaires nanocomposites à partir de matériaux biodégradables et bio-sourcés sûrs pour l'homme et l'environnement. Ce travail a été mené au sein de l'équipe ePOP (ecoefficient Polymeric and Organic Packaging) de l'UMR IATE (Ingénierie des Agro-polymères et Technologies Émergentes) à Montpellier. Cette thèse s'inscrit au sein du projet Emballage financé par le Labex SERENADE (Laboratory of Excellence for Safer Ecodesign Research and Education applied to NAnomaterial Development). Le Labex SERENADE fait partie du programme français Investissements d'avenir et a été créé en 2012. Les thématiques de recherche s'organisent autour des nanotechnologies dans des domaines variés tels que les peintures, les cosmétiques, les nouvelles technologies, la toxicité et les emballages alimentaires.

Elles se focalisent sur trois buts principaux :

-Développer des produits à base de nanomatériaux ou de matériaux nanostructurés en tenant compte des contraintes socio-environnementales (exposition et risque) tout au long de son cycle de vie.

-Concevoir et optimiser les paramètres de contrôle permettant aux entreprises de placer leurs produits sur le marché.

-Développer des procédés innovants sur les produits en fin de vie : recyclage, traitement des déchets, des effluents et des eaux superficielles.

Ce travail s'inscrit plus particulièrement dans le premier objectif de ce programme dont le contexte est présenté ci-après.

Depuis 1950, la production mondiale de matière plastique ne cesse d'augmenter. Elle a atteint 359 millions de tonne en 2018 contre 348 millions de tonne l'année précédente, consommant près de 10% de la production mondiale de pétrole. En Europe, c'est près de 62 millions de tonne qui ont été produites en 2018 (PlasticsEurope, 2019). Son grand succès est dû à son extrême polyvalence qui lui permet d'être utilisé dans divers domaines : emballage, construction, mobilité et transport, électronique, agriculture, soin et santé, loisir et sport, énergie, etc. Le domaine de l'emballage est le plus consommateur de matière plastique avec près de 40% de la demande. Pourtant, il s'agit du secteur pour lequel la durée d'utilisation du matériau est la plus courte, moins d'un an contre plusieurs décennies pour le secteur de la construction (Geyer et al., 2017). De plus, leur collecte après utilisation reste partielle. En Europe, en 2018, seulement 17.8 millions de tonne d'emballages plastiques ont été collectés pour être traités (42% ont été recyclés, 39.5% valorisés énergétiquement et 18.5% ont été enfouis). Cette grande utilisation des matières plastiques couplée à une collecte en fin de vie qui reste partielle, conduit à une accumulation exponentielle de ces déchets non biodégradables dans l'environnement qui mettent des siècles à disparaître. Si rien ne change, « il y aura plus de plastique dans l'océan que de poisson en 2050 », alerte une étude menée par la fondation Ellen

McArthur.

Face à ces préoccupations environnementales croissantes, des alternatives sont mises en place, encouragées par la Commission Européenne, pour le développement de matériaux biodégradables dans le but de substituer les plastiques d'origine fossile et non biodégradables.

Ainsi, de nombreux projets sont financés dans cet objectif tels que GLOPACK, Res Urbis et USABLE. Cependant, en plus de leur coût relativement élevé en comparaison des matériaux conventionnels, les polymères biodégradables ne possèdent pas toujours de bonnes propriétés fonctionnelles pour les applications envisagées. C'est pourquoi, ces dernières années de nombreux travaux ont été consacrés au renforcement de ces matériaux par diverses charges, végétale ou minérale. En plus de l'amélioration des propriétés fonctionnelles du matériau, elles peuvent en faire diminuer le coût puisque peu coûteuses et sont facilement accessibles. Dans le domaine de l'emballage alimentaire, une des préoccupations majeures liées à l'utilisation de ces nouveaux matériaux est d'avoir des propriétés barrières suffisamment élevées pour permettre une bonne conservation des aliments. L'utilisation d'argiles lamellaires est fortement répandue pour ce type de problématique. Ces particules de taille nanométrique présentent une grande surface spécifique et un facteur de forme élevé permettant de diminuer la perméabilité aux gaz des matériaux nanocomposites. Une bonne dispersion et exfoliation des nano-feuillets d'argile dans la matrice polymère créent un effet de tortuosité qui entraîne une augmentation du trajet de diffusion de la molécule et par conséquent, une diminution de la perméabilité.

Malgré ces bénéfices, l'utilisation de matériaux nanocomposites au contact d'aliments pose de nombreuses questions en ce qui concerne la sécurité du consommateur. La question est de savoir quelle est l'exposition du consommateur à ces nanoparticules elles-mêmes mais également aux autres additifs ajoutés intentionnellement ou non dans ces matériaux. En effet, nous pouvons nous demander si ces nanoparticules ont un effet délétère ou bénéfique sur la migration de ces additifs. Il s'agit de substances ajoutées lors de la formulation des emballages tels que des stabilisants UV, des plastifiants, des antioxydants ou encore des résidus de fabrication tels que des solvants. Un effet bénéfique de ces nanoparticules a été mis en évidence lorsqu'elles sont ajoutées dans des polymères de type polyéthylène, puisqu'elles entraînent une diminution du coefficient de diffusion d'un panel d'additifs et une diminution de leur migration (Nasiri et al., 2016). Mais cette tendance est-elle conservée dans le cas des polymères biodégradables dont la structure évolue au cours du temps ?

Les matériaux et objets destinés à entrer en contact avec des denrées alimentaires sont soumis à des réglementations. Les principales restrictions réglementaires européennes liées à l'utilisation de ces matériaux découlent du règlement (CE) 1935/2004 qui instaure le principe d'inertie. Les matériaux et objets doivent être fabriqués conformément aux bonnes pratiques de fabrication afin que, dans les conditions normales ou prévisibles de leur emploi, ils ne cèdent pas aux denrées alimentaires des constituants en une quantité susceptible de : (i) présenter un danger pour la santé humaine ou (ii) d'entraîner une modification inacceptable de la composition des denrées ou une altération des caractères organoleptiques de celles-ci. Pour évaluer l'aptitude au contact alimentaire de ces nouveaux matériaux et en absence de réglementations spécifiques pour les matériaux biodégradables et/ou nanocomposites, le règlement (CE) N°10/2011 établit pour les matériaux plastiques est appliqué. Ce règlement définit également les conditions d'essai des tests d'aptitude au contact alimentaire (migration globale et spécifique) ainsi que la liste positive des substances autorisées dans la formulation des matériaux destinés à entrer en contact avec les aliments et les limites de migration à ne pas dépasser pour chacune de ces substances. Concernant les nanomatériaux, seulement une substance est référencée dans cette réglementation : les nanoparticules de nitrure de titane (substance N°807). Deux autres substances sont autorisées mais ne sont pas listées comme nanomatériaux, le noir de carbone (substance N°411) et le dioxyde de silicium (substance N°504). Plus récemment, l'oxyde de zinc a également été autorisé au contact alimentaire (EFSA CEF Panel, 2015). Pour les autres substances, se présentant sous forme nanométrique, elles doivent être évaluées au cas par cas pour ce qui est des risques jusqu'à ce que davantage d'informations les concernant soient disponibles.

Ces nanomatériaux posent également d'autres questions en ce qui concerne les risques pour l'environnement. Après usage, ces matériaux seront collectés en vue d'être traités ou abandonnés dans la nature. Ils seront donc compostés industriellement ou biodégradés naturellement dans l'environnement. Mais que deviennent alors les nano-argiles ajoutées dans la formulation de ces matériaux ? Sont-elles un frein à la biodégradation ou au contraire la promeut-elle ? Quels impacts ont-elles sur l'environnement et plus particulièrement sur les plantes et le sol ?

Dans ce contexte, l'objectif de ce travail de thèse a été de :

-Développer des matériaux nanocomposites destinés à l'emballage alimentaire, formulés à partir de polymères biodégradables et bio-sourcés, sûrs pour l'homme et l'environnement.

-Comprendre l'impact de l'incorporation de nano-argiles sur les propriétés du matériau à tous les stades de son cycle de vie : après production, au cours de son usage au contact alimentaire et au cours de sa fin de vie.

-Etudier la balance bénéfice-risque liée à l'ajout de nano-argiles dans des matériaux biodégradables destinés à l'emballage alimentaire.

Dans ce contexte, la stratégie de recherche a été d'étudier deux polyesters biodégradables et bio-sourcés (partiellement ou totalement) présentant des propriétés de transfert différentes.

Le poly(butylene succinate-co-butylene adipate) (PBSA) et le poly(hydroxybutyrate-cohydroxyvalerate) (PHBV), ont donc été choisis. Le PHBV a relativement de bonnes propriétés barrières (Crétois et al., 2014) comparé au PBSA (Phua et al., 2013). Le PBSA est un polyester biodégradable dérivé du copolymère poly(butylène succinate) (PBS). Il est synthétisé par la réaction entre le 1,4-butandiol et des acides aliphatiques di-carboxyliques, acide succinique et acide adipique. Dans le passé, les monomères constitutifs du PBSA étaient exclusivement dérivés de ressources fossiles. Avec le développement de la bioraffinerie, ces monomères sont de plus en plus produits par voie fermentaire à base de matières premières renouvelables de deuxième génération (dextrose, glucose, saccharose, glycérol bio-sourcé et huile végétale) (Salomez et al., 2019). Les polyhydroxyalcanoates (PHAs), famille à laquelle appartient le PHBV, sont une série de polyesters biodégradables obtenus à partir de sucres et de lipides par fermentation bactérienne. Les PHAs possèdent 150 types de monomères différents ce qui permet une modulation de leurs propriétés physiques en fonction de la longueur de la chaîne aliphatique latérale sur le carbone chiral (Steinbüchel and Lütke-Eversloh, 2003). Les PHAs et plus particulièrement le copolymère PHBV possède des propriétés barrières à l'oxygène et à la vapeur d'eau intéressante, ce qui en fait un bon candidat pour des applications en emballage alimentaire. Pour renforcer ces matériaux, une nano-argile modifiée, la Cloisite 30B (C-30B) a été sélectionnée du fait de sa disponibilité, de son faible coût et de son interaction favorable avec le PHBV et le PBSA (Sinha Ray et al., 2008;Carli et al., 2011).

Ces travaux de thèse se sont organisés en différentes parties (Figure 1). La première étape a été de produire ces matériaux sous forme de films en utilisant le procédé d'extrusion.

Différentes méthodes ont été testées afin d'obtenir une structure exfoliée nécessaire à l'amélioration des propriétés barrières du matériau. Les techniques de diffraction des rayons X (XRD) et de microscopie électronique à transmission (TEM) ont permis d'étudier la structure des matériaux. Le taux de charge des matériaux nanocomposites a été déterminé par analyse thermogravimétrique (ATG). La cristallinité a été étudiée par calorimétrie différentielle à balayage (DSC). Les propriétés de transfert aux gaz et à la vapeur ont été étudiées dans le but de déterminer le bénéfice de l'ajout de nano-argiles dans ces matrices polymères. Par la suite, l'aptitude au contact alimentaire des matériaux vierges et nanocomposites a été étudiée (Figure 2) ainsi que l'impact des nano-argiles sur les propriétés de transfert et d'inertie de ces matériaux.

Pour cela, les tests de migration conventionnels utilisés dans le cas des matières plastiques ont d'abord été réalisés (Commission, 2011). Ces tests ont également été effectués sur les matériaux ayant subi un vieillissement accéléré visant à simuler l'exposition dans les supermarchés. Le but étant de connaître l'impact de l'évolution de leur structure dans les conditions d'utilisation sur leur aptitude au contact alimentaire. Pour finir, de nouveaux tests utilisant des simulants alimentaires microbiens ont été mis en place dans le but de déterminer si ces matériaux biodégradables peuvent être utilisés au contact d'aliments contenant des microorganismes tels que les fromages ou les yaourts. En effet, ces matériaux étant par définition instables dans le temps, il est intéressant d'avoir davantage d'informations concernant l'impact des microorganismes sur leurs propriétés de transfert et d'inertie. Une autre partie de cette thèse a été consacrée à l'étude de la fin de vie de ces matériaux. Ainsi, leur biodégradabilité en sol a été étudiée afin de déterminer l'impact de l'ajout de nano-argiles sur ce paramètre. Pour finir, des tests de désintégration ont été réalisés en vue de déterminer l'écotoxicité de ces nano-argiles résiduelles sur les plantes et le sol. Cette dernière partie a été réalisée au sein du CEA de Cadarache, spécialisé sur ces questions d'écotoxicité.

Le diagramme présenté en Figure 3 propose une vue d'ensemble du projet. Ce diagramme sera proposé à chaque début de chapitre pour mieux situer la partie qui y sera abordée. Afin de présenter ce travail, ce manuscrit de thèse a été décomposé en 5 chapitres.

Le premier chapitre est une synthèse de la bibliographie et traite des bénéfices et des risques liés à l'ajout de nano-argiles de la famille des 2:1 phyllosilicates dans des polymères biodégradables pour des applications d'emballages alimentaires.

Le deuxième chapitre présente les matériaux utilisés ainsi que les méthodes employées pour réaliser ce travail.

Le troisième chapitre aborde la question de l'aptitude au contact alimentaire de ces matériaux. Pour cela, l'évaluation des migrations globale et spécifique et l'étude des propriétés de transfert de ces matériaux ont été menées. L'impact de l'ajout des nano-argiles sur le transfert de petites molécules représentatives des additifs utilisés dans l'industrie de l'emballage a été étudié. Ces paramètres ont été étudiés dans une première partie, en suivant les tests définis par la réglementation puis ils ont été adaptés à notre système d'étude. Ainsi, dans une deuxième partie, ces tests de migration ont été effectués sur les matériaux après qu'ils aient subis un cycle de vieillissement aux UVs et dans une troisième partie, ils ont été mis en place en utilisant des simulants alimentaires microbiens.

Le quatrième chapitre vise à étudier les bénéfices et les risques liés à l'ajout des nanoargiles dans ces matériaux biodégradables. Comme nous l'avons dit précédemment, ces renforts ont été ajoutés pour améliorer les propriétés barrières des matériaux. Ainsi, dans la première partie de ce chapitre, la diminution de la perméabilité à l'oxygène obtenue grâce à l'ajout des nano-argiles sera utilisée pour (i) estimer un gain de durée de vie pour un aliment sensible à l'oxygène et (ii) estimer une économie matériau. Pour finir, le bénéfice en terme d'impact environnemental correspondant à ces deux cas de figure sera calculé afin de déterminer le scénario qui minimise le plus cet impact. Dans une deuxième partie, l'exposition du consommateur à un panel d'additifs sera étudiée en considérant la substitution de nos emballages alimentaires actuels par des matériaux bio-nanocomposites.

Pour finir, le cinquième chapitre s'intéresse à la fin de vie de ces matériaux. L'impact de l'ajout de nano-argiles dans ces matériaux sur leur biodégradabilité et leur écotoxicité pour les plantes et le sol sont abordés. Ainsi, les deux parties suivantes de cette synthèse bibliographique recensent les travaux existants sur les bénéfices et les risques décrits dans la deuxième partie de ce chapitre.

Benefits-Risks balance related to the addition of nanoclays in biodegradable polymers dedicated to food packaging 1 Bio-nanocomposites

Biodegradable polymers

In the last decades, biodegradable polymers are considered as a good alternative to replace conventional non-degradable synthetic polymers, particularly for short-term applications like food packaging (Avérous and Pollet, 2012). The terms biodegradable, compostable, biofragmentable or even oxo (bio)degradable are widely used as an argument to promote "environmental" materials but clearly meet very specific standards. A material can be labeled fulfills national or regional standards (i.e. and American Society for Testing and Materials (ASTM) International) distinctively appropriate for conditions that occur in an industrial composter or laboratory-based biodegradation tests using measurements of oxygen demand or carbon dioxide evolution.

There are several standard test methods, which are frequently used to assess the biodegradability of polymers by measuring molecular weight and molecular weight distribution, tensile properties, weight loss, extent of fragmentation, enzyme assays, biochemical oxygen demand, carbon dioxide production, and ecotoxicity [START_REF] Dean | Biodegradability characterization of polymer nanocomposites[END_REF].

The European standard for biodegradability is EN 13432, which sets out the requirements for packaging recoverable by composting and biodegradation. The standard NF EN 13432, accepted by decision of the Commission (2001/524/EC) and published in the Official Journal of the European Union, defines the composting and biodegradation (depending on whether the environment is aerobic or anaerobic) as: the decomposition under the action of microorganisms in the presence of oxygen of an organic chemical compound into carbon dioxide, water and mineral salts, other elements present (mineralization) resulting in appearance of a new biomass ; and decomposition in the absence of oxygen into carbon dioxide, methane, mineral salts and creation of a new biomass .

The definition of a biodegradable packaging material is specified in NF EN 13432:2000, which sets out the conditions for carrying out tests specifying that the tests must be carried out in a medium (compost, soil, fresh or marine water) over a period of maximum 6 months. The physical state and effects on the environment must also meet the following criteria: (i) the starting mass of the material must be degraded to 90%, (ii) the residues must therefore represent Since , when they appeared for the first time [START_REF] Carter | Poly(lactic acid) nanocomposites with various organoclays. I. Thermomechanical properties, morphology, and gas permeability[END_REF], layered silicates gave rise a remarkable interest. It was only in the 1990s that Toyota's first investigations into polyamide reinforced with 5% nanoclay primer demonstrated the full potential of nanocomposite materials with enhanced properties and opened up a vast field of research and development covering many fields of application, including the food packaging sector. The food packaging field is a dynamic market where innovation is a driver of growth and competitiveness. That is why, the nanotechnologies field is dominated, in term of commercial applications by the sector of food packaging [START_REF] Chaudhry | Applications and implications of nanotechnologies for the food sector[END_REF].

In the food packaging field, layered silicates are the widest commercial application of nanoparticles and constitute almost 70% of the market volume (Silvestre et al., 2011). This is not only due to their availability and low cost but mainly to their significant properties enhancements achieved at very low loadings, namely 1-10 wt% (Sorrentino et al. 2007). A wide range of layered silicate exists (Figure I.3).

Figure I.3: Classification of silicates (Rieder et al., 1999;Organization, 2005).

The most common layered silicates utilized to reinforce polymer matrix belong to the general family of 2:1 phyllosilicates. Their crystal structure consists of layers made up of two tetrahedrally coordinated silicon atoms fused to an edge-shared octahedral sheet of either aluminum or magnesium hydroxide (Figure I.4). The thickness of one silicate layer is about one nanometer and the lateral dimensions from 30 nm to several microns [START_REF] Alexandre | Polymer-layered silicate nanocomposites: Preparation, properties and uses of a new class of materials[END_REF] resulting in platelets with high aspect ratio of 100-500. To take maximum advantage of adding layered silicates in polymer matrices, their compatibility with the matrix have to be improved. Indeed, the surface of phyllosilicates, consisting essentially of oxygen atoms, is rather polar and hydrophilic whereas much of synthetic material (polyolefins, polyvinyl and several polyamide and polyesters) exhibits rather hydrophobic character. To fill this gap, the nanoclays are modified by ion-exchange reaction, the inorganic sodium ions being replaced by aliphatic organic cations like alkylammonium, which induce a widening between sheets allowing the insertion of polymer chains between them. Without modification, the interlayer distance is smaller than the radius of gyration of polymers that inhibits this insertion. In addition, the ion-exchange results in a modification of the nanoclays surface which becomes more hydrophobic and thus more compatible with the polymer (Pavlidou and Papaspyrides, 2008). The most common clay minerals used in food packaging applications are gathered in Table I.1 with their commercial name, the chemical structure of organic modifier, their concentration and the corresponding d-spacing, which represents the distance between two layers (Salam et al., 2015;[START_REF] Bumbudsanpharoke | Nanoclays in food and beverage packaging[END_REF]. Cloisites are widely used, they are montmorillonites (MMT) that differentiate by their organic modifier. Nanofil 804 Stearyl diethoxyamine 21 meq/100 g of clay 18

Biodegradable polymers based nanocomposite

Different synthesis methods are used to produce polymer nanocomposite [START_REF] Sundarram | Preparation and characterization of poly(ether imide) nanocomposites and nanocomposite foams[END_REF]:

(1) Solution blending method: it consists in the dissolution of the polymer in a suitable solvent. Nanoparticles are then mixed with the resulting solution. The composite material is obtained after evaporation of the solvent. This method avoid the high processing temperatures (Gloaguen and Lefebvre, 2007) Chapter I. State of the art

(3) Intercalated and flocculated nanocomposite, this structure is the same as intercalated nanocomposites, except for the formation of floccus due to hydroxylated edge-edge interaction of the silicate layers.

(4) Exfoliated nanocomposite, this structure results in silicate layers completely separated and dispersed in the polymer matrix. The exfoliation of the nanoclays platelets yields materials with the highest enhancement of properties. When the layered silicate is incorporated into polymer matrix, an exfoliated structure is desired. The bio-nanocomposite materials, particularly the exfoliated structures, exhibit enhanced properties compared to unfilled biopolymers, which allow to use them in a wide range of field like food packaging. Indeed, the addition of reinforcements in biodegradable polymer matrix lead to an improvement of mechanical properties (elongation at break, tensile strength, stress at break), thermal properties (thermal stability, heat resistance), optical properties (transparency) and barrier properties (gas permeabilities).

Structural factors affecting the properties of polymer matrices

The modification of the properties of the material induced by the addition of nanofillers is due to structural changes of the polymer matrix mainly (1) an increase of the diffusion pathway (tortuosity) and (2) a modulation of the crystallinity.

Tortuosity

The nanoclays platelets, impermeable to the permeate molecules act as an obstacle and create a tortuosity path that force molecules to get around impermeable fillers. The dispersion of fillers at a nanoscale, in a polymer matrix results in a greater enhancement of barrier properties compared to microcomposite and even more to unreinforced materials as the path tortuosity is greatly enhanced (Figure I.6). the distance travelled by the gas molecule of the length (L), width (W) and volume fraction ( ) of the layered silicate and expressed as the following equation [START_REF] Bharadwaj | Modeling the barrier properties of polymer-layered silicate nanocomposites[END_REF]:

(I.1)
The permeability is directly correlated to the tortuosity factor by the equation:

(I.2)
The enhancement of gas barrier and mass transfer properties in polymer layered silicate is influenced by the properties of the nanofillers but also by the architecture of the composite structure including the following structural properties [START_REF] Bharadwaj | Modeling the barrier properties of polymer-layered silicate nanocomposites[END_REF]Choudalakis and Gotsis, 2009) :

The volume fraction of the layered silicate in the polymer matrix. The commonly used volume fraction is low, most of the time inferior to 5 wt%, a high volume fraction leading sometimes to an opposite effect due to a percolation effect.

The morphology of polymer layered silicate in polymer matrix (phase separated, intercalated, flocculated or exfoliated). A poor dispersion state can also lead to an opposite effect due to the formation of preferential pathways.

The orientation of the layered silicate relative to the diffusion direction. A perpendicular orientation will be more efficient because it leads to a longer tortuosity path.

The aspect ratio of the layered silicate (L/W ratio). The higher the aspect ratio, the higher the gas barrier properties. That is why, a sheetlike morphology is particularly efficient compared to others filler morphologies such as spheres.

The properties of the interfaces between matrices and nanofillers. Poor cohesion creates diffusion channels that affect the barrier properties of the material.

The tortuosity is not the unique factor, which contributes to influence the properties of nanocomposite materials.

Crystallinity

The structure of a semi-crystalline polymer is composed of amorphous and crystalline phases. A two-phase model often describes the microstructure of a semi-crystalline polymer.

The crystallinity is assessed by the degree of crystallinity, which represents the percentage of these two phases, the crystalline phase acting as an obstacle to mass transfers according to the free-volume theory of diffusion proposed by Vrentas and Duda. On this basis, a high degree of crystallinity, which leads to a high percentage of crystalline phase, is positive for decreasing mass transfer phenomena (Dardmeh et al., 2015). However, a three-phase model including an additional fraction that connects the amorphous to the crystalline phase generally better replaces this model [START_REF] Esposito | From a three-phase model to a continuous description of molecular mobility in semicrystalline poly(hydroxybutyrate-cohydroxyvalerate)[END_REF]. This intermediate phase is composed of a mobile amorphous fraction (MAF) and a rigid amorphous fraction (RAF). In the MAF, when the polymer is in the rubbery state, the polymer chains are free to move. The RAF can be considered as amorphous segments of restricted mobility (Charlon et al. 2015). The formation of RAF has already been investigated in various polymers: poly(ethylene terephthalate) (PET) (Righetti et al., 2007;[START_REF] Chen | Vitrification and devitrification of rigid amorphous fraction of PET during quasiisothermal cooling and heating[END_REF][START_REF] Badia | The role of crystalline, mobile amorphous and rigid amorphous fractions in the performance of recycled poly(ethylene terephthalate) (PET)[END_REF], poly(trimethylene terephthalate) (PTT) (Ma et al., 2011), polyamide 6 (PA 6) [START_REF] Kolesov | The rigid amorphous fraction of cold-crystallized polyamide 6[END_REF], poly(hydroxybutyrate-cohydroxyvalerate) (PHBV) [START_REF] Esposito | From a three-phase model to a continuous description of molecular mobility in semicrystalline poly(hydroxybutyrate-cohydroxyvalerate)[END_REF], poly(butylene succinate) (PBS) and poly(butylene succinate-co-butylene adipate) (PBSA) [START_REF] Bandyopadhyay | Unique cold-crystallization behavior and kinetics of biodegradable poly[(butylene succinate)-co adipate] nanocomposites: A high speed differential scanning calorimetry study[END_REF]Charlon et al. 2015).

In polymer-layered silicate nanocomposites, the size and shape of nanoclays (Avella et al. 2005) and their content influence crystallization behaviors modifying crystallization kinetic, degree of crystallinity, size of spherulites [START_REF] Maiti | The effect of crystallization on the structure and morphology of polypropylene/clay nanocomposites[END_REF] and/or the percentage of RAF (Charlon et al. 2015). Indeed, the nanoparticles often act as nucleating agents [START_REF] Di Maio | Bionanocomposite polylactic acid/organoclay films: functional properties and measurement of total and lactic acid specific migration[END_REF] which affect the crystallization temperature and kinetic and the size of spherulites because they collide with each other during growth that limit their size. The nanoparticles can also lead to a delay in crystallization due to the confinement of the nanoclays layers (Xu et al., 2005). Concerning the impact on the degree of crystallinity, contrasted results were reported.

A decrease of the degree of crystallinity (Sinha Ray et al., 2005;Wang et al., 2005) can be explained by the full exfoliation of nanoclay platelets in the polymer matrix, which restricts the mobility of the polymer chains and thus prevents them from crystallizing (Krikorian and Pochan, 2003). On the other hand, an increase can also be observed, due to the role of the nanoclays as heterophase crystal nucleation agent (Panupakorn et al. 2013;Nasiri et al. 2016).

Moreover, the RAF occurs in the vicinity of the dispersed and exfoliated nanoclays, which explain the increase of this fraction with the addition of nanoclays. The opposite effect of nanoclays incorporation on the crystallization behaviors of polymers are related to the content, dispersion and exfoliation state of the nanoclays platelets in the polymer matrix. Anyway, the crystallinity is an important parameter to take into account and the study of the crystallinity on a macroscopic scale proved to be often insufficient to explain changes occurring in the material.

Regulation and suitability for food contact

As any material intended to be in contact with foods, which are called food contact materials (FCMs), biodegradable nanocomposite polymers must comply with the provisions of Regulation (EC) No 1935/2004, which establish the principle of inertia. It defines three requirements to ensure safe and quality food: FCM shall not transfer their components into the food in quantities that could endanger human health; FCM shall not change the composition of the food in an unacceptable way; and FCM shall not deteriorate the taste, odor or texture of the food. The regulation sets up a general safety requirement applicable to all possible FCMs and envisages a possibility for the adoption of specific safety requirements for seventeen FCMs.

Plastic materials are part of them and their suitability for food contact is evaluated according to Regulation (EC) No 10/2011 conditions but there is no specific requirements for biodegradable plastic materials. The emergence of these new materials modified the panorama of food packaging and could thus upset the regulatory principles to ensure the consumer safety. Given the diversity of physicochemical properties of biodegradable polymers and their diversity of use, a synthesis of current knowledge about migration phenomena of these materials is still difficult to establish. Many migration studies have focused on polylactic acid (PLA) and the release of lactic acid, lactide or oligomers [START_REF] Conn | Elaboration and characterization of nano-biocomposites based on plasticized poly(hydroxybutyrate-co-hydroxyvalerate) with organo-modified montmorillonite[END_REF][START_REF] Mutsuga | Migration of lactic acid, lactide and oligomers from polylactide food-contact materials[END_REF][START_REF] Dopico-Garcia | Commercial biodegradable material for food contact: Methodology for assessment of service life[END_REF][START_REF] Di Maio | Bionanocomposite polylactic acid/organoclay films: functional properties and measurement of total and lactic acid specific migration[END_REF]. However, the use of PLA as a food contact materials is considered as safe considering that migrating species are expected to convert to lactic acid for which no specific migration limit has been established by Regulation (EC) No 10/2011. In the same way, the migration of crotonic acid, compound resulting from thermal degradation of PHB and PHBV, is authorized with a specific migration limit of 0.5 mg/kg. These monomers being considered as safe for human health [START_REF] Clarke | Oral 28-day and developmental toxicity studies of (R)-3-hydroxybutyl (R)-3-hydroxybutyrate[END_REF], the question of the suitability for biodegradable polymers is more related to the alteration of the organoleptic properties of the packaged food. The suitability for food contact of these materials require specific provisions, which are different from those of the conventional polymers. Indeed, the high water sensitivity that often lead to an overall migration value greater than the limit set at Concerning the use of engineered nanomaterials (ENMs) in FCMs, little is known about their potential risks but based on the toxicological data the smaller nanoparticles are more toxic than larger ones due to their high specific area. A definition to determine whether a material may be still considered controversial and no harmonized definition between the different organizations has been chosen. More recently, zinc oxide nanoparticles was approved for use in food contact materials in a scientific opinion published by EFSA with a specific migration limit of 0.05 mg/kg (EFSA CEF Panel, 2015). Recently, the European Food Safety Authority has produced a guidance on human and animal health aspects (Part 1) of the risk assessment of the application of nanoscience and nanotechnologies in the food and feed chain (EFSA Scientific Committee, 2018). This guidance is aimed at providing a structured pathway for carrying out safety assessment of nanomaterials in the food and feed area. Regarding the risk assessment specifically recommended for the FCM and considering that the scientific knowledge remains too limited today to define a threshold for the migration of nanomaterials from FCM, may be made on a case-by-case basis if migration of a nanomaterial in particulate form is only

Explanation of the analysis approach

The present study focused on the benefits and the risks related to the addition of 2:1 phyllosilicate in biodegradable polymers dedicated to food packaging applications on the basis of peer-reviewed scientific journals published from 2000. Manufacturers in the sector of food packaging are currently developing and formulating nanocomposite materials in the hope of improving packaging performances, most often without having a clear vision of the added value provided by nanoparticles (NPs) or the potential risks associated with their incorporation.

Applied to the particular case of nanocomposite packaging, the analysis of the benefits-risks balance is all the more complicated because it combines two components that are assessed on different criteria and value scales and that fall within different time steps. As a first approximation, the incorporation of NPs into the packaging should be considered as generate benefits related to their performance in terms of barrier and mechanical properties. The expected benefits could then easily be expressed in terms of material and, consequently, cost savings.

Risks are more difficult to a priori anticipate and, above all, their assessment is performed within a regulatory framework that the authorities take several years to set. We have seen above that the current lack of specific guidelines applied to nanocomposite materials forces the user to undertake a case-by-case assessment for which it may be difficult to identify the contours.

We have seen previously that the addition of NPs in a material generates changes in the properties of the packaging likely to affect all stages of its life cycle. The consequences related to this incorporation are of course multiple and can have repercussions that are difficult to predict and measure (depletion of resources in the long term, impact on ecosystems or even consumer acceptability...). The exhaustive analysis of the benefits-risks balance associated with the incorporation of inorganic NPs therefore involves a multitude of technical, environmental, economic, social and even ethical components. It is necessarily based on a multi-criteria analysis that can only be put into practice through approaches such as "knowledge engineering". This ambitious objective is not unattainable in the long term provided that there is a sufficiently rich knowledge base (or "expert" data) for each of the dimensions taken into consideration.

With a more modest objective, it seems initially feasible to restrict this analysis to the scale of the packaging life cycle alone, with the assessment of risks and benefits to be established in relation to the performance of the virgin material considered as the reference for the study. In this way, the main steps that can be considered and integrated into the study are (1) use in contact with food and (2) end-of-life management of the material.

The benefits include publications related to:

(1) Oxygen, carbon dioxide and water vapor permeabilities. They are of great interest for food packaging applications. These gas permeability data were analyzed with respect to the polymeric matrix, the 2:1 phyllosilicate type (montmorillonite, hectorite, micas, modified or not), its percentage in the matrix and the process used to synthetize the material. All these data are gathered in Table I.2.

(2) Mechanical properties, they are an important parameter in the design of food packaging.

(E), tensile strength (TS) and elongation at break (EB) were analyzed as regard to the polymeric matrix, the 2:1 phyllosilicate type, its percentage in the matrix and the process used to synthetize the material. All these data are gathered in Table I.3.

The risks are focused on:

(1) The potential migrations of components from material to foods. It takes into account, the overall migration, the specific migration of additives and layered silicate modifier and the migration of constitutive elements of layered silicates, silicon, magnesium or aluminium.

(2) The impact of layered silicate on the biodegradability of polymer matrix.

(3) The ecotoxicity of layered silicate on the environment after biodegradation of the material.

Other parameters, which cannot be considered as a benefit or a risk, are important in the design of food packaging such as optical and thermal properties.

Benefits

Barrier properties

Many factors must be taken into account in designing food packaging, one of the important of them being the gas and vapor permeabilities [START_REF] Akbari | Improvement in food packaging industry with biobased nanocomposites[END_REF] which are involved in the preservation of packaged products and participate in the control of the respiratory intensity of fresh or low-processed products such as fruit and vegetables. The permeability is defined as the quantification of gas or vapor transmission through unit area per unit time. In contrast to glass materials, packaging made with plastics are more or less permeable, depending on polymer, to small molecules such as gases (oxygen, carbon dioxide, nitrogen, etc.), water vapor, aromas, flavors, etc. These molecules can migrate from external environment into the inside of the packaging and vice versa through the packaging film. The consequences being the contamination of the foods in the case of aromas or flavors or a reduction of the food shelf-life in the case of gas exchange (Siracusa, 2012). Indeed, some foods need sufficient impermeable packaging to gas to prevent deterioration. The cheese for example, requires low oxygen permeability food packaging to avoid fat oxidation and mold growth (Robertson, 2006). For other foods such as biscuits, the water vapor permeation should be avoided. It is also the case for fresh food products to prevent dehydration [START_REF] Siracusa | Biodegradable polymers for food packaging: a review[END_REF].

Impact of the addition of layered silicate

Gas permeability data of 64 publications were analyzed as regard to the polymeric matrix, the 2:1 phyllosilicate type (montmorillonite, hectorite, micas, modified or not), its percentage in the matrix, the process used to synthetize the material and the migrant molecule. These data are gathered in Table I.2. The analysis of these publications is presented in Figure I.8, which reports the permeability increase or reduction according to the percentage of 2:1 phyllosilicate. The variation rate of permeability is calculated by the following equation:

(I.3)
Where is the permeability of polymer nanocomposite and , the reference value of permeability measured on the neat polymer.

The result is positive when the addition of nanoclays decreases the gas or water vapor permeability and negative when an increase of permeabilities is obtained.

(1) Oxygen permeability (2) Carbon dioxide permeability

(3) Water vapor permeability The analysis showed that 38% of the publications are focused on oxygen (1), 12% on carbon dioxide (2) and 50% on water vapor (3) permeabilities. Water vapor and oxygen are the most studied because they are the two main permeants that affect the quality and the shelf life of the foods. However, the study of carbon dioxide is still important, especially for the modified atmosphere packaging technology used in the case of fresh products [START_REF] Siracusa | Biodegradable polymers for food packaging: a review[END_REF].

The result of this analysis indicates that globally, with some exceptions, the addition of layered silicate in a biodegradable polymer matrix is efficient to improve their barrier properties. The reduction of gas permeability can be explained by the presence of nanoclay platelets, which lead to structural changes of the polymer as discussed in Section 1.4. They are supposed to introduce a tortuous path for the diffusing molecules but it is also well established that there are indirect effects related to structural modifications.

Impact of the modification of layered silicate

In some cases, the addition of layered silicate in biodegradable polymer matrix has a negative impact on barrier properties. It is important to note that this is often the case when native sodium montmorillonites (MMT-Na + ) are used. The addition of MMT-Na + in wheat gluten matrix leads to an increase of the permeability of the films towards oxygen, until 18%, and carbon dioxide, until 20%, for a nanoclays content of up to 10 wt% [START_REF] Tunc | Functional properties of wheat gluten/montmorillonite nanocomposite films processed by casting[END_REF]. An increase of the oxygen permeability is also observed by Sanchez-Garcia & Lagaron (2010b) in their study focused on PHBV/mica, at a loading of 10 wt%. [START_REF] Sothornvit | Effect of nano-clay type on the physical and antimicrobial properties of whey protein isolate/clay composite films[END_REF], tested the VWP properties of PLA-based composite films reinforced with different nanoclays, unmodified montmorillonite, Cloisite 20A and Cloisite 30B. An increase of the water vapor permeability of 15% is obtained for the Cloisite Na + compared to the unfilled PLA. In contrast, the nanocomposite films compounded with organo-modified nanoclays show a decrease of the WVP of 36% for the Cloisite 20A and 6% for the Cloisite 30B, the same behavior being observed with PLA/MMT-Na + material [START_REF] Tang | A model for water vapor permeability reduction in poly(lactic acid) and nanoclay nanocomposites[END_REF]. However, a decrease of the permeability is also observed by replacing the MMT-Na + by either Cloisite 10A, 15A, 20A, 25A, 30B or 93A. Indeed, it is also the observation made by [START_REF] Chen | Preparation and characterization of nanocomposite of maleated poly(butylene adipate-co-terephthalate) with organoclay[END_REF] when they studied a film made of PBAT filled with different MMTs. This phenomenon can be explained by the incompatibility between the hydrophilic clays particles and the hydrophobic polymer matrix, which hinder the delamination of platelets to form exfoliated morphology. The functionalization of the polar clay surface with organic ammonium ions bearing long aliphatic chains allows to achieve a good dispersibility of nanoclay platelets and consequently better barrier properties (Duncan, 2011).

Impact of the layered silicate loading

A small loading of layered silicate is most of the time added in the polymer matrices. At low concentration, the nanoclays platelets were generally more uniformly dispersed in the matrix, which lead to a better exfoliation. The surface interface between nanoclays and polymer is therefore increased and better barrier properties are achieved [START_REF] Weizman | Modified whey protein coatings for improved gas barrier properties of biodegradable films[END_REF].

A high layered silicate loading can be responsible of a decrease of the barrier properties, as conclude on different publications. This is due to the tendency of the nanoclays to form aggregates, this phenomenon being even more marked in case of incompatibility between the nanoclay and the matrix. An expected impact of nanoclays is obtained on WVP, for a material made of zein, at low loading (up to 5 wt%) of modified MMT, whatever the processing method, solution blending or blown extrusion. On the other hand, at 10 wt%, an increase of 28% is observed [START_REF] Luecha | investigate the mechanisms of formation and loss of the rigid amorphous fraction[END_REF]. The same behavior is observed in two other studies conducted on PHBV. For the first one (Sanchez-Garcia and Lagaron, 2010), the polymer is reinforced with mica. At 5 wt% a decrease of the oxygen permeability of 30% is obtained while when the percentage is increased to 10 wt%, the permeability increases to 60%. The second study (Farmahini-Farahani et al., 2017) used Cloisite 30B as layered silicate. From 10 wt% and up to 40 wt% an increase of the WVP, going up to 140%, is observed.

However, an association of modified layered silicates and low loading can also lead to undesired behaviors. A study conducted on wheat starch filled with 3 and 6 wt% of native and organo-modified with cationic starch montmorillonite revealed, excepted for wheat starch reinforced with 6 wt% of native MMT, an increase of the WVP [START_REF] Chivrac | How does water diffuse in starch/montmorillonite nano-biocomposite materials?[END_REF]. Likewise, Crétois et al. (2014) obtained a decrease of both water vapor and carbon dioxide permeabilities when PHBV is reinforced with 2.5 wt% of Cloisite 30. The reinforcement of 2.5 to 7.5 wt% with native and modified MMT of fenugreek (Trigonella foenum-graecum) seed gum lead to an increase of the WVP [START_REF] Memis | Production and characterization of a new biodegradable fenugreek seed gum based active nanocomposite film reinforced with nanoclays[END_REF]. Recently, Akin and Tihminlioglu (2018), obtained an increase of 45% of the WVP for a PHBV/Cloisite 10A material filled with 5 wt%. The oxygen permeability is not spared by this observation since [START_REF] Weizman | Modified whey protein coatings for improved gas barrier properties of biodegradable films[END_REF] found an increase of the oxygen permeability of PLA coated with 8 wt% of whey protein when 0.5 or 1 wt% of Cloisite 15A or 1 wt% Cloisite 30B are incorporated. The creation of preferential pathways for diffusing molecules, in specific domains where the layered silicates were completely or partially aggregated and non-exfoliated or even absent can explained these observations.

Impact of the processing method

The process used to synthetize bio-nanocomposite films seems to have also an impact on the material transfer properties. The reduction of WVPs of zein film are similar at a MMT loading of 1 wt% whether solution blending method or blown extrusion method is used. At 3 wt%, the reduction of WVP is three time lower using the solution blending method [START_REF] Luecha | investigate the mechanisms of formation and loss of the rigid amorphous fraction[END_REF]. In addition, the films produced by solution blending method show better reduction in oxygen and WV permeabilities compared to melt blending method for PLA/MMT and PHBV/MMT materials [START_REF] Sanchez-Garcia | Novel clay-based nanobiocomposites of biopolyesters with synergistic barrier to UV light, gas, and vapour[END_REF]. This same investigation conclude that solution blending is more efficient to enhance the barrier properties of biopolyesters because there are no issues associated with filler-induced polymer hydrolysis [START_REF] Sanchez-Garcia | Novel clay-based nanobiocomposites of biopolyesters with synergistic barrier to UV light, gas, and vapour[END_REF]. Charlon et al. (2015) investigated the influence of extrusion conditions from films of PBS and PBSA reinforced with 5 wt% of native and organo-modified MMT (Cloisite 30B)

produced by a conventional process and those submitted to new extrusion conditions where water was introduced into the extruder barrel. They actually observed a difference in material barrier properties depending on the process conditions, the water-assisted extrusion process enhancing the carbon dioxide and WV permeabilities whatever the polymer and the MMT used.

The better exfoliation state of the nanoclays platelets in polymer matrix obtained using waterassisted extrusion would explain this improvement of barrier properties. The same conclusion was made by Rhim et al. (2006) who observed a difference of WVP values of PLA depending on the processing methods i.e. thermocompression or solvent casting. Moreover, even with using the same matrix Poly-L-lactide (PLLA, Biomer ® L9000; Mw=200 kDa, Mw/Mn=1.98) and process, the WVP values can be different probably due to the heterogeneity of the materials produced by solvent casting (Rhim et al., 2006b[START_REF] Sothornvit | Effect of nano-clay type on the physical and antimicrobial properties of whey protein isolate/clay composite films[END_REF]. Indeed, a precise comparison between barrier properties is difficult, even when the same polymer and method are used. Gas molecules transfers are strongly affected by various parameters such as the structure and composition of the biopolymer, the crystallinity [START_REF] Arvanitoyannis | Study of diffusion and permeation of gases in undrawn and uniaxially drawn films made from potato and rice starch conditioned at different relative humidities[END_REF], the type and content of plasticizer [START_REF] Enrione | Sorption behavior of mixtures of glycerol and starch[END_REF], the conditions of storage (Phan et al., 2005).

The conditions in which the determination of the permeability is made can affect also the results. [START_REF] Alves | Barrier properties of carrageenan/pectin biodegradable composite films[END_REF] determined the WVP of k-carrageenan/pectin filled with organomodified MMT and found, depending on the driving force imposed, very different values.

Moreover, it is important to specify the conditions under which the experiments are performed because the values can be very different. The water vapor transmission rate of neat PHBV varies from 12.1 g/m².day at 50 RH% and 23°C to 50.0 g/m².day at 90 RH% and 38°C (Farmahini-Farahani et al., 2017). The hydration state of the nanoclays proved to have a significant impact on water and oxygen permeability (Tenn et al., 2013). The incorporation of hydrated C-30B leads to a better intercalation of the nanoclay platelets and consequently improved barrier properties.

Mechanical properties

Another important parameter in designing food packaging is the mechanical properties.

They determine the behavior of the material when subjected to mechanical stress, how much it can be stretched, how much it can be bent, how hard or soft it is, how it behaves on the application of repeated load, etc. In this way, three main parameters are generally investigated:

(1) Elastic modulus or (E), which gives information on the stiffness of the material.

(2) Tensile strength (TS), which is related to the stretching of the material. It represents the stress required to induce the rupture of the sample.

(3) Elongation at break (EB), which translates the ductility of the material. It measures the percentage change in the length of the material before fracture.

The determination of these parameters is carried out thanks to the stress strain curve (Figure I.9).

Figure I.9: Tensile stress-strain curve for a semi-crystalline polymer.

Impact of the addition of layered silicate

(EB) data of 65 publications were analyzed as regard to the polymeric matrix, the 2:1 phyllosilicate type, its percentage in the matrix, the process used to synthetize the material and the property studied. These data are gathered in Table I The result is positive when the addition of nanoclays increases the mechanical properties and negative when a decrease is obtained.

Chapter I. State of the art

The results of this analysis show, above all, a very large disparity in the effects related to the incorporation of nanofillers in biodegradable polymers. An improvement of the mechanical at the same time is difficult to achieve [START_REF] Chang | Poly(lactic acid) nanocomposites with various organoclays. I. Thermomechanical properties, morphology, and gas permeability[END_REF]Sinha Ray et al., 2005;Bordes et al., 2008;[START_REF] Müller | Composites of thermoplastic starch and nanoclays produced by extrusion and thermopressing[END_REF][START_REF] Hadj-Hamou | Effect of cloisite 30B on the thermal and tensile behavior of poly(butylene adipate-co-terephthalate)/poly(vinyl chloride) nanoblends[END_REF][START_REF] Scarfato | Optical and mechanical properties of UV-weathered biodegradable PHBV/PBAT nanocomposite films containing halloysite nanotubes[END_REF][START_REF] Scarfato | Performance properties, lactic acid specific migration and swelling by simulant of biodegradable poly(lactic acid)/nanoclay multilayer films for food packaging[END_REF]Akin and Tihminlioglu, 2018;[START_REF] Issa | Sweet potato starchbased nanocomposites: development, characterization, and biodegradability[END_REF]. In most of nanocomposite structures, the addition of modulus, stress and hardness of materials but due to their stiffness a decrease of the elongation at break is often obtained with the addition of nanoclays.

(1) Elastic modulus or

(A) (B) (C) (2) Tensile strength (A) (B) (C) (3

Impact of the layered silicate loading and nature

s modulus (E)

The addition of layered silicate in biodegradable polymer matrices leads, in most cases, in (E) ), which means that the stiffness of the material is increased. The higher the E, the lower the strain for the same stress.

Most of the time, a low percentage of nanoclay, of the order of 5 or 10wt%, is added in the materials but some studies investigated the impact of the addition of higher nanoclay percentage [START_REF] Lee | Microstructure, tensile properties, and biodegradability of aliphatic polyester/clay nanocomposites[END_REF]. The E of a nanocomposite made of PHBV was increased from 65% with the addition of 1 wt% of Cloisite 15A to 165% with 5 wt% compared to the neat PHBV [START_REF] Bruzaud | Thermal degradation and (nano)mechanical behavior of layered silicate reinforced poly(3-hydroxybutyrate-co-3-hydroxyvalerate) nanocomposites[END_REF]. The addition of nanoclays promoted an increment in the E of 103% and 63% for the PHBV reinforced with 5wt% of Cloisite 30B and halloysite respectively (Carli et al., 2011). Others studies evidenced that this increase in E followed the increase of the nanoclay loading, for poly(vinyl alcohol) (PVA)/MMT-Na + [START_REF] Strawhecker | Structure and properties of poly(vinyl alcohol)/Na+ montmorillonite nanocomposites[END_REF], poly(butylene succinate-co-adipate) (PBSA)/Cloisite 30B (Sinha Ray et al., 2005), wheat gluten/Nanofil ® EXM 757 [START_REF] Tunc | Functional properties of wheat gluten/montmorillonite nanocomposite films processed by casting[END_REF] or brea gum/MMT-Na + [START_REF] Slavutsky | Preparation and characterization of montmorillonite/brea gum nanocomposites films[END_REF].

Other authors demonstrated that an optimal amount of organoclay can be achieved, which leads to the highest improvement of E [START_REF] Gloaguen | Biodegradable nanocomposites based on starch/polycaprolactone/compatibilizer ternary blends reinforced with natural and organo-modified montmorillonite[END_REF]Akin and Tihminlioglu, 2018).

However, a decrease of E is obtained in some nanocomposites. Singh et al. (2010), observed a slight increase for PLLA reinforced with 1 wt% of Cloisite 30B and then a decrease with the augmentation of nanoclay loading. Whey protein isolate/Cloisite Na + , Cloisite 20A or Cloisite 30B nanocomposite showed a decrease of 36%, 33% and 5% respectively with the addition of 5 wt% nanoclay [START_REF] Sothornvit | Effect of nano-clay type on the physical and antimicrobial properties of whey protein isolate/clay composite films[END_REF]. The authors explained this decrease by the increase of the film thickness with the addition of Cloisite Na + , while the significant impact of Cloisite 20A was attributed to their incomplete dispersion into the polymer matrix. The degree of crystallinity of materials can also play a role in the modulation of the mechanical properties. The lower E of the exfoliated PBAT/ODA-MMT nanocomposite with inorganic content of 3 wt% is attributed to its lower degree of crystallinity compared to the other studied nanocomposites, which reduces the stiffness of the material [START_REF] Someya | Nanocomposites based on poly(butylene adipate-co-terephthalate) and montmorillonite[END_REF].

On tensile strength (TS)

The addition of layered silicate in biodegradable polymer matrices leads to contrasted impact of TS ) results

in an increase of the TS. The TS is related to the stretching of the material, the higher the TS, the higher the stress to induce the rupture of the sample.

The TS appears to be highly impacted by the exfoliation state of the nanoclay platelets in the polymer matrix. Among the PBAT/organoclay nanocomposites, the exfoliated ODA-MMT material showed the highest strength at 3 wt% and 5 wt% compared to the intercalated DA-MMT and LEA-MMT materials. The occurrence of intercalation rather than exfoliation for ODA-MMT observed at 10 wt% of loading leads to similar TS between all materials [START_REF] Someya | Nanocomposites based on poly(butylene adipate-co-terephthalate) and montmorillonite[END_REF].

As for improvement of TS can be achieved. Above a certain percentage of nanoclay, the TS values of all nanocomposites started to decrease, mainly because of the agglomeration of nanoclay particles [START_REF] Chang | Poly(lactic acid) nanocomposites with various organoclays. I. Thermomechanical properties, morphology, and gas permeability[END_REF][START_REF] Sothornvit | Effect of clay content on the physical and antimicrobial properties of whey protein isolate/organo-clay composite films[END_REF]Xie et al., 2016).

On elongation at break (EB)

The addition of layered silicate in biodegradable polymer matrices leads, in most case, to a decrease of the EB ), translating the ductility of the material, the higher the EB, the higher the deformation before its rupture.

The EB of alginate-based films decreased with the addition of MMT-Na + and reached its lowest value at highest fillers content [START_REF] Abdollahi | Comparing physico-mechanical and thermal properties of alginate nanocomposite films reinforced with organic and/or inorganic nano fillers[END_REF]. Similar behaviors were observed in others studies using the same nanoclay (MMT-Na + ), for gelatin [START_REF] Bae | Effect of clay content, homogenization RPM, pH, and ultrasonication on mechanical and barrier properties of fish gelatin/montmorillonite nanocomposite films[END_REF], proteins [START_REF] Lee | Characteristics of soy protein isolate-montmorillonite composite films[END_REF][START_REF] Hassannia-Kolaee | Modification of functional properties of pullulan whey protein bionanocomposite films with nanoclay[END_REF], brea gum [START_REF] Slavutsky | Preparation and characterization of montmorillonite/brea gum nanocomposites films[END_REF], poly(vinyl alcohol) (PVA) [START_REF] Strawhecker | Structure and properties of poly(vinyl alcohol)/Na+ montmorillonite nanocomposites[END_REF]. The addition of organo-modified nanoclay lead to same behaviors, for PHBV/Cloisite 15A [START_REF] Bruzaud | Thermal degradation and (nano)mechanical behavior of layered silicate reinforced poly(3-hydroxybutyrate-co-3-hydroxyvalerate) nanocomposites[END_REF], PHBV/Cloisite 30B [START_REF] Choi | Preparation and characterization of poly(hydroxybutyrate-co-hydroxyvalerate)-organoclay nanocomposites[END_REF] or PLA/Cloisite 15A, 20A, 30B (Koh et al., 2008) nanocomposites. It is well known that the addition of stiff reinforcements can reduce the EB of polymeric matrix, since the reinforcements will cause stress concentrations.

The study of the mechanical properties evidenced the fact that there is an optimal amount of nanoclay needed in the nanocomposite to achieve tensile strength and elongation at break of the materials, the level of interface cohesion of the two phases being a structural parameter that is difficult to evaluate but well known to impact each of these mechanical characteristics.

Risks

Despite the benefits of the introduction of layered silicate in food packaging, this technology results in various potential risks. The commercial applications of biodegradable packaging materials have a still minor market share in Europe and their commercial development on a wider scale of production and distribution can only be achieved if the safety and sustainability issues associated with their use are scientifically approved. The risks associated with the incorporation of nanoclays can be considered at different stages of the packaging life cycle, i.e. at the stage of the manufacture (or production of the packaging material), during its contact with the food and, finally, in its end-of-life journey.

The management of risks related to the deployment of nanocomposite packaging should in principle includes the issue of worker exposure (from the extraction/purification/synthesis of the NPs to the production of the finished material). However, this concern is more a matter of industrial strategies aimed at confining equipment and cannot be integrated into a rational design approach for nanocomposite materials for packaging applications. Therefore, risks related to worker/operator exposure are voluntarily excluded from this study.

The first concern of public authorities and consumer associations is the potential migration of these nanoparticles (or their elementary component) into foodstuffs, which leads to their ingestion, and could be a hazard for human health. However, even if many researches are focused, particularly for the past ten years, on the impact of nanosized materials on the human health [START_REF] Esch | Body-on-a-chip simulation with gastrointestinal tract and liver tissues suggests that ingested nanoparticles have the potential to cause liver injury[END_REF], there is still a lack of knowledge in this field and it is still difficult today to establish generic laws given the great diversity of nanoparticles used in materials science. In addition to the question of the toxicity of the nanoclay itself, the presence of nanofillers in a polymeric matrix can also be considered as a positive option in terms of impact on consumer exposure to potentially toxic substances contained in packaging materials (additives and neoformed compounds) given their ability to limit the mobility of low molecular weight molecules. The impact of the presence of NPs in nanocomposite packaging on the migration of other non-nano and potentially harmful components incorporated in the materials is much less considered and very few studies have been conducted on this issue.

The risk assessment related to the addition of nanoclays in a biodegradable packaging necessarily includes an environmental component, which is closely related to the end-of-life material management. The biodegradability of bio-nanocomposite packaging therefore raises two fundamental questions:

(1) what is the influence of the incorporation of nanoclays on the stability of biodegradable materials and consequently on their degradation kinetics? and ( 2) what changes follow the nanoparticles resulting from these materials in the natural environment and what are their impacts on the soil? The first question has been a concern in the packaging community for several years and a number of investigations aimed at characterizing the influence of nanoparticles on material inertia have highlighted the complexity of the effects produced by the incorporation of nanoparticles on the structural properties of biodegradable polymers. Furthermore, the biocompliance of residual nanoparticles after total degradation of the packaging materials and their impact on ecosystems is a critical issue that must be taken into account in an environmental assessment.

Use in food contact

As a material dedicated to a food application, bio-nanocomposite materials must comply with the prerogatives laid down in the European framework Regulation EC/1935/2004 which introduces the principle of inertia, which guarantees that any material or article intended to come into contact with foods must be manufactured in accordance with good manufacturing practices. Beyond this generic definition of the principle of inertia, which must apply to all packaging materials, nanocomposite materials and, a fortiori, bio-nanocomposite materials are not subjected to any specific directive or regulation describing the conditions of controlling their compliance. As consequence, and by default, the suitability for food contact applications of such materials is carried out according to the conditions defined for plastic materials in the European Regulation (EC) No 10/2011. One of the critical issues in the use of materials for food packaging application is the contamination according to a migration process, which consists in the unintentional transfer of packaging components into the food [START_REF] Honarvar | Nanocomposites in food packaging applications and their risk assessment for health[END_REF]. In this way, the assessment of plastic materials is based on two separate measures i.e.

the overall migration and the specific migration both associated with threshold values. In the specific case of nanocomposite material, the evaluation of the migration of nanoparticles or/and their constituents must necessarily complement this approach developed for continuous homogeneous materials.

Overall migration

The overall migration represents the total amount of non-volatile substances released from a material into food or food simulant. The European Commission (European Commission, 2011) defined an overall migration limit (OML) of 10 mg.dm -², not to be exceeded.

The respect of this limit is primary when materials are intended to be in contact with food.

That is why, it is investigated in many publications.

The overall migration of biodegradable polymers reinforced with layered silicate was studied and it is on this type of reinforcement that the following study focuses. The use as food packaging of a bio-nanocomposite material, PLA reinforced with 5% Cloisite 30B was investigated [START_REF] Schmidt | Combining asymmetrical flow field-flow fractionation with light-scattering and inductively coupled plasma mass spectrometric detection for characterization of nanoclay used in biopolymer nanocomposites[END_REF]. The results demonstrated that, even if the amount of migration is higher in the nanocomposite than in the pure PLA, the materials comply with the overall migration limit of 10 mg.dm -². The suitability for food contact of a novel protein-based material made of wheat gluten and montmorillonite was also studied [START_REF] Iglesias | Wheat gluten nanocomposite films as foodcontact materials: migration tests and impact of a novel food stabilization technology (high pressure)[END_REF]. The authors determined the overall migration in three different aqueous food simulants and in olive oil. In contact with fatty food simulant, the overall migration was below the limitation, whereas these ones conducted in aqueous food simulants revealed the necessity to develop migration test suitable for water-sensitive materials. In another study, the overall migration of PLA plasticized with 15 wt% and 20 wt% of polyadipate and blended with 3wt% of organo-modified montmorillonite was determined. In water simulant, the overall migration limit fixed to 10 mg.dm -² by the Commission Directive 2007/19/EC (now repealed by the Regulation EC/10/2011) was respected while only the materials with the lower plasticizer content fulfilled the requirements in olive oil simulant [START_REF] Martino | Correlation between composition, structure and properties of poly(lactic acid)/polyadipate-based nano-biocomposites[END_REF]. The overall migration in 3% acetic acid and 50% ethanol was additionally measured from unfilled and PLA films added with different amount of Cloisite 30B. In all cases, a low overall migration level and above the regulation limit was evidenced [START_REF] Di Maio | Bionanocomposite polylactic acid/organoclay films: functional properties and measurement of total and lactic acid specific migration[END_REF]. The PLA reinforced with unmodified bentonite and four types of organo-modified bentonites has also been studied

. The determination of overall migrations were conducted on distilled water, 3% acetic acid and 95% ethanol food simulants. The migration study results revealed that the migration values were again well below the authorized limit. More recently, a study showed that PHBV bio-nanocomposites with natural and modified vermiculite were possibly inert in contact with food since the results of the overall migration were less than 5.0 mg/kg as recommended by ANVISA Resolution 105/99 in Brazil [START_REF] Da Costa Reis | Biodegradability of and interaction in the packaging of poly(3-hydroxybutyrate-co-3-hydroxyvalerate)-vermiculite bionanocomposites[END_REF]. As conclusion, all these works mainly directed at biopolyesters, suggests that the incorporation of nanoclays does not have a detrimental impact on the inertia of materials that meets the regulatory prerogatives on this single criterion of overall migration. It shows also in some cases, the necessity to provide specific requirements for these type of materials, especially for leads to an overall migration value greater than the limit.

Nanoparticle migration

In the last few years, several studies focused on the migration from polymer nanocomposites were published but they mainly investigate on the migration of AgNPs largely used for their antimicrobial properties (Duncan, 2011;[START_REF] Maillard | Silver as an antimicrobial: Facts and gaps in knowledge[END_REF].

The migration of nanoclays was, in a lower extent, studied but it is difficult to highlight their migration in nanoforms since their migration were most of the time assessed by the quantification of their constitutive elements, silicon, aluminum and magnesium. The main researches started with conventional polymers, PET bottles [START_REF] Farhoodi | Migration of aluminum and silicon from PET/clay nanocomposite bottles into acidic food simulant[END_REF], PP and PA6 [START_REF] Xia | Release of nanoclay and surfactant from polymer-clay nanocomposites into a food simulant[END_REF] or LDPE (Echegoyen et al., 2016). These studies confirmed the migration of Chapter I. State of the art a low amount of nanoclays. From a purely theoretical point of view and according to an evaluation based on a modelling approach, the nanoparticles completely embedded in the polymer matrix with a non-altered surface, do not have the potential to migrate into food (Störmer et al., 2017). In this case, the biodegradable polymer for which the structure evolved over time due, for example to hydrolysis, raises questions.

The specific case of nanoclays migration from biodegradable polymers was investigated in some studies. Firstly, a biodegradable starch/clay nanocomposite films was tested to pack lettuce and spinach and the results showed that this material can be put in contact with vegetables (Maurizio Avella et al., 2005). [START_REF] Huang | Experimental and mathematical assessment of migration from multilayer food packaging containing a novel clay/polymer nanocomposite[END_REF] determined by ICP-OES, on a biaxially oriented polypropylene/poly(vinyl alcohol)/MMT material, the concentrations of silicon and aluminum, the main constituents of nanoclays (36 wt% and 8.6 wt%, respectively). The results show that the migration level of aluminum was in most of the experiments below the detection limits whatever the temperature and the food simulant. Concerning the silicon, the highest migration values were obtained with vegetable oil at 70°C but they were below the limit fixed by the Regulation. These observations are in line with the conclusions of [START_REF] Iglesias | Wheat gluten nanocomposite films as foodcontact materials: migration tests and impact of a novel food stabilization technology (high pressure)[END_REF], who observed a particularly high migration of aluminum into acetic acid, due in particular to the high solubility of this element in an acidic environment. In most studies, the quantification of chemical elements results in concentration values in the food that differ from the respective proportion in the initial particle. These results suggest that the migration of nanoclays does not take place in a particulate form but rather implies a release of individual elements after solubilisation.

Specific migration

The specific migration defined the amount of a given substance transferred from a material into foods. To be used in food packaging application in EU, these substances must be on the Union list of authorized substances that can be used in the manufacture of the plastic layers of food contact plastic materials and articles covering monomers, starting substances, additives and polymer processing aids (European Commission, 2011). Indeed, as for the overall migration, a maximum permitted amount was defined as the Specific Migration Limit (SML) specified for each substance on the basis of toxicological data ensuring that the food contact material does not induce any risk for human health.

Very few studies have been conducted on the specific migration of plastic additives from nanocomposite material and the impact of the introduction of nanoparticles in materials on the migration remain an issue little addressed in the literature. The studies conducted in this area are mainly focused on conventional polymers such as LLDPE (Nasiri et al. 2016;[START_REF] Dudefoi | Nanoscience and nanotechnologies for biobased materials , packaging and food applications: New opportunities and concerns[END_REF], PP [START_REF] Xia | Release of nanoclay and surfactant from polymer-clay nanocomposites into a food simulant[END_REF][START_REF] Otero-Pazos | Determination of partition coefficients of selected model migrants between polyethylene and polypropylene and nanocomposite polypropylene[END_REF], PET [START_REF] Farhoodi | Migration of aluminum and silicon from PET/clay nanocomposite bottles into acidic food simulant[END_REF], PA (Pereira de Abreu et al., 2010;[START_REF] Xia | Release of nanoclay and surfactant from polymer-clay nanocomposites into a food simulant[END_REF]. All these works demonstrated the ability of nanoclays to reduce the diffusivity of low molecular weight molecules, regardless of the nature of the food simulant used, and agrees that the incorporation of such nanoparticles tends to limit migration in the case of high-barrier polymers. The effect of the nanoclays on migration from low-barrier polymers such as polyolefins is more contrasted. In aqueous simulants, the presence of inorganic nanofillers is not associated with any deleterious effects and induces a significant decrease in the migration values. In fatty and alcoholic simulants (95% ethanol), the influence of the addition of Cloisite 20A is minor but in some cases results in an increase in the level of migration. This observation, which is opposed to the generally accepted idea of a beneficial effect of the addition of NPs, justifies the relevance of this concern in terms of consumer exposure.

To the best of our knowledge, no study are reported on the specific migration of additives from biodegradable nanocomposite materials. Only few studies are dedicated to the specific migration of lactic acid, lactide (constitutive monomer) or lactoyllactic (linear dimer of lactic acid) from PLA materials. Conn et al. were the first to be interested in the migration of lactic acid, lactide and lactoyllactic. Their approach revealed a limited migration. Another study focused on the determination of the specific migration of lactic acid in 3% acetic acid and 50% ethanol from neat and nanocomposite films with 3 and 6 wt% of Cloisite 30B [START_REF] Di Maio | Bionanocomposite polylactic acid/organoclay films: functional properties and measurement of total and lactic acid specific migration[END_REF], showed that the lactic acid migration is higher in 50% ethanol and in presence of nanoclays and demonstrated that the release increases with the nanoclays content while remaining below the limit. More recently, a study was conducted on the specific migration of lactic acid in 50% ethanol in multilayer films to show the impact of the film thickness. The authors confirm that the lactic acid migration is higher in presence of nanoclays and specified that this amount increases with the augmentation of the thickness of the nanocomposite layer, the specific migration value remaining below the limit defined by the Regulation [START_REF] Scarfato | Performance properties, lactic acid specific migration and swelling by simulant of biodegradable poly(lactic acid)/nanoclay multilayer films for food packaging[END_REF].

End-of-life

The impact of nanoparticles introduction on the materials end-of-life can be studied according to their biodegradation and their ecotoxicity on the environment. However, there is also a lack of knowledge concerning the potential effects of engineered nanoparticles on the environment . In fact, as the liberation and the fate of nanoparticles in the environment after use is not fully understood, it is difficult to predict their impact on the environment, bioaccumulation and/or contamination [START_REF] Souza | Nanoparticles in food packaging: Biodegradability and potential migration to food-A review[END_REF].

Biodegradability

The biodegradation of plastics can be assessed according to various simulation tests at the laboratory scale and the degradation might be monitored in different media i.e. soil, compost, sea-water, sludge and sewage placed in a controlled reactor [START_REF] Müller | Biodegradability of polymers: regulations and methods for testing[END_REF]. Most of the time, the biodegradation of a material is quantified by monitoring its mass loss. Respirometric methods can also be applied to follow the amount of the carbon dioxide produced or the oxygen demand originated by microorganisms containing in the medium (Kumar Thakur and Kumari Thakur, 2015). Biodegradability has been extensively studied on a wide variety of polymers and in different media [START_REF] Torres | Poly(lactic acid) degradation in soil or under controlled conditions[END_REF][START_REF] Tsuji | Blends of aliphatic polyesters. III. Biodegradation of solution-cast blends from poly(L-lactide) and poly(e-caprolactone )[END_REF][START_REF] Ho | Degradation of polylactic acid (PLA) plastic in Costa Rican soil and Iowa State University compost rows[END_REF][START_REF] Ghorpade | Laboratory composting of extruded poly(lactic acid) sheets[END_REF].

It is legitimate to investigate the impact of inorganic nanoparticles on the biodegradability of nanocomposite structures since they are strategically introduced into the packaging to increase their stability in contact with aqueous foods. The results of the studies conducted in this way lead to controversial conclusions depending on the polymer. Biodegradability data of 20 publications were analyzed as regard to the polymeric matrix, the 2:1 phyllosilicate type, its percentage in the matrix, the process used to synthetize the material and the biodegradability evaluation method. These data are gathered in Table I.4. The analysis of these publications is presented in Figure I.11, which reports the augmentation or reduction in biodegradation rate according to the 2:1 phyllosilicate content.

For a better readability, the biodegradation rate reached for the neat polymer was set at 1. Then, the biodegradation rate obtained for nanocomposite materials was weighted by the value obtained without nanoclays. A value greater than 1 means that the addition of nanoclays increases the biodegradation rate of the polymer, while a value below 1 indicates a decrease in biodegradability.

The result is positive when the addition of nanoclays increases the biodegradation and negative when a decrease is obtained. The result of this analysis (Figure I.11) shows contrasted results, in some cases the addition of nanoclay leads to an increase of the biodegradation rate while in other case a reduction is observed.

The impact of layered silicate on the biodegradation of PCL, one of the first developed biodegradable petroleum-based materials, has been extensively studied. 25% of the publications studied in this review are dedicated to this polymer with contrasted conclusions.

Its biodegradability, reinforced with 1 wt%, 2 wt% and 4 wt% of unmodified Na + -MMT was determined in compost and in 0.1 mol/L phosphate buffered solution containing 10 mg/L lipase (Wu et al., 2009). The results show that the Na + -MMT hinders the biodegradation of PCL in both cases. The higher the Na + -MMT percentage, the lower the material weight loss. The biodegradation rate in compost decreases from 80 wt% for neat PCL to 55 wt% for PCL-4 wt% Na + -MMT. The same conclusion was reported by Fukushima et al. (2010) 2009) who observed a decrease of the biodegradability with the addition of C-Na + . It is important to note that the impact of the intercalating agent that determines the degree of dispersion of MMT is also variable depending on the nature of the medium. Singh et al. (2010) studied the biodegradability of PCL in different media: enzyme, pure microorganisms (fungi), composted organic manure, Ganges water and alkaline buffer and observed a significant enhancement of the biodegradation rate with the incorporation of organo-modified MMT whatever the environment. The C-30B leads to the highest biodegradation in all media, presumably due to the greater interaction between C-30B and PCL and to the presence of excess hydroxyl ions presents in the organic modifier of C-30B. In composted organic manure, where the biodegradation rate is the highest, the material weight loss after 60 days of composting is 40% and 23% higher for PCL/C-30B and PCL/C18 respectively compared to neat PCL. On the other hand, the biodegradation rate is low for all materials in Ganges water.

In contrast, the impact of the addition of nanoclays on the biodegradability of PLA leads to the same tendency that is an increase of the biodegradability. A significant enhancement of 35% of the biodegradability under composting conditions is observed after incorporation of 4 wt% of organically modified synthetic fluorine mica (Sinha Ray et al., 2003). Similar (2003) who measured the biodegradation rate of neat PHB at 55 wt% and at 39 wt% and 36 wt% for PHB-fluoromica and PHB-MMT, respectively. In the same way, the biodegradation of PHBV nanocomposites filled with 3 wt%, 5 wt% and 10 wt% was investigated in soil suspension by Wang et al. (2005) who observed after 350h a significant decrease in the biodegradation of materials due to the addition of an increasing quantity of nanoclays. In contradiction, Maiti et al. (2007) observed an enhancement of the biodegradation rate in compost of PHB in the presence of organically modified nanoclays.

Other materials were studied but to a lesser extent. The biodegradability in activated soil of a biodegradable aliphatic polyester (APES) filled with various percentages of two kinds of nanoclays, Cloisite 30B and Cloisite 10A [START_REF] Lee | Microstructure, tensile properties, and biodegradability of aliphatic polyester/clay nanocomposites[END_REF] shows a decrease with an increase of the amount of nanoclays. This phenomenon can be explained by the increase of the barrier properties of the material induces by the nanoclays. Moreover, a lower biodegradability was obtained for APES/Cloisite 30B compared to APES/Cloisite 10A due to the higher degree of intercalation of Cloisite 30B in the APES matrix. [START_REF] Swain | Study of thermal, oxygen-barrier, fire-retardant and biodegradable properties of starch bionanocomposites[END_REF] studied the biodegradability in sludge water by weight loss of starch/clay bio-nanocomposite materials prepared with various concentrations of Cloisite 30B. The authors observed a decrease of the biodegradation rate with the increase of the concentration of nanoclays, which could be due to the hydrophobic nature of nanoclays. The same behavior was observed by [START_REF] Issa | Sweet potato starchbased nanocomposites: development, characterization, and biodegradability[END_REF] on -amylase from sweet potato. In soil burial degradation, the weight loss is complete for neat sweet potato starch material after 60 days. In contrast, the weight loss of the nanocomposite material was 78 wt%.

The enzymatic degradation leads to a weight loss of 49 wt% for neat polymer against 23 wt% for the nanocomposite polymer. The biodegradation of poly(butylene adipate-co-terephthalate) (PBAT), grafted with maleic anhydride or not, prepared with natural MMT modified by either octadecylamine (ODA) or dihexylamine (DHA) was examined [START_REF] Chen | Preparation and characterization of nanocomposite of maleated poly(butylene adipate-co-terephthalate) with organoclay[END_REF]. While no impact of MMT addition is clearly demonstrated for pure PBAT, the grafting of maleic anhydride appears to improve the degradability of PBAT. This is due to the anhydride groups of maleic anhydride, which (i) increase the hydrophilicity of the sample and (2) improve the dispersion and exfoliation state of the nanoclays, and therefore it degradability.

The diversity of the results described here makes difficult to identify aggregate trends summarizing the effect of nanoclays on the materials biodegradability. The degradation appears as a multi-factorial process, which is influenced by different parameters such as (Guaras et al.,

2016):

(1) The type of polymer and its chemical structure, especially the presence of functional groups and hydrophilicity hydrophobicity balance

(2) The type of layered silicate and its concentration in the polymer matrix

(3) The polymer/layered silicate compatibility or the dispersion and exfoliation state of the layered silicate in the polymer matrix (4) The environment used for the degradation i.e. soil, compost, marine substrates (sludge, water), and sewage. Each environment contains different microorganisms (in terms of species diversity and population), different physical and chemical parameters (in terms of temperature, moisture, pH, aeration, and nutrients) that influence the rates of microbial activity and affect in turn the rate of degradation [START_REF] Dean | Biodegradability characterization of polymer nanocomposites[END_REF].

The improvement of the biodegradability is explained by several factors. The presence of hydroxyl groups on the edges of the clays can catalyze the hydrolytic degradation of the matrix polymer (Maiti et al. 2007;Singh et al. 2010). Most of the added nanoparticles are hydrophilic compared to polymer matrices, which are more hydrophobic, leading to higher absorption of water. Moreover, in some cases, due to a poor affinity between these two constituents, spaces created by a non-cohesive interface generate a porous structure in which water and enzymes can be easily penetrate and diffuse. Organic compounds that are used for the modification of nanoclays exhibit some reactive groups that can catalyze the enzymatic hydrolysis. Moreover, the addition of nanoclays proved to have an impact on the polyesters degree of crystallinity (XC). In the case of a decrease of the XC, the polyesters become more amorphous and the water can be more easily absorbed and increases the hydrolysis rate [START_REF] Dean | Biodegradability characterization of polymer nanocomposites[END_REF].

The delay of biodegradability can be explained in different ways. First, the nanoclays improve the barrier properties of the material, which reduce their mass transfer properties and consequently the penetration of water [START_REF] Lee | Microstructure, tensile properties, and biodegradability of aliphatic polyester/clay nanocomposites[END_REF]Wu et al., 2009). Moreover, the incorporation of impermeable nanofillers prevent the penetration and the diffusion of enzymes and microorganisms involved in the polyester hydrolysis. Another reason is the high surface volume and the nucleating effect of nanoparticles, which increases, in some cases, the degree of crystallinity of polymers. Since crystalline parts are less susceptible to hydrolysis, the rate of nanocomposites becomes lower compared to neat polymers.

Ecotoxicity

Another important concern when discussing the incorporation of nanoclays into biodegradable polymers is the fate of these nanoparticles and the effects on its deposition into the environment [START_REF] Klaine | Paradigms to assess the environmental impact of manufactured nanomaterials[END_REF]. Ecotoxicity tests are necessary to determine the risks on the environment of the introduction of these nanomaterials. In fact, until now most of the studies focused on the impact of nanomaterials on the human health [START_REF] Oberdorster | Correlation between particle size, in vivo particle persistence[END_REF][START_REF] Brown | Increased inflammation and intracellular calcium caused by ultrafine carbon black is independent of transition metals or other soluble components[END_REF][START_REF] Nemmar | Passage of inhaled particles into the blood circulation in humans[END_REF] and to the best of our knowledge, only few studies are dedicated to the impact of the nanoclays on plants and soil. [START_REF] Tullio | Physical and chemical characterization of a natural and modified nanoclays and their ecotoxicity on a freshwater algae species (Chlamydomonas reinhardtii)[END_REF] The diversity of the results show that toxicity of a particular type of nanoclay is likely dependent on its physicochemical properties (e.g., surface charge, shape, state of dispersion, particle size and size distribution) and shows once again the difficulty of drawing general trends. des matériaux puis une dernière partie décrit les méthodes utilisées pour étudier la fin de vie de ces matériaux.

Conclusion

1 Materials

Polymeric matrices

In view of the diversity of their nature and transfer properties, two distinct biodegradable and bio-sourced (totally or partially) polymers i.e. poly(butylene succinate-co-butylene adipate)

(PBSA, PBE 001, density 1.24 g.cm -3 ) (Figure II.1A) and poly(hydroxybutyrate-cohydroxyvalerate) (PHBV, PHI 002, density 1.23 g.cm -3 ) (Figure II.1B) were selected. This choice allows to cover different behavior in contact with food simulant, the PHBV being considered as a high-barrier polymer (Crétois et al., 2014) compared to PBSA (Phua et al., 2013). The polymers in form of pellets were supplied by NaturePlast and were stored away from air and light as well as from source of heat. The main properties of these two polymers

given by the supplier are presented in Table II.1.

Nanoclay reinforcement

Cloisite 30B (C-30B) was chosen as filler because of its favorable interaction with PBSA (Sinha Ray et al., 2008) and PHBV (Carli, Crespo, & Mauler, 2011 ;Iggui et al., 2015). It is an organo-modified montmorillonite clays by a methyl bis-2-hydroxyethyl ammonium quaternary salt with a cation exchange capacity (CEC) of 90 meq/100 g. The C-30B powder was supplied by BYK additives & instruments, Germany.

Additives

An array of solid and liquid additives were chosen. These molecules must represent all general categories of chemical compounds with a variety of chemical and physical properties as described in the FDA and EFSA Regulations (Food and Drug Administration, 2006;

European Food Safety Authority, 2011). These additives include volatile polar organic substances, volatile non-polar organic substances, non-volatile polar organic substances and non-volatile non-polar organic substances. Three high molar mass additives and eight low molar mass additives, all procured to Sigma-Aldrich, are presented in Table II.2. oxygen free environment and a CO2 atmosphere.

These microbial food simulants require preparation before being used. First, a pre-culture is necessary for the use of the microorganisms, the powder was rehydrated in an enriched solution (16h at 5°C for P. Candidum and 2h at 37°C for L. Lactobacillus). Then, the solution was spread on a culture medium (Sabouraud Chloramphenicol agar plate during 5 days at 25°C

for P. Candidum and MRS agar plate during 3 days at 37°C under anaerobiose for L.

Lactobacillus). At the end of this time, a colony was taken and inoculated on PDA agar plates (for P. Candidum) or MRS agar plates (for L. Lactobacillus). Three days at 25°C (for P.

Candidum) or 37°C (for L. Lactobacillus) were necessary for the growth of the microorganism before using the agar plates.

Production of bio-nanocomposite materials

PBSA and PHBV films containing 5 wt% of C-30B were synthetized by melt extrusion process using a co-rotating twin-TM , Germany) with a L/D ratio of 40 and a screw diameter of 16 mm. A calendar die was assembled to the extruder to produce films. Polymer pellets were dried at 60°C during 15 hours before using to remove residual moisture. Contrariwise, nanoclays were used as-received, a better intercalation of the nanoclay platelets in PLA matrix was observed when C-30B is hydrated (Tenn et al., 2013).

PBSA films were produced within three steps following the same protocol used by Charlon et al. (Charlon et al. 2015). This procedure was first described by Soulestin et al. [START_REF] Soulestin | Mechanical and Optical Properties of Polyamide[END_REF] who got a good dispersion and exfoliation of montmorillonites within PA6 matrix.

First, a masterbatch of 15 wt% nanoclays was prepared. The polymer pellets were introduced in the zone 1 of the extruder (Figure II.2) at a flow rate of 0.85 kg.h -1 using a volumetric feeder (Brabender single screw DSR28). Nanoclays were introduced in the zone 2 of the extruder at a flow rate of 0.15 kg.h -1 using a gravimetric feeder (Brabender loss-in-weight, twin-screw feeder DDW-MD1-MT2). A temperature profile from 120°C to 160°C was registered from the feed to the die. At the extrusion die, the emerging pellets of polymer were cooled down in a water bath, pelletized and oven dried at 60°C during 15 hours. Then, the masterbatch was diluted with neat polymer to obtain 5 wt% nanoclay pellets. A flow rate of 1.0 kg.h -1 and a temperature profile from 120°C to 180°C were used. The resulting pellets were also cooled down, pelletized and oven dried during 15 hours. Finally, films were produced from these pellets using a flow rate of 1.0 kg.h -1 and a temperature profile from 100°C to 135°C. All steps were processed at a screw speed of 200 rpm. Films were oven dried during 15 hours before storage.

PHBV films were produced within two steps following the same protocol used by Iggui et al. (2015). Fisrt, a masterbatch of 15 wt% nanoclays was prepared in the same way as the PBSA and a screw speed of 100 rpm was used. The temperature profile settings ranged from 140°C to 180°C. Finally, the masterbatch was diluted with neat PHBV to obtain films containing 5 wt% nanoclays. They were produced using a screw speed of 210 rpm, a flow rate of 1.0 kg.h -1 and the same temperature profile as previously. Films were oven dried during 15 hours before storage.

-screw profile used are given in Figure II.2 and Table II.4.

The control samples of neat PBSA and PHBV were prepared following the same protocol used for nanocomposites in order to respect the same thermal processes.

Transmission Electron Microscopy (TEM)

The experiments were performed in the Electronic and Analytical Microscopy Platform (MEA) of the University of Montpellier. A transmission electron microscope JEOL 1200EX2 with a 100 kV acceleration voltage equipped with a EMSIS Olympus camera was used to analyze film samples and observe level of dispersion and exfoliation of nanoclays in the polymer matrix. The film samples were included in the resin and cut with a Leica UC 7 ultramicrotome before observations.

Thermal characterization

Differential Scanning Calorimetry (DSC)

A differential scanning calorimeter (DSC series Q200, TA Instruments) was used to study the crystallinity of the materials. All measurements were performed under a nitrogen atmosphere. Specimens of 5-8 mg weighed using a microbalance (Sartorius micro PRO 11)

were placed in aluminum hermetic sample pans. The thermal protocol was defined according to the polymer. The PBSA samples were cooled from 40°C to -35°C, heated to a temperature of 150°C and maintained for 5 min at this temperature. The samples were then recooled to cooled from 40°C to -30°C, heated to a temperature of 200°C and maintained for 5 min at this temperature. The samples were then recooled to -30°C, reheated to 200°C and finally cooled to 40°C. Temperatures were adjusted at a rate of 10°C.min -1 . The apparent melting enthalpy TA Instruments.

The degree of crystallinity (XC) of the PBSA and the PHBV matrix and corresponding nanocomposites was determined by the equation:

(II.1)

-1 ) is the melting enthalpy of the polymer matrix, w is the polymer weight fraction (PBSA or PHBV) in the sample and is the theoretical melting enthalpy of the PBSA or PHBV polymer assumed to be 100% crystalline, = 146.0 J.g -1 (Barham et al., 1984) for PHBV and = 116.9 J.g -1 (Nikolic and Djonlagic, 2001) for PBSA.

Thermogravimetric Analysis (TGA)

The percentage of nanoclays in PBSA and PHBV was determined using Mettler TGA2 apparatus (Schwerzebbach, Switzerland) equipped with a XP5U balance (precision of 0.0001 mg). Specimens of around 10 mg weighed using a microbalance (Sartorius micro PRO 11) were heated from 25°C to 800°C at a rate of 10°C.min -1 under air flow of 50 mL.min -1 . The percentage of nanoclay present in the film was calculated using the remaining residue (R) obtained at the end of the thermal cycle, for neat polymer, nanocomposite polymer and nanoclays according to the following equation:

(II.2)

Gas mass transfer characterization

Oxygen permeability

The O2 permeability of films was determined in triplicate at 23°C and 50 % of relative humidity using an O2 permeation cell equipped with a PSt6-type sensor spot to detect the concentration of O2, manufactured by PreSens Precision Sensing GmbH (Regensburg, Germany) (Figure II.3). Films were placed in the middle of the cell and the lower and upper chambers were purge with nitrogen. Once the concentration of O2 in the cell is zero, a constant flow of 50 mL.min -1 of O2 was introduced in the lower chamber and the percentage of O2 crossing the film was directly measured in the upper chamber. O2 permeability (mol.m/(m 2 .s.Pa)) was calculated from the Eq. (II.3).

(II.3)

Where: J (mol.s -1 ) is the flux of gas that pass through the film, e is the film thickness (m), A, the permeation area (m²) , the gas (O2 in this case) pressure differential between the upper and lower chambers of the cell (Pa).

Carbon dioxide permeability

The CO2 permeability of films was determined in triplicate at 23°C and 42 % of relative humidity through an isostatic and dynamic method using gas phase chromatography. The film samples were placed inside sealed stainless steel permeability cells. The lower and upper chambers were each spread by a 5 mL.min -1 flux of permeant gas (CO2) and vector gas (helium), respectively. The flow rates were controlled and monitored by digital flowmeters (Bronkhorst, Germany). The permeability cells were coupled to a gas chromatograph with thermal conductivity detector (GC-TCD, Agilent 7890A, USA), equipped with an automatic multiposition injection valve (Vici, Valco Instruments). This enabled to sequentially analyze online the evolution of CO2 gas concentration in the upper chamber (crossing the film from the lower chamber). The GC-TCD was equipped with two capillary columns -PoraPlot U (25 m × 0.530 mm id, Agilent) and HP Molesieve (30 m × 0.535 mm id, Agilent) -able to separate CO2, O2 and N2. Data were collected and processed using the ChemStation OpenLab Software (Agilent) and SRA Prochem interface (SRA Instruments). The CO2 permeability, PCO2

(mol.m/(m 2 .s.Pa)), of the material was determined following the Eq. II.3.

Water vapor permeability

The water vapor permeability of films was gravimetrically determined using a modified ASTM procedure, at 23°C and a relative humidity gradient of 0 -100 % (ASTM E96 Standard test methods for water vapor transmission of materials). The free film surface was 9.08 cm² per sample. Film samples were hermetically sealed in glass permeation cells containing distilled water, which were placed in a desiccator containing silica gel to have 0 % RH.

(II.4)

Where: WVTR is the water vapor transmission rate (g.m -2 .s -1 ), e, the film thickness (m), , the water vapor pressure differential between P1 et P2. P1 is the water vapor pressure at 23°C

(P = 2809 Pa) and P2 is assumed to be zero. w is the slope of the weight loss versus time (g.s -1 ),

A, the permeation area (m²) and , the water molar mass (g.mol -1 ).

Sorption of food simulant

The food simulant uptake by the films was determined by gravimetric measurements using a precision balance (10 -4 g) before and after subjecting films to contact with food simulants at 40°C for 10 days under magnetic agitation. At the end of this procedure, excess food simulant was removed with tissue paper before re-measuring. The percentage of solvent uptake was obtained by the following equation:

(II.5)

Where: mi and mt are weights of samples before and after sorption, respectively.

Evaluation of the properties of transfer and inertia of materials

Overall migration

Overall migration (OM) level was gravimetrically determined after the total immersion of samples in the selected food simulants based on the standardized testing conditions set out for long term storage (40°C for 10 days) in Regulation (EC) No 10/2011. The samples were cut in disks of 2.5 cm diameter and kept in a vacuum oven at 60°C for 2 days. The polymer disks with the total area of 60 cm 2 were weighed using a microbalance (0.1mg resolution) prior to the migration test and then placed in a 100 mL migration cell (DURAN® screw thread tubes) with rod and glass spacers and PTFE-sealed caps. Before filling, the migration cells were blown out with nitrogen to prevent any dust contamination. The bottles were then filled with the respective food simulants, sealed and stored in a 40°C oven. Each sample/simulant combination was prepared in triplicate. After 10 days of contact, the bottles were ultrasonicated for 2 min to detach any potential particles from the sample surface. The samples were then removed and placed in a vacuum oven at 60°C for 2 days. After evaporation of the simulant, the specimens were weighed again and overall migration was calculated in milligrams per square decimeter of the surface area of sample.

Specific migration

Spiking of neat and nanocomposite films

The procedure used for the spiking of films depends on additive molar mass. High molar mass additives were introduced into the films during the extrusion process because of their excellent thermal stability (Mauricio Iglesias, 2009). Low molar mass additives were introduced into the films through contamination at 40°C for 7 days under rotary agitation. The theoretical additive amount was 500 mg.kg -1 for all molar mass additives except for toluene, which was introduced at 1000 mg.kg -1 .

Determination of additive concentration

Gas chromatography with flame ionization detector (GC-FID)

me ionization detector was used to determine the concentration of low molar mass additives (< 400 g.mol -1 ) in food simulants. The components were separated using a HP-5 (5%phenyl)methylpolysiloxane capillary column of 32 mm ID and 30 m length. The thermal protocol used for the oven was as follows: an initial temperature of 40°C maintained for 5 min, heated at a rate of 6°C.min -1 to 270°C, maintained for 15 min. The injector temperature was 250°C. An external calibration was made in each food simulant in the range of 1.25 -40 mg.L -1 .

High performance liquid chromatography (HPLC)

An Alliance HPLC system (Waters) equipped with an Alltima C18 column (250 mm × 2.1 mm, 5-determine the concentration of high molar mass additives in food simulants. Compounds were separated with 55% acetonitrile at a flow rate of 0.3 mL.min -1 at 50°C. Absorbance was recorded at 230 nm to quantify the additive concentration. An external calibration was made in each food simulant with concentrations in the range of 1.25 to 20 mg.L -1 .

According to EU Regulation

The migration of additives from film samples to food simulant was studied by desorption tests. For that, the contaminated films were put in contact with food simulants in a surface/volume ratio of 6 dm 2 .L -1 . The bottles were stored under magnetic agitation at 40°C for 10 days, except for PHBV films contaminated by high molar mass additives, for which storage was increased to 30 days. Over specific periods of time, an aliquot of each food simulant was collected and analyzed by chromatography (GC-FID for low molar mass additives or HPLC for high molar mass additives). The samples of isooctane were evaporated under nitrogen and redissolved in 100% ethanol before HPLC analysis.

On UV aged materials

The neat and nanocomposite films were then subjected to accelerated aging in a Q-SUN Xe-3 xenon arc chamber (Q-LAB, France) equipped with Window Q filter. The conditions were as follows: 35°C, 50% RH and an irradiance of 1.44 W.m -2 at 420 nm for 30 days.

In both cases, the relative migration, which represents the percentage of an additive that migrates from polymer to food simulant, can be calculated. For that, the mass of additive recovered in the food simulant after 10 days of contact was divided by the initial mass of additive in the film sample measured after two consecutive extractions in 20 mL of n-hexane.

Estimation of the Dapp

The Dapp of additives from the films into food simulants was calculated from experimental -dimensional infinite plane sheets, given that diffusion via the borders of the films is negligible. Applying he following one dimensional diffusion equation:

(II.6)
Where: C is the additive concentration, t, the contact time, x, the position in the film and , the apparent diffusion coefficient.

The Eq. (II.6) was solved using the following initial (Eq. II.7) and boundary (Eq. II.8) conditions:

(II.7)

(II.8)
is the additive concentration in food simulant at time t and L, the film half thickness.

This diffusion model can be used provided that the following assumptions are verified (Helmroth et al., 2002): (i) additive contamination of the film is homogeneous, (ii) there is no noticeable swelling caused by interaction between the food simulant and the polymer, (iii) there is no concentration gradient in the food simulant, and (iv) the film thickness is homogeneous.

In the case of an infinite plane sheet suspended in a stirred solution of limited volume, the Eq.

(II.6) can be solved analytically as described by Crank (Crank, 1975):

(II.9)

With:

(II.10)

Where: Mt is the total amount of additives in food simulant at time t and is the total amount of additives in food simulant at the steady state, is the polymer volume and the food simulant volume, , the positive roots of the equation and , the partition coefficient of the additive in the polymer/food simulant system.

The numerical simulations were carried out using Matlab software and its lsqnonlin function to estimate the Dapp. To evaluate the correlation between experimental and estimated data, the percentage of the root mean-square error (% RMSE) was calculated using the following equation:

(II.11)

Where: is the initial mass of additive in the polymer film and is the mass of additive in the food simulant at time t.

Migration tests using microbial solid food simulant

The films were contaminated by the array of low molar mass additives as indicated in Section 4.2.1. After a quick rinsing with ethanol to eliminate additives on the surface, contaminated materials were put in contact with agar plates at 25°C under hermetic environment (for P. Candidum) or 37°C under anaerobiose (for L. Bulgaricus) for 10 days.

Over specific periods of time:

-Films were recovered, rinsed with distilled water and ethanol and dried with absorbent -Agar plate was recovered and extracted with 30 mL of dichloromethane at 23°C during 24h under magnetic agitation in order to dose the additives that migrated from the films to the solid food simulant. After filtration, the solution was analyzed by GC-FID. simulants.

The study system possess three compartments: the film sample, the agar plate and the headspace (Figure II.5). The additives present in the film can stay in the film, migrate into the agar plate or in the headspace. The mass of additives remaining in the film sample was followed over time as well as the additives that migrated from the film to the agar plate. However, the migration of the additives to the headspace was not determined. The relative migration is calculated as described in Section 4.2.4.

17556: 2012 as methodological standard and NF EN 17033: 2018 as specification standard.

The method measures the conversion of organic carbon in CO2 over time, this later being proportional to the percentage of biodegraded substrate. Microorganisms assimilate polymer chains and ultimately convert them aerobically into biomass, water and CO2.

The soil used was a vegetal soil displaying the following characteristics: pH 6.8 (H2O), 2.3 wt% of organic matter, 16.85 wt% of clay, 26.85 wt% of lime and 56.3 wt% of sand. Barium chloride-dihydrat, sodium hydroxide, hydrochloric acid (Grosseron, France), thymolphtalein (Alfa Aesar, Germany), and microcrystalline cellulose (Merck, Germany) were used for biodegradation tests.

After manually removal of large inert objects, the soil was dried and sieved discarding 2 mm to obtain homogeneous medium.

The materials in form of pellets were cooled down in liquid nitrogen before being grinded into a powder of 500 nm using an Ultra Centrifugal Mill Retsch ZM-200.

The respirometric equipment consisted of a hermetic 1L glass reactor containing three flasks as previously described (Chevillard et al., 2012;Lammi et al., 2019). One of the flasks contained 25 g of dried soil mixed with 50 mg of equivalent carbon of the grounded samples.

The water content was adjusted to reach 80% of the soil water retention capacity. The second flask contained distilled water to maintain a constant relative humidity of 100%. The last flask contained a solution of 0.2 N hydroxide sodium (NaOH), which allows capturing the CO2 released by the soil microorganisms when degrading the material. The reactors were incubated in the dark at 28°C ± 2°C. Experiments included blank sample, which contained soil without material and control sample, which contained soil mixed with cellulose as positive reference material. According to ISO DIS 17556 methodological standard, the two following criteria should have been fulfilled to consider the test as valid: i) at least 60% biodegradation reached in less than 2 years for the positive reference and ii) the amount of carbon dioxide evolving from the three blank samples should be within 20 % of the mean at the plateau phase or at the end of the test. According to NF EN 17033: 2018 specification standard, the material is considered as biodegradable if its degree of biodegradation is at least equal to 90% in absolute, or relative to that of the positive reference, after less than 2 years of incubation. All samples were run in triplicate.

To evaluate the biodegradation, titrimetric assays of the NaOH solutions were performed over time to determine the CO2 released by microorganisms. For that, 5 mL of a solution of barium chloride (20% w/v in distilled water) was added in the NaOH solutions before titration to ensure that all the Na2CO3 formed during incubation was precipitated. The excess of NaOH was then titrated back with 0.1N hydrochloric acid using thymolphtalein (0.1% prepared in 95% ethanol) as color indicator.

The percentage of biodegradation (%B) is calculated from the ratio of the cumulative amount of CO2 produced by the sample related to the maximal theoretical amount of CO2 that could be released (Eq. II.13).

(II.13) With (II.14)

Where: C, is the amount of carbon (mg) of the sample introduced in the soil, and , the molar mass of carbon dioxide (44 g.mol -1 ) and carbon (12 g.mol -1 ), respectively.

After each dosage, a new NaOH solution (0.2 N) was placed in the hermetic glass reactor.

The flasks containing soil were weighted and the water content was adjusted to keep the soil at 80% of its total water-holding capacity.

Finally, the experimental degradation data were modelled using Hill (for cellulose) and Boltzmann (for the tested materials) equations (Deroiné et al., 2015).

Disintegration

Mature green compost was collected in the waste management center of Aspiran (France) and sieved through 5 mm meshes. Solid synthetic waste was prepared by mixing 10% of mature green compost, 40% sawdust, 30% rabbit food, 10% corn starch, 5% saccharose, 4% corn oil and 1% urea. The water content was adjusted to 55% of the total weight.

The method was already described by (Balaguer et al., 2016). 1 kg of the solid synthetic waste was introduced in polypropylene reactors, which were perforated to facilitate the gas transfer. 100 g of polymer, 90% (w/w) in powder form with a particle size of 500 µm and 10%

(w/w) in the form of 25 mm × 25 mm films were mixed with the solid synthetic waste. The reactors were closed and incubated in the dark at 58 ± 2°C during 90 days. Samples were run in triplicate. Over specific periods of time, water was added to maintain adequate moisture conditions and solid synthetic waste was carefully mixed to maintain aerobic conditions. After 90 days, the compost resulting was dried in open air and sieved through 5 mm and 2 mm sieves.

Residual film samples were recovered, cleaned to eliminate stuck compost and dried at 40°C until constant weight was reached. Then, the degree of disintegration, D was calculated (Eq. II.15).

(II.15)

Where: is the weight of dry material in pieces of 25 mm × 25 mm introduced initially and is the weight of the clean and dry fraction of films recovered after sieving.

Ecotoxicity

The ecotoxicity tests were carried at CEA Cadarache specializing in these questions of ecotoxicity.

The soil used is a low-carbon calcareous soil (C/N = 10.45). A mixture of 25% of solid synthetic waste (described in Section 6.2.1) and 75% of soil was used for the plant cultivation.

Winter forage rapeseed (Mosa type) was selected as crop model as this plant is particularly demanding in nitrogen, phosphorus and sulfur during the vegetative stage.

The resulting compost of the disintegration step was then used to amend a low-carbon calcareous soil (25% compost) for the culture of a winter forage rapeseed. Plant were grown in climate-controlled growth chamber (250 µmol photon.m -2 .s -1 intensity), temperature (19-22°C thermoperiod), air moisture (70-80%), with a 8h:16h day:night period. Water was dispensed once a day to set the soil moisture near 20% humidity. The biocompatibility of the residual nanoparticles was assessed using the response of the plant, soil and microbiota as proxy. On the plant side, the tests measured the immediate toxicity of the compost (germination inhibition) or the latent toxicity (growth inhibition), the shoot biomass and the number of leaves by plant. On the soil side, the tests monitored the enzyme activity and the microbiota composition.

Introduction

The growing global production and consumption of plastic materials engenders serious environmental concerns [START_REF] Kirwan | Plastics in food packaging[END_REF], primarily the need to implement ecoefficient end-of-life treatments. For many years, alternative materials based on bio-sourced and biodegradable polymers have been considered as innovative and promising materials for future food packaging applications (Tharanathan, 2003;Sorrentino et al., 2007). However, the substitution of plastic in all its various uses in food packaging remains difficult given the broad spectrum of functionality offered by petroleum-based polymers. In addition to their high water sensitivity, the properties of biodegradable materials make them uncompetitive in comparison with those of conventional plastic polymers, particularly in terms of barrier properties. The addition of reinforcing fillers such as clay nanoparticles, giving rise to the formation of a bionanocomposite material, is widely considered as the solution to this problem [START_REF] Perumal | Effects of multiscale rice straw (Oryza sativa) as reinforcing filler in montmorillonite-polyvinyl alcohol biocomposite packaging film for enhancing the storability of postharvest mango fruit (Mangifera indica L[END_REF].

The introduction of nanotechnologies to the field of food packaging raises new issues related to consumer safety. Research has focused on the exposure of consumers to nanoparticles through contamination of the food in contact with nanocomposite materials. There is a consensus in the research on the migration of nanoclay that its limited diffusion under a nanoform in a polymeric matrix prevents its migration into food . Like any material in contact with food, nano-and bio-nanocomposite materials are subject to the European framework regulation UE 1935/2004. In the absence of specific regulations covering their use in contact with food, the suitability for food contact applications of nanocomposite packaging are assessed according to the recommendations established for plastic materials specified in the regulation EU/10/2011.

Given the impact of nanoclays on the transfer properties of materials [START_REF] Muñoz-Shugulí | Cetylpyridinium bromide-modified montmorillonite as filler in low density polyethylene nanocomposite films[END_REF], it is logical to investigate their impact on the migration of property-enhancing plastics additives such as plasticizers, UV-stabilizers, anti-oxidants, and anti-static agents [START_REF] Figge | Migration of components from plastics-packaging materials into packed goods -test methods and diffusion models[END_REF]. The impact of nanoparticles on the potential migration of low molar mass additives has received little attention, the only studies having focused on plastic-based nanocomposite materials. These studies, performed on nanocomposite materials, specifically:

PETs [START_REF] Farhoodi | Migration kinetics of ethylene glycol monomer from PET bottles into acidic food simulant: effects of nanoparticle presence and matrix morphology[END_REF] From a safety standpoint, the barrier effect provided by the nanostructure of packaging film against additives could reduce the exposure of consumers to toxic compounds and their potential adverse health effects, depending on the individual barrier properties of polymers.

Although nanoclays have a positive effect on high barrier polymers such as PET, the decrease in the diffusion coefficient has no effect on the migration value for low-barrier polymers, for which the migration values are more related to the partition coefficient [START_REF] Farhoodi | Migration kinetics of ethylene glycol monomer from PET bottles into acidic food simulant: effects of nanoparticle presence and matrix morphology[END_REF].

The reasons why the presence of nanoparticles decreases the mobility of the diffusing molecules are multiple. While the tortuosity effect is generally cited [START_REF] Paul | Polymer nanotechnology: nanocomposites[END_REF]Duncan, 2011), the mechanisms of diffusion in nanocomposite matrices imply additional essential factors. In the case of polymer-clay nanocomposites, the interactions of organic molecules with a decrease in their Dapp by sorption or adsorption mechanisms (Nasiri et al. 2016). In addition to their high cation exchange capacity, clays can exchange other chemical bonds, such as van der Walls interactions, or hydrogen bonding, with migrants, delaying their transport in the nanocomposite structure [START_REF] Aguzzi | Use of clays as drug delivery systems: possibilities and limitations[END_REF]. It is also well established that the presence of clay nanoparticles structurally modifies polymer networks, having nucleating effects, and their ability to increase the crystallization rate of semi-crystalline thermoplastic polymers [START_REF] Ke | Crystallization, properties, and crystal and nanoscale morphology of PET-clay nanocomposites[END_REF][START_REF] Yuan | Nonisothermal crystallization behavior of melt-intercalated polyethylene-clay nanocomposites[END_REF]. At a finer scale, crystallinity gradients in the vicinity of nanoparticles can have a modulating effect on the transfer properties of plastic-based nanocomposites [START_REF] Wurm | Retarded crystallization in polyamide/layered silicates nanocomposites caused by an immobilized interphase[END_REF]. This combination of effects emphasized nanocomposite bioplastics (Charlon et al. 2015b), but to our knowledge, no research has measured and characterized migration from bio-nanocomposite packaging. Such materials, by definition unstable, may behave differently than synthetic polymers in contact with food, and their inertial properties remain undemonstrated. This study aims to characterize the food contact suitability of bio-nanocomposite packaging films. In view of their emergence on the market and their environmental benefits [START_REF] Rajan | Polyhydroxybutyrate (PHB): a standout biopolymer for environmental sustainability[END_REF][START_REF] Hongsriphan | Properties of agricultural films prepared from biodegradable poly(butylene succinate) adding natural sorbent and fertilizer[END_REF], two distinct polymers: poly(butylene succinate-co-butylene adipate) (PBSA) and poly(hydroxybutyrate-cohydroxyvalerate) (PHBV) were selected on the basis of their differing transfer properties, in order to cover different behaviors in contact with food simulants, the PHBV being a high-barrier polymer (Crétois et al., 2014) contrary to PBSA (Phua et al., 2013) Organo-modified montmorillonite was added to these materials, which were spiked with a panel of target migrating substances and submitted to migration tests following the protocol for testing conventional plastic packaging. The approach aims to measure and identify the influence of nanoclays on the diffusion properties of these emerging packaging materials.

Materials and Methods

Materials

Poly(butylene succinate-co-butylene adipate) (PBSA, PBE 001, density 1.24 g.cm -3 ) and

Poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV, PHI 002, density 1.23 g.cm -3 ) pellets were commercially procured from Natureplast, France.

Cloisite 30B (C-30B), an organo-modified montmorillonite clay containing a methyl bis-2-hydroxyethyl ammonium quaternary salt with a cation exchange capacity (CEC) of 90 meq/100 g, were supplied by BYK additives & instruments, Germany. C-30B was chosen

Chapter III. Impact of nanoclays on the properties of transfer and inertia of materials because of its favorable interaction with PBSA (Sinha Ray et al., 2008) and PHBV (Carli et al., 2011;Iggui et al., 2015).

An array of solid and liquid additives was chosen. These molecules represent a range of categories of chemical compounds with a variety of chemical and physical properties as described in FDA and EFSA regulations (Food and Drug Administration, 2006;European Food Safety Authority, 2011). The additives chosen include: volatile polar organic substances, volatile non-polar organic substances, non-volatile polar organic substances, and non-volatile non-polar organic substances. Three high molar mass additives and eight low molar mass additives were procured from Sigma-Aldrich (Table III.1).

Preparation of bio-nanocomposite films

PBSA and PHBV films with 5 wt% C-30B were synthetized by a melt extrusion process using an EuroLab 16XL co-rotating twin-TM , Germany) with a L/D ratio of 40 and a screw diameter of 16 mm. The extruder produced films using a calendar die. Polymer pellets were dried at 60°C for 15 hours before use to remove residual moisture.

Chapter III. Impact of nanoclays on the properties of transfer and inertia of materials Nanoclays were used without drying, because hydrated nanoclay platelets intercalate better with the polymer matrix (Tenn et al., 2013).

PBSA films were produced in three stages (Charlon et al. 2015a). First, a masterbatch of 15 wt% C-30B was prepared. The polymer pellets and nanoclays were introduced in the extruder at a flow rate of 0.85 kg.h -1 and 0.15 kg.h -1 , respectively. The feed to die temperature profile was 120°C to 160°C. At the extrusion die, the emerging polymers were cooled in a water bath and pelletized. The masterbatch was then diluted with neat polymer to obtain 5 wt% nanoclay pellets. Finally, films of 180 ± 10 µm for PBSA and 220 ± 25 µm for PBSA nanocomposite (PBSA NCP) were produced from these pellets using a flow rate of 1.0 kg.h -1 and a 100°C to 135°C temperature profile. All steps were processed at a screw speed of 200 rpm.

PHBV films were produced in two stages (Iggui et al., 2015). A masterbatch of 15 wt% C-30B was prepared in the same way as for PBSA, however with a temperature profile of 140°C to 180°C. The masterbatch was diluted with neat polymer to obtain PHBV films containing 5 wt% nanoclay. The films of 145 ± 20 µm for PHBV and 140 ± 10 µm for PHBV NCP were produced using a screw speed of 210 rpm and a flow rate of 1.0 kg.h -1 . PBSA and PHBV pellets or films were oven dried at 60°C for 15 hours after each stage and before storage.

The control samples of neat PBSA and PHBV were prepared following the same protocol used for nanocomposites in order to respect the same thermal processes.

Spiking of neat and nanocomposite films

The procedure used for the spiking of films depends on additive molar mass. High molar mass additives were introduced to films during the extrusion process because of their excellent thermal stability (Mauricio Iglesias, 2009). Low molar mass additives were introduced to films through contamination at 40°C for 7 days under rotary agitation. The theoretical additive amount was 500 ppm for all low molar mass additives apart from toluene at 1000 ppm.

Food simulants

Two different food simulants were chosen to evaluate the Dapp of the selected additives:

95% ethanol, and isooctane, which both simulate fatty foods behaviors according to the European Commission for food contact materials (EU 10/2011) (European Commission, 2011).

The experiments were also performed using 3% acetic acid to simulate acidic food behavior, but the migration of high molar mass additives in this solvent was less than the limit of detection.

Desorption tests

Desorption tests were carried out by putting contaminated films in contact with food simulants in a surface/volume ratio of 6 dm 2 .L -1 . The bottles were stored under magnetic agitation at 40°C for 10 days, except for PHBV films contaminated by high molar mass additives, for which storage was increased to 30 days. Over specific periods of time, an aliquot of each food simulant was collected and analyzed by chromatography (GC-FID for low molar mass additives or HPLC for high molar mass additives). The samples of isooctane were evaporated under nitrogen and re-dissolved in 100% ethanol before HPLC analysis.

Small angle X-ray diffraction (SAXS)

exfoliation of the nanoclays in the polymer matrix. A high brightness low power X-ray tube, coupled with aspheric multilayer optic (GeniX 3D from Xenocs) delivered an ultralow divergent beam (0.5 mrad; flux: 20 MPhotons.s 1 transmission configuration was used and the scattered intensity determined by a 2D pixel the case of nanoclay powder, while for films, the beam passed through five stacked film thicknesses. The obtained intensities were corrected by transmission and the empty cell contribution was subtracted. The interlayer spacing relative to nanoclay platelets was determined at a diffraction angle in the range of 0.5° to 10°.

Transmission electron microscopy (TEM)

A JEOL 1200EX2 transmission electron microscope with 100 kV acceleration voltage equipped with an EMSIS Olympus camera was used to analyze film samples and observe nanoclay dispersion and exfoliation in the polymer matrix. For that, the film samples were included in a resin and cut with a Leica UC 7 ultramicrotome.

Differential scanning calorimetry (DSC)

A differential scanning calorimeter (DSC series Q200, TA Instruments) was used to study the crystallinity of the films. Experiments were carried out on neat and nanocomposite films, before and after 10 days of contact with 95% ethanol or isooctane. All measurements were performed under a nitrogen atmosphere. Specimens of 5-8 mg, weighed using a microbalance (Sartorius micro PRO 11), were placed in aluminum sample pans. The thermal protocol was defined according to the polymer. The PBSA samples were: heated from ambient temperature to 40°C, cooled to -35°C, heated to 150°C, maintained at 150°C for 5 min, cooled to -35°C, heated to 150°C, and finally cooled to 40°C. In the case of PHBV, samples were: heated from ambient temperature to 40°C, cooled to -30°C, heated to 200°C, maintained at 200°C for 5 min, cooled to -30°C, reheated to 200°C, and finally cooled to 40°C. Temperatures were adjusted at a rate of 10°C.min -1 . The apparent the DSC curves using the Universal Analysis 2000 software by TA Instruments. The crystallinity degree (XC) of neat and nanocomposite polymers was determined by the equation:

(III.1)
in the sample and is the theoretical melting enthalpy of the polymer assumed to be 100% crystalline, = 146.0 J.g -1 for PHBV (Barham et al., 1984) and = 116.9 J.g -1 for PBSA (Nikolic and Djonlagic, 2001).

Sorption of food simulants

The food simulant uptake by the films was determined by gravimetric measurements using a precision balance (10 -4 g) before and after subjecting films to contact with food simulants at 40°C for 10 days under magnetic agitation. At the end of this procedure, excess food simulant was removed with tissue paper before re-measuring. The percentage of solvent uptake was obtained by the following equation:

(III.2)

Where: mi and mt are weights of samples before and after sorption, respectively.

Gas chromatography with flame ionization detector (GC-FID)

detector was used to determine the concentration of low molar mass additives (< 400 g.mol -1 ) in food simulants. The components were separated using a HP-5 (5%phenyl)methylpolysiloxane capillary column of 32 mm ID and 30 m length. The thermal protocol used for the oven was as follows: an initial temperature of 40°C maintained for 5 min, heated at a rate of 6°C.min -1 to 270°C, maintained for 15 min. The injector temperature was

250°C. An external calibration was made in each food simulant in the range of 1.25

High performance liquid chromatography (HPLC)

An Alliance HPLC system (Waters) equipped with an Alltima C18 column (250 mm × 2.1 mm, 5-Alltima C18 (7.5mm × 2.1 mm, 5determine the concentration of high molar mass additives in food simulants. Compounds were separated with 55% acetonitrile at a flow rate of 0.3 mL.min -1 at 50°C. Absorbance was recorded at 230 nm to quantify the additive concentration. An external calibration was made in each food simulant with concentrations of 1.25 to 20 mg.L -1 .

2.12 Estimation of the Dapp 2.12.1

The Dapp of additives from the polymer films into food simulant was calculated from one-dimensional infinite plane sheets, given that diffusion via the borders of the films is dimensional diffusion equation:

(III.3)
Where: C is the additive concentration, t, the contact time, x, the position in the film and , the apparent diffusion coefficient.

The Eq. (III.3) was solved using the following initial (Eq. III.4) and boundary (Eq. III.5) conditions:

(III.4)

(III.5)
Where: is the additive concentration in food simulant at time t and L, the film half thickness.

This diffusion model can be used provided that the following assumptions are verified (Helmroth et al., 2002): (i) additive contamination of the film is homogeneous, (ii) there is no noticeable swelling caused by interaction between the food simulant and the polymer, (iii) there is no concentration gradient in the food simulant, and (iv) the film thickness is homogeneous.

In the case of an infinite plane sheet suspended in a stirred solution of limited volume, the Eq.

(III.3) can be solved analytically as described by Crank (Crank, 1975):

(III.6)
Chapter III. Impact of nanoclays on the properties of transfer and inertia of materials With:

(III.7)

Where: Mt is the total amount of additives in food simulant at time t and is the total amount of additives in food simulant at the steady state, is the polymer volume and the food simulant volume, , the positive roots of the equation and , the partition coefficient of the additive in the polymer/food simulant system.

The numerical simulations were carried out using Matlab software and its lsqnonlin function to estimate the Dapp. To evaluate the correlation between experimental and estimated data, the percentage of the root mean-square error (% RMSE) was calculated using the following equation:

(III.8)
Where:

is the initial mass of additive in the biopolymer film and is the mass of additive in the food simulant at time t.

Piringer correlation

The Piringer correlation (1994) estimates the worst case additive diffusivity in a given polymer by correlating the diffusion coefficient with the molar mass of additives and the temperature.

(Eq. III.9) can be refined to not depend on experimental data [START_REF] Brandsch | Migration modelling as a tool for quality assurance of food packaging Migration modelling as a tool for quality assurance of food[END_REF].

(III.9)

With: = 10 4 cm².s -1 = 1 m².s -1 and

Where the parameter is linked to the polymer and describes the basic diffusion behavior of the polymer matrix in relation to the migrants, M is the molar mass of the additive, T is the apostrophe ' indicates the parameter is temperature independent.

The parameters III.2 [START_REF] Begley | Evaluation of migration models that might be used in support of regulations for food-contact plastics[END_REF]. In the absence of specific parameters for PBSA and PHBV, those for LDPE and PET were used for, respectively, low and high barrier property polymers.

Microstructure of composite material

Because the interaction between polymers and nanoclays determines the formation of exfoliated nanocomposite materials, clay modification has been extensively studied [START_REF] Muñoz-Shugulí | Cetylpyridinium bromide-modified montmorillonite as filler in low density polyethylene nanocomposite films[END_REF]. Exfoliated nanoparticles reduce diffusion by creating tortuosity effect and results in higher transfer properties (Nasiri et al. 2016).

The XRD revealed a diffraction peak at 4.9°, corresponding to an interlayer spacing of 1.8 nm [START_REF] Bharadwaj | Structure -property relationships in cross-linked polyester -clay nanocomposites[END_REF]. The analysis of neat biopolymers exhibited no diffraction peaks in the ocomposites revealed one diffraction peak with a shift towards the smallest angles compared to C-30B powder, 2.7° for PBSA NCP and 2.2° for PHBV NCP, which respectively corresponds to a d-spacing of 3.3 nm and 4.0 nm.

Similar values were reported for PBSA NCP (Charlon et al., 2015a) and PHBV NCP (Bordes et al., 2008). This increase in the interlayer distance reflects the formation of intercalated structures, confirmed by TEM observations which indicated both intercalated and exfoliated structures for nanocomposite materials.

Comparison of the Dapp of additives in neat and nanocomposites materials

The estimation of the Dapp of each additive from biopolymer into food simulant is reported in Fig. III.3, with the respective impacts of: polymer type, food simulant, and the effect of nanoparticle incorporation.

The differences in the Dapp in the various systems, illustrated by the differing scales of the histograms, reflect the influences of the polymer type and the food simulant in contact. As in previous works that highlight the poor gas barrier properties of PBSA (Phua et al., 2013), the diffusivity values measured in PBSA are between five and twenty times lower than those in PHBV, which normally has good barrier properties (Crétois et al., 2014).

Contact with 95% ethanol seems to have a dramatic impact: producing diffusion values nearly ten times higher than for isooctane contact. The very peculiar behavior of polyesters in contact with ethanol was already reported for PET (Begley and Hollifield, 1990), PLA (Jamshidian et al., 2012), and PHBV (Chea et al., 2015).

For PBSA and PHBV matrices with nanoparticles in contact with 95% ethanol and isooctane, the Dapp varies according to additives. The impact of nanoparticles is not significant if compared to the impact of the biopolymer type or food simulant in contact. For PBSA in contact with 95% ethanol, nanoparticles decreased the Dapp for all additives. In contact with isooctane, nanoparticles increased the Dapp, especially for low molar mass additives. For PHBV, nanoparticles increased the Dapp significantly whatever the food simulant in contact.

These results contradict previous conclusions on conventional synthetic materials. For polyurethane, there is a negative correlation between C-30B (concentrations 0 -50 wt%) and the diffusivity of three volatile organic compounds: toluene, decane, and butanol (Herrera-Alonso et al., 2009). Nasiri et al. (2016) measured the diffusivities in LLDPE with a wide range of additives exposed to four food simulants: Dapp decreased with the addition of Cloisite regardless of the nature of the diffusing substance. Therefore, whereas plastic-based nanocomposite materials meet inertia criteria regarding the migration of low molar mass substances, the behavior of biodegradable materials raises new concerns relating to compliance with regulations and consumer safety. Given the limited available data, it is difficult to establish behavioral laws to describe the specific impact of nanoclays on the evolution of the Dapp of low molar mass additives. In addition to the tortuosity effect demonstrated in section 3.1, these results suggest that nanoparticles modify polymer structures (size, shape, or arrangement of spherulites, and degree of crystallinity) and thus their transfer properties.

Predicting the Dapp of additives according to molar mass

The Dapp of additives is related to their specific volume and, consequently, to their molar mass: D M with a scaling parameter that can be related to the transport mechanism [START_REF] Lodge | Reconciliation of the molecular weight dependence of diffusion and viscosity in entangled polymers[END_REF].

Impact of the sorption of food simulant

The contact of polymers with food simulating liquids can modify their transport properties [START_REF] Figge | Migration of additives from plastics films into edible oils and fat simulants[END_REF], and the presence of nanoparticles is likely to impact these interactions. The sorption of food simulant by the semi-crystalline biopolymer usually induces swelling in the biopolymer, which accelerates additive migration and increases the diffusion coefficient value (Reynier et al., 2001;Barnes et al., 2007).

The solvent uptake of PBSA, PHBV and their corresponding nanocomposites after 10 days of contact with 95% ethanol and isooctane at 40°C were summarized in Table III. In the case of PBSA based materials, significant ethanol uptakes of 3.5% (neat) and 4.7%

(nanocomposite) were recorded, although there was no measurable change in film thickness.

The 95% ethanol sorption value of PHBV is in accordance with the value reported by Chea et al. (2015) on the same matrix/food simulant system. As mentioned in section 3.3, the peculiar interaction of 95% ethanol with polyesters was already proven with PET (Begley and Hollifield, 1990;Widén et al., 2004;Kim and Lee, 2012), PLA (Jamshidian et al., 2012) and PHBV (Chea et al., 2015).

For neat and nanocomposite PBSA in contact with isooctane, no solvent uptake was detected. Neat and nanocomposite PHBV in contact with isooctane had a < 1.5%, solvent uptake.

The incorporation of nanoclays in the polymer matrix leads to different behaviors depending on the nature of the food simulant. 95% ethanol uptake increased with nanoclays whatever the polymer. In PHBV, addition of nanoclay reduced isooctane uptake. Nanoclays are hydrophilic and therefore soluble in ethanol [START_REF] Ho | Effects of solvent solubility parameters on organoclay dispersions[END_REF] which leads to a greater solvent uptake. However, we cannot attribute the increase in Dapp values observed for all additives solely to solvent sorption.

Impact of the semi-crystalline structure of bio-nanocomposites

Bio-nanocomposite transfer properties are influenced by factors such as the degree of crystallinity of the polymer: high crystallinity limits movement and therefore limits the diffusion of migrating substances [START_REF] Hedenqvist | Diffusion of small-molecule penetrants in polyethylene: free volume and morphology[END_REF].

The degrees of crystallinity of neat and nanocomposite biopolymers evaluated by DSC experiments on films without contact and after 10 days in contact with food simulants at 40°C

were summarized in Table III.4.

PHBV has a high degree of crystallinity (Chea et al., 2015): almost twice that of PBSA, which may explain the difference between Dapp values for these two materials.

The degree of crystallinity of polymers proved to be food simulant dependent. In contact with 95% ethanol, the degree of crystallinity decreases by 1.3% for PHBV, and by 3.9% for PBSA. These results are similar to previous research conducted on these polymers and food simulants (Chea et al., 2015;[START_REF] Siracusa | Poly(butylene succinate) and poly(butylene succinateco-adipate) for food packaging applications: gas barrier properties after stressed treatments[END_REF]. The decrease in crystallinity could be attributed to the degradation of polymer chains due to hydrolysis reactions induced by the 95% ethanol. However, in contact with isooctane, there was no change in the degree of crystallinity for PHBV, a 1.3% increase for PHBV NCP and a close to 2% increase for neat and nanocomposite PBSA. This result confirms the findings of Chea et al. (2015): there was no structural change to PHBV in contact with isooctane.

C-30B decreases the degree of crystallinity of the two polymers by nearly 10%. This has previously been observed for PBSA/C30B (Sinha Ray et al., 2005), and for PHBV/OMMT (Wang et al., 2005). It can be explained by the full exfoliation of nanoclays platelets in the polymer matrix (see section 3.1), which restricts the mobility of the polymer chains and thus prevents them from crystallizing (Krikorian and Pochan, 2003). This decrease results in an increase of the amorphous phase in which the diffusivity of additives is facilitated, this tendency is observed in all cases apart from PBSA in 95% ethanol (Fig. III.3).

Although the degree of crystallinity of materials is known to impact the transport properties of semi-crystalline polymers, strict correlation between crystallinity and diffusivity cannot be concluded in the case of biodegradable packaging materials. For instance, Dapp of additives in PBSA are three times higher in contact with 95% ethanol than with isooctane, but the crystallinity of both materials is similar. The relationship between diffusion and crystallinity on a macroscopic scale remains obscure. Nanoclays generate localized crystalline gradients [START_REF] Wurm | Retarded crystallization in polyamide/layered silicates nanocomposites caused by an immobilized interphase[END_REF] which could modify the transport properties of materials.

Conclusion

The incorporation of nanoclays in biodegradable materials raises new questions related to their food contact suitability. While the few studies carried out on synthetic plastic materials agree that the inclusion of nanoclays decreases diffusivity, this work demonstrates that this is not the case for biodegradable materials. The presence of nanoparticles affects diffusion variously, depending on the nature of the migrating substance and particularly depending on the nature of the food simulant. As previously observed for biopolyester based materials, contact with ethanol, whose diffusion activation mechanisms remain unclear, is the worst-case scenario. This particular sensitivity of biopolyesters to ethanol, especially when they incorporate inorganic nanoparticles such as nanoclays, underscores the problem of applying existing recommended testing conditions for the evaluation of conventional plastic materials to these new materials. Ethanol sorption, which promotes the crystallization of synthetic polyesters and thus prevents the mobility of low molar mass molecules, appears to be enhanced by the incorporation of clay nanoparticles. Being necessarily cautious we cannot, with sufficient accuracy, predict migration levels from bio-nanocomposite packaging using diffusion models generally applied to food contact material considered as continuous homogeneous phase.

licable way to overestimate migration and guarantee consumer safety. This model would be more effective with the creation and inclusion of database of the diffusion coefficients of low molar mass molecules in these materials.

Introduction

In face of growing environmental concerns, new alternative materials must be proposed especially for short-term applications like food packaging. In this context, biodegradable polymers made with bio-sourced resources appear as promising materials. Among the biodegradable polymers, aliphatic polyesters like poly(butylene succinate-co-butylene adipate)

(PBSA) and poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) appear as interesting candidates to replace conventional non-degradable polymers. In addition to their biodegradability, these two polymers have the advantage to be fully (PHBV) or partially (PBSA) bio-sourced. However, to make them competitive towards non-degradable petroleumbased polymers, particularly in term of barrier properties, their blend with reinforcing nanofillers appear as a promising alternative widely described in the literature but also already placed on the market exclusively in synthetic matrices until now.

The introduction of nanofillers in materials intended to food contact raises new questions in term of consumer safety. The migration of nanoclays from nanocomposite material into food or food simulant has been already studied [START_REF] Handy | Toxic effects of nanoparticles and nanomaterials: Implications for public health, risk assessment and the public perception of nanotechnology[END_REF][START_REF] Xia | Release of nanoclay and surfactant from polymer-clay nanocomposites into a food simulant[END_REF][START_REF] Duncan | dissolution, and desorption[END_REF]Echegoyen et al., 2016). They conclude that their migration will take place in the case of very small nanoparticles with a radius in the order of 1 nm, from polymer matrices that have a relatively low dynamic viscosity, and that do not interact with the nanoparticles . Recently, investigations were performed in the same way on nanocomposite materials exposed to thermal, chemical, and mechanical stress followed by mechanical abrasion of their surface and conclude that even under dynamic stress conditions, nano-laponites, considered as exhibiting a higher potential to be released from a polymer matrix than other clay minerals, do not contaminate the food under a nano-form (Bott and Franz, 2019).

In addition to the risks related to the release of nanoparticles to food, the impact of the addition of nanoclays in polymer matrix on the migration of non-nano substances is much less studied. The few studies carried out on conventional plastic materials concluded that the incorporation of nanoclays positively affect the diffusion of migrating substances (Pereira de Abreu et al., 2010;Aida Nasiri et al., 2016;[START_REF] Farhoodi | Migration kinetics of ethylene glycol monomer from PET bottles into acidic food simulant: effects of nanoparticle presence and matrix morphology[END_REF]. In the case of biodegradable plastic materials this is not always verify depending on the nature of the migrating substance, the polymer type and the nature of the food simulant in contact (Lajarrige et al., 2019).

Moreover, biodegradable polymers which are, by definition, instable over time are subject to degradation. These polymer degradations can be classified according to the nature of the causing agents as photo-oxidative degradation, thermal degradation, ozone-induced degradation, mechanochemical degradation, catalytic degradation and biodegradation [START_REF] Singh | Mechanistic implications of plastic degradation[END_REF]. These degradations lead to a modification of their structure and consequently to their properties (Rydz et al., 2015). Most of the time, the degradation affects the thermal stability, color, mechanical properties, crystallinity and lamellar thickness distribution [START_REF] Ramis | Thermal degradation of polypropylene/starch-based materials with enhanced biodegradability[END_REF]. These changes may be desirable as for the end-of-life of the polymer, or undesirable, such as changes during its use in contact with food [START_REF] Speight | Chapter 14 -Monomers, Polymers, and Plastics[END_REF].

From a safety standpoint, as any materials intended to food contact, bio-nanocomposite materials are subject to the European framework Regulation (EC) No 1935/2004. In absence of specific regulations dedicated to nanocomposite and bio-nanocomposite, their food contact suitability is assessed following the recommendations for non-degradable plastic materials specified in the European Regulation (EC) No 10/2011. However, in the case of biodegradable nanocomposite materials, for which the structure evolves over time, their food contact ability has to be investigated throughout their use in food contact. In practice, their compliance can be validated after the production of the packaging but can evolve during an extended storage period under detrimental conditions of relative humidity associated with uncontrolled exposure to light. That is why it is important to determine the impact of the polymer degradations on the properties of transfer and inertia of materials.

In this context, this work aims to evaluate the food contact suitability of two distinct biodegradable polyesters (PBSA and PHBV) after accelerated aging (UV, humidity and temperature) and to determine if the recommendations established for non-degradable materials can be applicable to the particular case of biodegradable materials. Neat and nanocomposite materials reinforced with 5 wt% of organo-modified montmorillonite were considered to determine the impact of the reinforcing nanofillers on the aging of materials and consequently on their properties of transfer and inertia. In this way, overall and specific migrations using a panel of additives representative of possible substances added in the polymer formulation were assessed, according to the conditions of test recommended for plastic material, as well as their diffusivity in the polymer matrix.

Materials and Method

Materials

Poly(butylene succinate-co-butylene adipate) (PBSA, PBE 001, density 1.24 g.cm -3 ) and

Poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV, PHI 002, density 1.23 g.cm -3 ) pellets were commercially procured from Natureplast, France.

Cloisite 30B (C-30B), an organo-modified montmorillonite clay containing a methyl bis-2-hydroxyethyl ammonium quaternary salt with a cation exchange capacity (CEC) of 90 meq/100 g, were supplied by BYK additives & instruments, Germany.

An array of eight low molar mass molecules with a variety of chemical and physical properties as described in FDA and EFSA Regulations (Food and Drug Administration, 2006;

European Food Safety Authority, 2011) was selected as representative plastic additives. The additives chosen include: chlorobenzene, methyl salicylate, biphenyl, phenyl cyclohexane, benzophenone, methyl stearate and DEHA, all provided by Sigma-Aldrich.

Acetic acid, ethanol and 2,2,4 trimethyl pentane (isooctane) were commercially provided by Sigma-Aldrich with a purity > 99% and were used to prepare the food simulants for migration experiments: 3% acetic acid, 95% ethanol and isooctane.

Preparation of materials

PBSA and PHBV films containing 5 wt% C-30B (PBSA NCP and PHBV NCP) were synthetized by a melt extrusion process using an EuroLab 16XL co-rotating twin-screw TM , Germany) with a L/D ratio of 40 and a screw diameter of 16 mm as described previously (Lajarrige et al., 2019). The control samples of neat polymers were prepared following the same protocol used for nanocomposites in order to respect the same thermal processes.

The neat and nanocomposite films were then subjected to accelerated aging in a Q-SUN Xe-3 xenon arc chamber (Q-LAB, France) equipped with Window Q filter. The conditions were as follows: 35°C, 50% RH and an irradiance of 1.44 W.m -2 at 420 nm for 30 days.

Differential scanning calorimetry (DSC)

The crystallinity of the materials was studied using a differential scanning calorimeter (DSC series Q200, TA Instruments). Experiments were carried out on neat and nanocomposite films, before and after 30 days of accelerated aging. All measurements were performed under a nitrogen atmosphere (50 mL.min -1 ). Specimens of 5-8 mg, weighed using a microbalance (Sartorius micro PRO 11), were placed in aluminum sample pans (Tzero Aluminium Hermetic pan, TA Instruments). The thermal protocol was defined according to the polymer. The PBSA samples were heated up to 150°C, maintained at 150°C for 5 min, cooled to -35°C and finally reheated to 150°C. In the case of PHBV, samples were heated up to 200°C, maintained at 200°C in the sample and is the theoretical melting enthalpy of the polymer assumed to be 100% crystalline, = 146.0 J.g -1 for PHBV (Barham et al., 1984) and = 116.9 J.g -1 for PBSA (Nikolic and Djonlagic, 2001).

Overall migration testing

Overall migration (OM) level was gravimetrically determined after the total immersion of samples in the selected food simulants based on the standardized testing conditions set out for long term storage (40°C for 10 days) in Regulation (EC) No 10/2011. The samples were cut in disks of 2.5 cm diameter and kept in a vacuum oven at 60°C for 2 days. The polymer disks with the total area of 60 cm 2 were weighed using a microbalance (0.1mg resolution) prior to the migration test and then placed in a 100 mL migration cell (DURAN® screw thread tubes) with rod and glass spacers and PTFE-sealed caps. Before filling, the migration cells were blown out with nitrogen to prevent any dust contamination. The bottles were then filled with the respective simulants, sealed and stored in a 40°C oven. Each sample/simulant combination was prepared in triplicate. After 10 days of contact, the bottles were ultrasonicated for 2 min to detach any potential particles from the sample surface. The samples were then removed and placed in a vacuum oven at 60°C for 2 days. After evaporation of the simulant, the specimens were weighed again and overall migration was calculated in milligrams per square decimeter of the surface area of sample.

Specific migration testing

Spiking of films and desorption tests

The films were contaminated by the array of additives by impregnation at 40°C during 7 days under rotary agitation with a theoretical additive amount of 500 mg.kg -1 for each of them.

After a quick ethanol rinsing to eliminate surface additives, between 0.5 g and 1 g of contaminated materials were immersed in 100 mL of food simulants in a surface/volume ratio of 6 dm².L -1 at 40°C for 10 days under magnetic agitation. At the end of the contact time, an aliquot of food simulant was collected and analyzed by GC-FID using an Agilent 7890A GC were separated using a HP-5 (5%-phenyl)methylpolysiloxane capillary column of 32 mm ID and 30 m length. The thermal protocol used for the oven was as follows: an initial temperature of 40°C maintained for 5 min, heated at a rate of 6°C.min -1 to 270°C, maintained for 15 min.

Chapter III. Impact of nanoclays on the properties of transfer and inertia of materials

The injector temperature was 250°C. An external calibration was made in each food simulant in the range of 1.25 to 40 mg.L -1 .

Relative migration

The relative migration represents the percentage of an additive that migrates from polymer into food simulant. It was calculated by dividing the mass of additive recovered in the food simulant after 10 days of contact by the initial mass of additive in the film sample measured after two consecutive extractions in 20 mL of n-hexane.

Statistical treatment

Variance analysis were performed using XLSTAT software. Effects of the addition of nanoclays and the UV treatment (n = 42 for each material-food simulant couple) were studied by variance analysis regardless of each other and the nature of the additive.

Estimation of the Dapp

The Dapp of additives from the polymer films into food simulant was calculated from second law (Crank, 1975):

(III.11)

With:

(III.12)

Where: Mt is the total amount of additives in food simulant at time t and is the total amount of additives in food simulant at the steady state, is the polymer volume and the food simulant volume, , the positive roots of the equation and , the partition coefficient of the additive in the polymer/food simulant system.

The numerical simulations were carried out using Matlab software and its lsqnonlin function to estimate the Dapp.

Results and discussion

Semi-crystalline structure of materials

The crystallization behaviour was determined from a heat-cool-heat cycle using DSC for neat, nanocomposite and aged materials. The crystallization temperature (Tc), melting temperature (Tm) and the degree of crystallinity (XC) were gathered in Table III.5. The data indicated that the aging significantly affected the thermal properties of the materials with roughly similar effects on both polyesters. In the case of PBSA materials, the Tm do not appear significantly modified after accelerated aging. In contrast, the crystalline transition was significantly impacted with a decrease and the XC increases significantly.

Concerning the PHBV materials, the Tm and Tc decrease significantly after aging. The XC tends to increase but to a lesser extent compared to PBSA materials. The effect of irradiation on PBSA [START_REF] Zhang | Photodegradation behavior of poly(butylene succinate-co-butylene adipate )/ZnO nanocomposites[END_REF][START_REF] Cai | Study on the photodegradation stability of poly(butylene succinate-co-butylene adipate)/TiO2 nanocomposites[END_REF] and PHBV [START_REF] Oliveira | Gamma irradiation effects on poly(hydroxybutyrate)[END_REF][START_REF] Rosario | Effect of gamma radiation on the properties of poly(3-Hydroxybutyrate-co-3-Hydroxyvalerate)/Poly(e-Caprolactone) blends[END_REF][START_REF] Iggui | The effects of accelerated photooxidation on molecular weight and thermal and mechanical properties of PHBV/Cloisite 30B bionanocomposites[END_REF] has already been studied and similar results were obtained. The decrease of the Tc, observed with both materials, is attributed to chain scission mechanism leading to the formation of shorter polymer chains. This reduction in chain size should result in an increase in mobility (and therefore freedom of movement) that allows structural rearrangement of polymers in a crystalline state at a lower temperature compared to long polymer chains [START_REF] Oliveira | Gamma irradiation effects on poly(hydroxybutyrate)[END_REF][START_REF] Hermida | Gamma irradiation effects on mechanical and thermal properties and biodegradation of poly(3-hydroxybutyrate) based films[END_REF]. Regarding the Tm, for PHBV, a decrease is observed and double melting peaks appear which indicate changes in the crystal structure of polymers. The decrease of Tm may also be attributed to the decrease in molecular weight of polymer chains, this hypothesis being supported in several studies by the determination of the number average molecular weight ( ) by size-exclusion chromatography (SEC) [START_REF] Mitomo | Radiation-induced degradation of poly(3-hydroxybutyrate) and the copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate)[END_REF][START_REF] Zembouai | Gamma irradiation effects on morphology and properties of PHBV/PLA blends in presence of compatibilizer and Cloisite 30[END_REF][START_REF] Zembouai | The influence of gamma irradiation on the chemical structure and crystallinity of PHBV/PLA/Cloisite 30B nanocomposites[END_REF][START_REF] Iggui | The effects of accelerated photooxidation on molecular weight and thermal and mechanical properties of PHBV/Cloisite 30B bionanocomposites[END_REF]. The increase of the degree of crystallinity observed is initiated by the scission of polymer chains particularly in amorphous regions, which are more accessible for degradation. The higher mobility of these shorter chains allows them to recrystallize, this phenomenon being more marked in the case of PBSA whose degree of crystallinity is initially lower.

Finally, it is important to point out that the increase of the degree of crystallinity was visually confirmed since the materials have become very brittle compared to unaged control materials (Figure III.5). The addition of nanoclays does not seem to have any effect on the impact of accelerated aging, neither on crystallization and melting temperatures nor on the degree of crystallinity. From a mechanical point of view, they seem to play a reinforcing role by slightly preserving the ductility of the materials, this effect being more noticeable on the PBSA.

Figure III.5: Pictures of the materials before and after accelerated aging.

Overall migration measure

According to Article 3 of Regulation (EC) No 1935/2004, the release of substances from food contact materials should not bring about unacceptable changes in the composition of the food. That is why, the overall migration limit, which represents the maximum permitted amount of non-volatile substances released from a material into foods or food simulants, was set to 10 mg of substances per 1 dm² of surface area of plastic material.

The results of the overall migration measurements of PBSA, PBSA NCP, PHBV, PHBV NCP and the corresponding aged materials are reported in Figure III.6 and III.7.

All the studied materials showed an overall migration value lower than the authorized threshold of 10 mg.dm -2 . On this criterion, these materials meet the level of requirement that to the selected food simulants (3% acetic acid, 95% ethanol and isooctane).

Relative migration measurement

The results of the relative migration of additives after 10 days of contact with food simulants are gathered in The relative migration values are generally high, reaching 100% in several cases, which reflect a high level of contamination in the case of contact with fatty foods. The values are substantially similar in both types of solvents and very close to the values measured with the same target molecules from synthetic materials such as polyolefins (Nasiri et al 2016). Overall, the level of migration obviously depends on the nature of the substance, but each compound behaves differently depending on the type of material and the food simulant in contact. The sorption of such polar simulants is known to induce the swelling of the polymeric network and, consequently, modify the transport properties of the material. In addition, the sorption of nonpolar solvent is likely to significantly modify the affinity of the compound for the packaging film and thus impact its migration level.

The impact of the presence of nanoclays is difficult to extract and does not follow any particular pattern. The analysis of variance reveals a variable influence of nanofillers depending on the nature of the material and the simulant. In the particular case of PBSA, the addition of nanoclay has no significant impact when the packaging is in contact with ethanol, whereas it induces a decrease in the level of migration (p = 0.027) as a result of contact with isooctane.

On the other hand, the presence of nanoclays does not seem to produce any significant effect This result appears in good coherence with the increase of the degree of crystallinity previously observed (Table III.5) since as a first approximation and according to the free volume theory describing the diffusion in semi-crystalline polymeric system [START_REF] Sharma | Diffusion in polymeric systems A review on free volume theory[END_REF], a higher degree of crystallinity, which leads to a higher percentage of crystalline phase, is positive for decreasing the migration phenomena (Dardmeh et al., 2015).

On the other hand, it is important to note that no significant interaction (0.4 p-value 0.8) was evidenced between the UV treatment and the addition of nanoclays, which implies that the effect of aging treatment is not dependent on the presence of the nanoclays. From a practical point of view, these results provide the basis of the recommendations related to the assessment of migration from biodegradable materials. While the migration test conditions defined for plastic materials may seem inappropriate for biodegradable and bio-composite materials, this study demonstrates that the food simulants and contact conditions established under Regulation (EC) No 10/2011 are potentially applicable to these particular materials.

Dapp of additives in neat and nanocomposite aged materials

The estimation of the Dapp of each additive from biopolyester into fatty food simulants is reported in Figures III.11 The differing scales of the histograms highlight the impact of the polymer type and the food simulant, this observation having already been established and commented on a previous study performed on untreated material (Lajarrige et al., 2019). The accelerated aging does not impact this range of scales, the apparent diffusion coefficient ranking as follow: PBSA/95% ethanol > PBSA/isooctane > PHBV/95% ethanol > PHBV/isooctane. However, contrasted results due to the treatment of materials were obtained depending on polymer type and food simulant in contact.

The effect of the presence of nanoclays in the PHBV is very significant (n < 0.0001), leading to a dramatical increase in the diffusivity values of almost all additives, whatever the food simulant in contact. However, this effect does not apply to the particular case of higher molar mass molecules for which the influence of nanofillers is more difficult to identify. This increase in the diffusivity of low molar mass molecules may appear in opposition to an expected tortuosity effect due to the presence of nanofillers and an increase in crystallinity induced by UV treatment. The most tangible hypothesis to explain this behaviour is the macroscopic damage of the films induced by the aging process. It is indeed likely that the accelerated aging of materials produces microcracks generating diffusion channels particularly for the most modify the assembly of the polymer network, a refined observation of materials on a nanoscale seems necessary to characterize the diffusion mechanisms that take place in these structures.

Conclusion

The use of nanocomposite materials as food packaging raises new questions in term of consumer safety. This is even more true with the use of biodegradable polymers for which the structure is, by definition, instable over time. It was not a priori advisable to reproduce the conditions of migration testing developed and applied to plastic materials in the same way. This study was performed to verify the food contact suitability of bio-nanocomposite materials after critical accelerated aging following the recommendations of European Regulation (EC) No 10/2011 defined for non-degradable polymers. The structure of materials proved to be affected by the treatment but the consequences on the properties of transfer and inertia of materials are not deleterious regarding the safety issues of such material. The increase of the degree of crystallinity induced by the UV irradiation leads to a decrease of the overall migration. Except for a few rare cases, a lower specific migration of additives was also observed after aging, regardless of the additive, the nature of the polymer or the food simulant in contact. This decrease in migration is not related to a decrease in the diffusivity of additives, which is, in most cases, subject to an increase despite the increase in the crystallinity of the material measured and the presence of impermeable nanoclays likely to exert a barrier effect. As consequence, these results do not call into question the food contact suitability of these materials, even as they age. From a practical point of view, this study demonstrates that the recommendations provided by Regulation (EC) No 10/2011 are potentially applicable to the particular case of biodegradable materials, the critical point remaining rather at the level of the mechanical properties of the materials after aging, which become brittle and difficult to use for packaging applications.

Introduction

Since their first appearance in 1950, the production of plastic materials continues to grow in the food packaging market. The packaging is the largest plastic market with nearly 40% of the demand in 2017 in Europe (PlasticsEurope, 2018). This growing plastic consumption leads to an exponential accumulation of these non-biodegradable materials in the environment. To face the environmental issues, alternative are proposed to substitute these materials with biodegradable plastics, whether they derive from renewable resources or not. However, these materials remain poorly competitive towards conventional plastics considering their low mechanical and barrier properties and their high water sensitivity. One of the main strategy to improve their performances is the incorporation of inorganic nanoparticles, which act as impermeable sheets and/or mechanical reinforcement agents. This application can partly explain the emergence of nanotechnologies in the packaging area [START_REF] Bumbudsanpharoke | Nano-food packaging: an overview of market, migration research, and safety regulations[END_REF]) -Enabled Packaging For Food and Beverages: Intelligent Packaging to Witness Highest Growth -enabled packaging market for food and beverages was estimated to be $6.5 billion in 2013 and will grow at a compound annual growth rate of 12.7% to reach about $15.0 billion in 2020.

Despite the large number of conducted research programs, the incorporation of engineering nanomaterials in biodegradable food packaging is still at the development stage. The emergence of novel biodegradable food packaging materials with nanocomposite structures implies to clearly target specific requirements. Indeed, concerning functional properties (e.g. transport properties) it is necessary to verify the potential migration into food for ensuring consumer safety issues.

The safety issue of such material in term of human exposure cannot be restricted to the unique study of nanoparticles migration. In fact, their incorporation in the polymeric matrix might induce modifications of its transport properties and consequently affect the migration of other substances (i.e. monomers, additives or processing aids) which can potentially demonstrate a toxic character. The complete evaluation of the relevance of any bionanocomposite materials for contact with foods must necessary include these two investigations.

In addition and considering their possible degradation by microorganisms, the application of bio-nanomaterials as food packaging raises new interrogations about their inertia during the contact with fermented foodstuff. This concern that is not be considered for conventional plastic materials, required the development of adequate protocols for migration assessment according to specific conditions of contact with medium simulating foods containing microorganisms. In the particular case of biodegradable materials, their stability in contact with food that may contain microorganisms must be viewed with caution since these materials are, by definition, subject to biotic degradation pathways.

This study aims to determine if biodegradable polymer matrices can be used for the packaging of foods containing microorganisms such as dairy products. In this way, the migration from material into food simulant of a panel of additives representative of potential migrating substances from food packaging was investigated. The liquid food simulants recommended by Commission Regulation No 10/2011 for plastic materials were substituted by agar plate inoculated with two different microorganisms, Penicillium Candidum and Lactobacillus Bulgaricus to basically mimic the behavior of cheese and yogurt, respectively. In addition to the apprehension of the behavior of the biodegradable polymer matrix, this work aims to understand the impact of the incorporation of reinforcing nano-fillers in such materials on their stability and related inertial properties. The investigation was focused on poly(butylene succinate-co-butylene adipate) (PBSA), an aliphatic polyester considering as having mechanical properties similar to those of polyolefins (Phua et al., 2013). PBSA reinforced or not with 5 wt% of organo-modified montmorillonite was selected as model packaging assumed to be representative of the materials most sensitive to biotic degradation reactions. This work therefore demonstrates the need to pay particular attention to the ways in which such materials are used in food packaging applications.

Materials and Methods

Materials

A biodegradable and partially bio-sourced polymer, the poly(butylene succinate-cobutylene adipate) (PBSA, PBE 001, density 1.24 g.cm -3 ) was commercially procured from Natureplast, France. Dichloromethane and nadditives from agar plate and films, respectively.

Preparation of bio-nanocomposite materials

Materials in form of films with 5 wt% C-30B were produced by a melt extrusion process using an EuroLab 16XL co-rotating twin-TM , Germany) with a L/D ratio of 40 and a screw diameter of 16 mm as previously described (Lajarrige et al., 2019).

Films of 205 ± 4 µm for PBSA and 250 ± 10 µm for PBSA NCP were produced.

Preparation of agar plates

-PDA agar plates: a pre-culture is necessary for the use of P. Candidum. First, the powder was rehydrated in an enriched solution during 16h at 5°C. Then, the solution was spread on a culture medium (Sabouraud Chloramphenicol) during five days at 25°C. At the end of this time, a colony was taken and inoculated on PDA agar plates. Three days at 25°C was necessary for the growth of the microorganism before putting the film in contact. The optimal growth conditions were found in previous studies (Le [START_REF] Le Dréan | Quantification of Penicillium camemberti and P. roqueforti mycelium by real-time PCR to assess their growth dynamics during ripening cheese[END_REF][START_REF] Kalai | Modelling the effect of temperature, pH, water activity, and organic acids on the germination time of Penicillium camemberti and Penicillium roqueforti conidia[END_REF]. The petri dishes were closed hermetically with parafilm and aluminum, to prevent any loss of additives.

-MRS agar plates: a pre-culture is necessary for the use of L. Lactobacillus. The powder was rehydrated in an enriched solution during 2h at 37°C. The solution was spread on MRS agar plate during three days at 37°C under anaerobiose. A colony was taken and inoculated on MRS agar plates. Three days at 37°C was necessary for the growth of the microorganism before putting the film in contact. The optimal growth conditions were found in previous studies [START_REF] Silva | Effect of the pH of growth on the survival of Lactobacillus delbrueckii subsp. bulgaricus to stress conditions during spary-drying[END_REF][START_REF] Mel | Optimization of Lactobacillus bulgaricus growth conditions for probiotic production[END_REF]. In this case, the petri dishes were stored in a plastic bag with an Anaerocult ® P sachet to maintain anaerobic conditions, which allow also to prevent any loss of additives.

Spiking of films and migration

The films were contaminated by the array of additives by impregnation at 40°C during 7 days under rotary agitation with a theoretical additive amount of 500 mg.kg -1 for each of them except for toluene, which was introduced at 1000 mg.kg -1 . After a quick rinsing with ethanol to eliminate additives on the surface, contaminated materials were put in contact with agar plates at 25°C (for P. Candidum) or 37°C under anaerobiose (for L. Bulgaricus) for 10 days as described in Section 2.3. Over specific periods of time:

-Films were recovered, rinsed with distilled water and ethanol and dried with absorbent paper.

The concentration of additives remaining in films was evaluated by extraction in 20 mL of nhexane at 23°C during 24h under magnetic agitation. The solution was analyzed by GC-FID.

-Agar plate was recovered and extracted with 30 mL of dichloromethane at 23°C during 24h under magnetic agitation in order to dose the additives that migrated from the films to the solid food simulant. After filtration, the solution was analyzed by GC-FID.

An Agilent 7890A GC equipped with a 7693A Automatic Sample detector was used for the analysis of the solutions. The components were separated using a HP-5 (5%-phenyl)methylpolysiloxane capillary column of 32 mm ID and 30 m length. The thermal protocol used for the oven was as follows: an initial temperature of 40°C maintained for 5 min, heated at a rate of 6°C.min-1 to 270°C, maintained for 15 min. The injector temperature was

250°C. An external calibration was made in n-hexane and dichloromethane in the range of 1.25 to 40 mg.L -1 .

Determination of the material balance of additives

The material balance of additives between film and agar plate was determined after 10 days of contact. It includes the percentage of additives that migrated from polymer into food simulant (agar plate) and the percentage of additives remaining in the film. They were calculated by dividing the mass of additives recovered in agar plate or film after 10 days of contact by the initial mass of additives in the film measured after two consecutive extractions in 20 mL of nhexane.

Evaluation of the desorption rate of additives from the film

The desorption rate of the additives from the film was estimated by reporting the mass of additives remaining in the film according to the square root of time. The results of the first five hours were used to obtain a linear regression. For better readability, the mass of additives recovered at each time was divided by the initial mass of additives.

Chapter III. Impact of nanoclays on the properties of transfer and inertia of materials

Evolution of materials in microbial medium

Mass loss measurement

The mass loss of PBSA and PBSA NCP was evaluated over time under microbial conditions. The materials were put in contact with the microbial solid food simulant and stored:

-Under aerobic conditions at 25°C for PDA agar plates inoculated with P. Candidum.

-Under anaerobic conditions at 37°C for MRS agar plates inoculated with L. Bulgaricus.

Over specific periods of time, the films were recovered, rinsed with distilled water and ethanol and dried at 50°C during 48h before being weighed. The film mass remaining was calculated using the following equation:

(III.14)
Where: is the initial mass of the film and is the mass of the film at time t.

Scanning electron microscopy (SEM) observations

The surface of the film in contact with agar plate inoculated or not with microorganisms was observed using a Phenom ProX scanning electron microscope (SEM) (Thermo Fisher Scientific) with an acceleration voltage of 5 kV, to evidence any degradation of the surface of the material.

SEM observations

The surface of polymer film was observed after 10 days of contact with agar plate inoculated or not with microorganisms in order to evidence microscopic modifications induced by the microorganisms. The SEM image A, showing the surface of PBSA after 10 days of contact with uninoculated agar plate, evidenced that PBSA presents a surface made of asperities. It was already observed in another study, where the AFM experiments of PBSA evidenced a surface made of numerous asperities of a few micrometers height (Salomez et al., 2019). The surface of materials after 10 days of contact with microorganisms was presented in

Evolution of the migration of additives from the film to the agar plate

The evolutions of the mass of additives (chlorobenzene, methyl salicylate, biphenyl, phenyl cyclohexane and benzophenone) remaining in films (Figure III.17 A) and the mass of additives that migrated into the agar plate (Figure III.17 B) over the 10 days of experiment were presented for PBSA NCP in contact with P. Candidum as an example.

The migration of additives follows the same tendency with a rapid transfer during the first hours. A rapid decrease was observed from the film and a rapid increase into the agar plate. The equilibrium state seems to be reached after 24h of contact but it continues to evolve to a lesser extent up to 10 days. It is particularly highlighted for methyl salicylate in agar plate (Figure III.17 B) for which the mass dramatically decreased between 24h and 10 days probably due to exchange between the three compartments of the study system. The mass of additives at the equilibrium varies according to their physical properties such as polarity and volatility. For chlorobenzene, the most volatile additive presented on this graph, the mass recovered at 10 days in the film was very low in comparison to the gain measured in the agar plate, which also decreases over the same 10-day period. The losses of the most volatile compounds are therefore probably related to their desorption and vaporization into the headspace of the experimental system. The additional incidence of degradation of these same compounds (by oxidation reactions in particular) over such a long period of time cannot be excluded.

Study of the material balance of additives after contact with fermented food simulant

Regarding the results of migration tests performed with Lactobacillus Bulgaricus as microorganism in fermented food simulant, the material balance shows a high retention of all additives in the film except for the most volatile compounds (i.e. toluene and chlorobenzene) which are not released into the culture medium but are desorbed into the headspace (data not shown). The relative migration values of all compounds do not exceed 10%, which represents levels of migration far from the results obtained in liquid simulants (Lajarrige et al., 2019). In view of these results, the impact of the presence of L. Bulgaricus on the migration can be considered as negligible and demonstrates that the presence of this microorganism in the food does not raise additional concerns in terms of consumer exposure.

The mass balance of additives between film and agar plate after 10 days of contact with PDA medium inoculated or not (control) with P. Candidum was reported in Figure III.18 for Chapter III. Impact of nanoclays on the properties of transfer and inertia of materials each additive. The percentage of additives in films represents the mass of additives remaining in the film while the percentage of additives in agar plate represents the mass of additives that migrated from the film into the agar plate. As this second percentage is low, the values were reported in the graph. In order to assess the respective impact of the incorporation of nanoclays and of the presence of the microorganism, four case studies were reported including neat (1) and nanocomposite (2) polymer in contact with inoculated agar plate, neat (3) and nanocomposite (4) polymer in contact with uninoculated agar plate (control).

Benzophenone

Methyl stearate

DEHA

It is important to bear in mind that these material balances are established after a long contact time, which implies that they reflect the partitioning of the different additives into the two considered compartments without integrating the quantities present in the gas phase.

Therefore, the very low percentage of toluene and chlorobenzene, the two most volatile additives, recovered in material balance suggests that they are most likely released into the headspace. For the others additives, the most important mass was measured in the film and a small percentage, inferior to 3.2% migrated to the solid food simulant except for toluene and methyl stearate for which a higher percentage was found. These migration values are very different from those obtained in the case of contact with liquid food simulants since the migration is almost total in the case of a fatty food simulant (ethanol and isooctane) and generally higher than 20% in the case of contact with aqueous food simulants (ethanol 10% or acetic acid 3%) (Article 4). Therefore, the degradation of the film by P. Candidum observed on its surface in contact does not have a significant impact on the migration process.

The presence of nanoclays appears to be associated with a higher mass of additives recovered in film sample. The addition of nanoclay therefore tends to retain the additives in the film which agrees with the hypothesis of a torturous effect induced by the presence of fillers but can also be related to a sorption/adsorption of the diffusing molecules at the surface of the nanoclay (Nasiri et al., 2017). This trend was in addition observed whether agar plate was inoculated or not.

Some specific additives including methyl salicylate, methyl stearate and DEHA are characterized by very contrasted behavior depending on whether the film is in contact with inoculated culture medium or uninoculated agar plate. In presence of microorganism, the mass of these additives recovered in the film was largely lower compared to the control suggesting that P. Candidum metabolizes some of the additives, especially those with ester linkages. In their study, [START_REF] Yang | An esterase from Penicillium decumbens P6 involved in lignite depolymerization[END_REF] evidenced the key role of esterase for Penicillium decumbens P6 in hydrolysing carboxyl within the lignite structure. It is therefore likely that the consumption of the esters by the microorganisms generates a concentration gradient between the two compartments (i.e. film and culture medium) that favors the diffusion and therefore the migration of the compound in the fermented food simulant.

Desorption rate of additives from the film

With respect to the negligible influence of L. Bulgaricus on previously observed migration levels, the study of the influence of the degradation of the materials on the transfer rates of additive to fermented food simulants was only undertaken on films placed in contact with media inoculated by using P. Candidum. The desorption rate of additives from the film to other compartments (agar plate and headspace) was estimated using the mass of additives recovered in the film sample over time (Figure III.19). In order to better evaluate the release rates of the different compounds, the values obtained during the first five hours were reported according to the square root of time according to a decreasing linear function, the slope of the regression reflecting the contamination rate.

The first significant fact concerns the differences in apparent desorption rate, which differ according to the nature of the additives. The most volatile compounds are released more quickly due to their desorption in the gas phase. The other compounds exhibit similar desorption rates that do not appear correlated to their molar mass. This first observation suggests a transfer that does not follow a purely Fickian behavior but which involves probably more complex mechanisms, the films having a porous structure on their surface due to the degradation by microorganisms.

Except for some rare cases, a significant influence of the presence of nanoclays was evidenced whether agar plates were inoculated or not. The barrier effect of nanofillers on the diffusion of the additives in the film seems therefore to be exerted whatever the level of degradation of the material, which can be interpreted as a positive effect in term of consumer exposure for very short contact times before the equilibrium state.

Regarding the impact of the microorganism, no clear behavior was highlighted. For PBSA, the results obtained with and without P. Candidum are similar while for PBSA NCP the presence of microorganism accelerates the desorption of the additives. As the nanoclays slow down the migration of additives, the microorganism may play a role in nanocomposite polymer, while in absence of nanoclays the speed is already high and their impact is therefore less obvious.

(A)

Chapter III. Impact of nanoclays on the properties of transfer and inertia of materials Chapter III. Impact of nanoclays on the properties of transfer and inertia of materials

Study of the impact of Penicillium Candidum on the migration of additives in the fermented food simulant

The impact of the microorganism P. Candidum on the migration of additives from films into agar plate was reported in Figure III.20 using a representation of the ratio between the mass of additive at 10 days that migrated into agar plate inoculated with microorganism and the mass of additive measured in same conditions in uninoculated agar plate. The result is positive when the presence of microorganisms exacerbates the migration of additives and negative when they decrease the migration. On this basis, the results show a very contrasting effect depending on the additive with a migration-enhancing effect of the contact with P. Candidum for methyl stearate and to a lesser extent for biphenyl, phenyl cyclohexane, benzophenone and DEHA. On the contrary, the migration of chlorobenzene and methyl salicylate is lower in presence of microorganism. The mechanisms of action of microorganisms and their influence on the migration of different compounds remain difficult to identify. In the particular case of ester compounds, their consumption by P.Candidum could explain the accelerated migration produced in the presence of microorganisms. In any event, the influence of such a microorganism is likely to promote the migration of some compounds and must necessarily be considered when defining the conditions of use of biodegradable polyester-based packaging.

The influence of the addition of nanoparticles also varies according to the additive. While the presence of nanofillers is an element that prevents the diffusion of all of the compounds into the material, their influence on the level of migration at 10 days (in a state of equilibrium)

proved to be detrimental for the contamination of fermented food by specific additives including biphenyl and phenyl cyclohexane and deserves a particular attention in the development of nanocomposite materials for food packaging applications.

Chapter III. Impact of nanoclays on the properties of transfer and inertia of materials

Conclusion

In a context of transition in the food packaging sector, a conversion aimed at limiting the use of petro-sourced materials while considering the emergence of nanotechnologies is beginning. The wide-scale use of bio-nanocomposite materials needs an evaluation of potential risks for health consumer. Such issue must take into account the migration of the nanoreinforcements themselves but also of the additives and contaminants, potentially toxics, used in the formulation of the packaging. Based on the hypothesis of market conversion to bionanocomposite packaging, it is important to consider all the scenarios of use of these materials.

This work provides a first overview on the possibility of using bio-nanocomposite materials for the packaging of dairy products. The addition of nanoclay in a biodegradable matrix in contact with such type of food simulant leads to a positive effect, which is a decrease of the level of migration of additives from the film sample. However, even if the migration of additives into the microbial food simulant proved to be low, the contact with microorganisms, mainly P.

Candidum, prove to be harmful due to their involvement in the degradation of materials. As consequence, this study highlights all the difficulties in using biodegradable polymers for a contact with foods containing microorganisms especially since the transfer mechanisms in the matrices that have been damaged are difficult to describe and therefore to predict.

Funding

The project leading to this publication has received funding from Excellence Initiative of Aix-Marseille Universityassociated Labex SERENADE project.

Conclusion

Si la présence de nano-argiles a tendance à diminuer la diffusivité et la migration des additifs dans des matériaux plastiques conventionnels, leur impact sur les propriétés de transfert En suivant les recommandations définies par le règlement européen N° 10/2011, un impact non conventionnel de la présence des nano-argiles a été mis en évidence dans la plupart des cas -à-PHBV) et simulant alimentaire au contact (éthanol 95% ou isooctane).

-

pas

Abstract

In face of growing environmental concerns, biodegradable and bio-sourced polymers are considered as a good alternative to replace conventional non-degradable synthetic polymers, particularly for short-lived applications such as food packaging (Avérous and Pollet, 2012).

However, their applications in this field is limited because mainly of their poor barrier properties. To make them competitive, their blend with nanomaterials appears as promising alternative but no without raise questions in term of consumer safety.

The present work aims to evaluate the consumer exposure to additives when foods are packed with two different biopolyesters incorporating or not nanoclays. As model additives, methyl salicylate, benzophenone, DEHA, Uvitex OB and Irganox 1076, authorized as additives and polymer production aids in the formulation of food packaging, were selected. The choice of the packaging material had a negligible impact on the consumer exposure. On the other hand, a non-homogeneous impact of the addition of nanoclays was evidenced depending on biopolyester used. However, these results are rather positive since the consumer exposure to additives is lower than the limit set by the Regulation, except for benzophenone for which the initial concentration in material have to be decreased. Keywords: bio-nanocomposite, food packaging, migration, additives, consumer exposure

Introduction

Packaging is essential for the protection of foods from deterioration induced by the external environment (Barnes et al., 2007). That is why the packaging sector is the largest consumer of plastic with nearly 40% of the demand in Europe in 2017 (PlasticsEurope, 2018) and the primary waste producer with more than half of plastic wastes (Geyer et al., 2017). In face of growing environmental concerns due to the production and consumption of plastics, materials based on bio-sourced and biodegradable polymers appear as promising alternative (Tharanathan, 2003). However, the market deployment of these materials is still hampered by their lack of competitiveness, especially in terms of barrier properties and, for some of them, their high water sensitivity. The addition of reinforcing fillers is a commonly invested strategy to address these technological deficiencies. Due to their availability, low cost and efficiency to significantly enhance the material properties at very low loadings (1-10 wt%) (Sorrentino et al., 2007), layered silicates are the widest commercial reinforcing fillers use in food packaging area (Silvestre et al., 2011).

Despite the expected benefits of the introduction of fillers in biodegradable polymer matrices, the emergence of nanotechnologies in the field of packaging applications raises many questions about potential risks for consumer safety. The packaging is a well-known source of contamination of foods by the transfer of substances originally present in the material in contact.

As all materials and articles intended to come into contact with food, biomaterials and bionanocomposite materials use for food packaging applications are required to comply with the framework Regulation (EC) No 1935/2004 without appearing in the list of groups of materials and articles which may be covered by specific measures. As consequence and in absence of specific regulation or directive, the control of their suitability for food contact application must be carried out by default following the compliance testing requirements defined for plastic material in the Regulation (EC) No 10/2011. The inertia criteria required and set out in the regulation is based on two distinct thresholds: (1) the overall migration limit (OML) that sets the maximum permitted amount of non-volatile substances released from a material into food simulants and (2) the specific migration limit (SML) defined for all individual substances (belonging to the positive list of authorized substances) according to the evaluation of their migration extent and their toxicological properties. In the particular case of nanocomposite materials, the migration of nanoparticles or their potentially toxic constituents is an additional concern that must be considered in a comprehensive risk assessment. This issue is the subject of a guidance recently published the European Food Safety Authority (EFSA) which introduces the notion of nano-specific risk assessment including attention to nanomaterials used in packaging (EFSA Scientific Committee, 2018). This document specifies that exposure to nanomaterials must be assessed on the basis of the experimental data on their migration or transfer from a food contact material (FCM) to food including a qualitative and quantitative analysis of the migrating substances in order to establish whether they are in nanoparticulate or solubilized/degraded form. Previous work on the evaluation of nanoparticle migration, whether from experimental measurements or theoretical studies, agrees towards the conclusion that migration of nanoparticles from plastic food packaging materials in general is not possible according to Fickian Law of diffusion as long as the particles are larger than 3-4 nm in diameter [START_REF] Šimon | Migration of engineered nanoparticles from polymer packaging to food -a physicochemical view[END_REF]Bott et al., 2014;Störmer et al., 2017;Bott and Franz, 2019).

If several investigations therefore seem to rule out the idea of consumer exposure to nanoparticles, the impact of the incorporation of nanoparticles on the migration of non-nano substances usually added in polymers as processing aids or properties enhancing is much less studied. Moreover, these studies were still focused on synthetic polymers like polyethylene (Helmroth et al., 2002;Nasiri et al., 2016), polyamide (Pereira de Abreu et al., 2010), polyurethane (Herrera-Alonso et al., 2009) or PET (Begley et al., 2004;Widén et al., 2004). All these works lead to the same conclusion about the positive effect of the nanoparticles that prevent the migration of contaminants. In the case of plastic materials, the non-organic nanoclays were considered as impermeable obstacles inducing a tortuosity of the diffusion path and/or the retention by adsorption of diffusing substances. The extrapolation of these observations to the particular case of biodegradable materials appears, however, to be hazardous. Previous investigations on the effect of nanoclays have already demonstrated their contrasting influence on the transport properties of biodegradable materials such as the poly(butylene succinate-co-butylene adipate) (PBSA) and the poly(hydroxybutyrate-cohydroxyvalerate) (PHBV) with, in particular, an increase of the apparent diffusivity of low molecular weight additives induced by the presence of montmorillonite in packaging film in contact with fatty food simulant (Lajarrige et al., 2019). These results suggest that the migration could be exacerbated with the use of biodegradable polymers, which are by definition, instable over time and for which the presence of nanoclays could produce detrimental effects.

On these bases, this study aims to determine the risks in term of consumer exposure towards potentially toxic additives related to the addition of nanoclay in biodegradable polymers dedicated to food packaging application. Two bio-nanocomposite packaging distinctly composed of PBSA and PHBV were selected as study models on the basis of their differing transfer properties, the PHBV being considered as high-barrier polymer (Crétois et al., 2014) in contrast to the PBSA (Phua et al., 2013). Neat and nanocomposite materials reinforced with 5% (w/w) of organo-modified montmorillonite were spiked with an array of additives, representative of potential contaminants, in order to measure their migration when subjected to contact with various food simulants. For demonstration of compliance for plastic materials not yet in contact with food, six food simulants (3% acetic acid, 10% ethanol, 20% ethanol, 50% ethanol, vegetable oil and poly(2,6-diphenyl-p-phenylene oxide) were assigned by the Regulation (EC) No 10/2011 with the objective to be representative of all general food categories (acid, aqueous, alcoholic, fatty and dry). The consumer exposure to additives was evaluated on the basis of a study on the consumption and eating habits of the French population published in 2017 by the French Agency for Food, Environmental and Occupational Health & Safety (ANSES). In this way, each category of foods consumed has been associated with a food simulant according to Regulation (EC) No 10/2011 (Annex I). t is of course acknowledged that the food packaging market will not switch to the "all nano" and that the use of bionanocomposite materials, although promised to economic growth, will only have a small market share in the medium term. This work therefore aims to establish a first inventory of a worstcase situation in terms of risk for the consumer according to scenarios that involve very overestimated levels of exposure.

Experimental

Materials

Two biodegradable and bio-sourced polymers, poly(butylene succinate-co-butylene adipate) (PBSA, PBE 001, density 1.24 g.cm -3 ) and poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV, PHI 002, density 1.23 g.cm -3 ) were commercially procured from Natureplast, France.

Cloisite 30B (C-30B), a montmorillonite clay modified by a methyl bis-2-hydroxyethyl ammonium quaternary salt was supplied by BYK additives & instruments, Germany.

An array of three low molar mass additives: methyl salicylate, benzophenone, DEHA and two high molar mass additives: Uvitex OB and Irganox 1076, was selected, all provided from Sigma-Aldrich. These molecules are referenced in the union list of authorized substances as additive or polymer production aid in food packaging with a specific migration limit (SML) as indicated in the Regulation (EC) No 10/2011. The SML values applicable for each additive are gathered in Table IV.1.

Table IV.1: List of selected additives and their corresponding specific migration limit (SML).

Additive

CAS number M (g.mol -1 ) SML (mgadditive/kgfood) Acetic acid, ethanol and 2,2,4 trimethyl pentane (isooctane) were commercially provided by Sigma-Aldrich with a purity > 99% and were used to prepare the food simulants for migration experiments. Following the recommendations of the Regulation (EC) No 10/2011, the selected food simulants were: 3% acetic acid to simulate acidic food behavior (simulant B), 10% ethanol for aqueous foods (simulant A), 20% ethanol for alcoholic foods with an alcohol content of up to 20 % (simulant C), 50% ethanol for alcoholic foods with an alcohol content of above 20 % and for oil in water emulsions (simulant D1), 95% ethanol and isooctane both used to simulate fatty food behavior. 95% ethanol and isooctane were employed as substitutes of simulant D2 for compliance testing in contact with fatty food. Poly(2,6-diphenyl-p-phenylene oxide) was supplied by Sigma-Aldrich under the trade name Tenax (60/80 mesh) and was used as solid food simulant (simulant E).

Production of bio-nanocomposite films

PBSA and PHBV films containing 5 wt% C-30B (PBSA NCP and PHBV NCP) were synthetized by a melt extrusion process using an EuroLab 16XL co-rotating twin-screw TM , Germany) with a L/D ratio of 40 and a screw diameter of 16 mm as described previously (Lajarrige et al., 2019). The control samples of neat polymers were prepared following the same protocol used for nanocomposites in order to respect the same thermal processes.

Overall migration testing

Overall migration (OM) level, which represents the total amount of non-volatile substances transferred from the food-contact material to the food (EU 10/2011) was determined by a gravimetric method after the total immersion of uncontaminated samples in the selected food simulants based on the standardized testing conditions set out for long term storage (40°C for 10 days) in EU 10/2011. Measurements were performed exclusively in 3% acetic acid, 95% ethanol and isooctane considered as the most severe conditions. The samples were cut in disks of 2.5 cm diameter and then kept in a vacuum oven at 60°C for 2 days. The polymer disks with the total area of 60 cm 2 were weighed using a microbalance (0.1mg resolution) prior to the migration test and then placed in a 100 mL migration cell (DURAN® screw thread tubes) equipped with glass rod, glass spacers and PTFE-sealed caps. Before filling, the migration cells were blown out with nitrogen to prevent any dust contamination. The bottles were then filled with the respective simulants, sealed and stored in a 40°C oven. Each sample/simulant combination was prepared in triplicate. After 10 days of contact, the bottles were ultrasonicated for 2 min to detach any potential particles from the sample surface. The samples were then removed and placed in a vacuum oven at 60°C for 2 days. After evaporation of the simulant, the specimens were weighed again and eventually overall migration was calculated in milligrams per square decimeter of the surface area of sample.

Relative migration testing

Spiking and desorption

The films were first contaminated by the array of additives with a theoretical additive amount of 500 mg.kg -1 for each of them. The methods used to contaminate materials were described previously (Lajarrige et al., 2019). Then, for liquid food simulants, between 0.5 g and 1 g of contaminated materials were immersed in 100 mL of food simulants in a surface/volume ratio of 6 dm².L -1 at 40°C for 10 days under magnetic agitation. At the end of the contact time, an aliquot of food simulant was collected and analyzed by chromatography (GC-FID for low molar mass additives or HPLC for high molar mass additives). For solid food simulant, a film of 3 × 2 cm was placed into Petri dish and 0.24 g of Tenax (4 g.dm -1 ) was distributed over the film to cover it completely. The Petri dish was closed and stored at 40°C for 10 days. At the end of the contact time, the Tenax was transferred into a test tube and two successive extractions of 24h with 5 mL of n-hexane were performed. The whole extract was filtered and analyzed by GC-FID. The relative migration represents the percentage of an additive that migrates from polymer into food simulant. It was calculated by dividing the mass of additive recovered in the food simulant after 10 days of contact by the initial mass of additive in the film sample.

Quantification of migration

The concentration of additives in food simulants was determine using chromatography.

Solutions containing low molar mass additives were analyzed using gas chromatography with flame ionization detector (GC-FID) while solutions containing high molar mass additives were analyzed using high performance liquid chromatography (HPLC).

For GC-FID analysis, an Agilent 7890A GC equipped with a 7693A Automatic Sampler

The components were separated using a HP-5 (5%phenyl)methylpolysiloxane capillary column of 32 mm ID and 30 m length. The thermal protocol used for the oven was as follows: an initial temperature of 40°C maintained for 5 min, heated at a rate of 6°C.min -1 to 270°C, maintained for 15 min. The injector temperature was 250°C. An external calibration was made in each food simulant in the range of 1.25 to 40 mg.L -1 .

For HPLC analysis, an Alliance HPLC system (Waters) equipped with an Alltima C18 column (250 mm × 2.1 mm, 5--of the values are out of consideration as far as they are significantly below the authorized threshold. The influence of the nature of the simulant is also remarkable with, in particular, a detrimental effect linked to contact with ethanol with all of film samples.

Contact with ethanol induced a significantly higher migration extent for both materials and can be considered as the "worst case" condition. The high sensitivity for ethanol was previously pointed out on many other biodegradable and non-biodegradable polyesters such as PET (Begley and Hollifield, 1990;Widén et al., 2004;Kim and Lee, 2012), PLA (Jamshidian et al., 2012) and PHBV (Chea et al., 2015). The reason generally given for this increase in the overall migration value is that the polyester chains are shortened by a transesterification process, the short chains thus released being likely to be released into the food (or simulant).

Regarding the impact of nanoclays addition, nanocomposite materials are characterized by an overall migration rate higher than that of virgin polymer. This trend is respected for PBSA and PHBV based-materials. The low mobility of nanoclays in the polymeric matrix (already mentioned above) suggests that this increase is not related to the release of the nanoparticles themselves but rather to an indirect effect that they may produce on the material structure. These modifications can occur during the heat treatment of the polymer [START_REF] Annamalai | Can clay nanoparticles accelerate the environmental biodegradation of polyolefins?[END_REF] or during the contact of the film with the food simulant (by enhancing the sorption of the food simulant). In any case, even if the presence of nanoclays tends to increase the overall migration, these values remain very close to those measured on the standard film and do not question the suitability for food contact of the materials any way.

Relative migration of additives from films into food simulants

The relative migration of additives from neat and nanocomposite biopolyesters after 10 days of contact at 40°C with 3% acetic acid, 10% ethanol, 20% ethanol, 50% ethanol, 95% The results of the relative migration appear to be highly dependent on the food simulant in contact with the materials with an apparent activation of the migration induced by the hydrophobicity of the solvent in contact. Contact with 3% acetic acid produced low migration values for low molar mass additives and no migration was detected for high molar mass additives. In contrast, contact with fatty simulants induces the release of almost all additives regardless of their nature, molar mass or polarity. Indeed, contact with 95% ethanol and isooctane produced migration values higher than 80% or even 90%, except for high molar mass additives added in PHBV and PHBV NCP in contact with isooctane. Contact with Tenax produced also, contrary to what was expected, high migration values for volatile additives.

However, similar migration values were obtained for benzophenone and DEHA additives in polyethylene [START_REF] Rubio | The behaviour of Tenax as food simulant in the migration of polymer additives from food contact materials by means of gas chromatography/mass spectrometry and PARAFAC[END_REF]. For high molar mass additives, no migration in Tenax was detected, except a small percentage of Irganox 1076 from PBSA and PBSA NCP materials.

Regarding the results obtained with ethanolic food simulants, the higher the percentage of ethanol, the higher the migration values. As mentioned above, ethanol probably induces structural changes in materials that may alter their inertial properties but these results can be additionally explained by the affinity of the additives towards the food simulants. The migration of additives from a polymer into a food simulant breaks down into two phenomena: (1) the diffusion of additives (kinetic parameter) through the polymer matrix and (2) the desorption of additives (thermodynamic parameter) from the polymer surface into the food simulant [START_REF] Ferrara | Diffusion coefficient and activation energy of Irganox 1010 in poly(propylene-co-ethylene) copolymers[END_REF]. The kinetic parameter can be estimated by the determination of the diffusion coefficient while the thermodynamic parameter, which reflects the affinity of the diffusing substance for each of the compartment is expressed by the partition coefficient (i.e. the ratio between the concentration of solute in polymer matrix and the concentration of solute in food or food simulant). Therefore and in the case of low-barrier polymers, the migration level evaluated under the standard conditions imposed by the Regulation (EC) No 10/2011 (after 10 days of contact at 40°C) is measured in an equilibrium state where the diffusivity does not impact the migration extent. In the specific case of PBSA and PHBV based material, a previous study (Lajarrige et al., 2019) demonstrated that the equilibrium state is reached after only few hours of contact regardless of the tested food simulant. The migration values obtained in these experiments therefore reflect the partition coefficient of each additive between the two compartments (packaging material/food simulating liquid) and a high partition coefficient indicates a low solubility of additive in the food simulant. This statement explains why migration levels decrease with the polarity of solvents used as simulants and is in agreement with the already widely established finding on synthetic polymers (Nasiri et al., 2017).

This statement additionally explains why the polymer type have a little impact on the relative migration even if the values obtained for PHBV are significantly higher than for PBSA in the case of contact with less "aggressive" simulants (3% acetic acid and 10% ethanol). These results confirm that even if PHBV has good barrier properties compared to PBSA (Crétois et al., 2014;Charlon et al., 2015), the affinity between the additives with the two phases, i.e.

biopolyester and food simulant is predominant.

In this way, it is surprising to point out a dependence of the molar mass of additives on the relative migration particularly observed in 10%, 20% and 50% ethanol. The dependence of the diffusion coefficient on the size has well been demonstrated (Reynier et al., 2001;[START_REF] Martinez-Lopez | Practical identifiability analysis for the characterization of mass transport properties in migration tests[END_REF]Lajarrige et al., 2019) and could appear in contradiction with the fact that the relative migration of additives is exclusively impacted by the affinity of the components for the food simulant without any influence of the diffusion properties.

Concerning the effect of inorganic nanoclays, it is easily accepted that their incorporation into a packaging film is associated with a positive effect on migration, particularly because of the tortuous effect they bring [START_REF] Yano | Synthesis and properties of polyimide-clay hybrid[END_REF]. However, this assumption applies to packaging based on high-barrier polymers for which transport kinetics directly impact migration. In the case of PHBV-based materials for which the level of migration is more directly related to thermodynamic consideration, the presence of nanoclay is likely to modify the affinity of additives for the packaging film and, on this basis, cannot be considered as systematically beneficial. The results in Figure IV.2 show the minor influence of the presence of the nanoclays on migration values. Detailed analysis of the results independently for each compound does not make it possible to identify any constitutive relation (or generic behavior law) allowing to describe the effects of the nano-fillers on the migration extent. When their impact is significant, it induces, most of the time, a decrease of the migration of additives. However, it is important to note that, in some cases and particularly for high molar mass additives, the presence of clays induces a significant increase in migration. These contrasting effects, as weak as they are, raise questions about their impact on the consumer's level of exposure.

Consumer exposure

The consumer exposure results from the combination of the food consumption data with the contamination level of the food. In the case of contaminants from FCM, the concentration of contaminant corresponds to the migration level of substances approved for food contact in the relevant safety regulations with associated specific migration limits (SML) or not. In food is basically assessed by the equation:

(IV.4)

Where: Ci is the consumption rate of a particular food i, Pi, the relative packaging usage of a given FCM for a particular food i, Mi, the migration rate from a given FCM into a particular food i and Cn, the consumption rate of foodstuffs considered for the exposure estimation.

As consequence, the risk assessment related to the use of the evaluated packaging material is based on the comparison of the level of exposure with the available toxicological data and, more particularly, with the tolerated exposure thresholds for each contaminant (i.e. additives).

The tolerated consumer exposure to selected additives determined according to Regulation is reported in Table IV.2. The tolerated consumer exposure depends on additives since it is set according to toxicological data in a way that there is no adverse effect on the consumer health. That is why, benzophenone and Uvitex OB have a low tolerated exposure value compared to methyl salicylate. Moreover, since the SML is expressed in mgadditive.kg -1 food and that the amount of food consumed daily is higher for adults, the tolerated consumer exposure values are significantly higher for adults compared to children.

The assessment of exposure related to the use of new packaging not yet placed on the market must also be based on a priori projections (or prospects) of market share. In terms of target market, it is now difficult to predict the level of deployment of the new biomaterials selected. While no restrictions are, to our knowledge, in force about the ways and conditions of use of PBSA-based materials, a recent opinion published by EFSA emphasizes that PHBH is not of safety concern for the consumer if it is used either alone or blended with other polymers in contact with all kinds of food, under contact conditions of more than 6 months at room temperature or below, including hot-fill or a short heating up phase.

Following a precautionary principle, the current evaluation is deliberately based on three distinct scenarios, all three being part of an overestimated risk assessment:

(1) All consumed food and beverage would be packed either in standard biopolyesters (PBSA/PHBV) or in the corresponding bio-nanocomposites (PBSA NCP/PHBV NCP).

(2) All the foods except beverages would be packed in standard biopolyesters (PBSA/PHBV) or in the bio-nanocomposite materials (PBSA NCP/PHBV NCP).

(3) The foods consumed are packed in standard biopolyesters (PBSA/PHBV) or in the bionanocomposite materials (PBSA NCP/PHBV NCP) only when it is realistic according to foreseeable usage i.e. for dry foods, dairy products, meat products, fish products or low hydrated solid foods.

The foods considered in each scenario are gathered in Annex II.

This approach aims to establish, on the basis of a comparative measure, whether the incorporation of nano-fillers is likely to generate new risks for the consumer regardless of exposure to the nanoparticles themselves (not considered in this study).

The level of consumer exposure to each additive established according to the "worst case" scenario (1) was presented in Table IV.3. The consumer exposure to methyl salicylate, DEHA, Uvitex OB and Irganox 1076 additives are lower, regardless of the age range and the material used, to the limit calculated on the basis of SML specified in the Regulation (Table IV.2). On the other hand, the exposure to benzophenone is higher than this health-based limit in all cases.

It is recognized that this value remains of little concern since it depends on the initial concentration of the materials arbitrarily set at 500 ppm for each additive for this study. In fact, this additive has a low specific migration limit and a high migration in food simulants, especially in 50% ethanol, 95% ethanol, isooctane and Tenax.

The consumer exposure is similar whether PBSA or PHBV is used for the packaging.

However, it is important to note that the addition of nanoclays affects differently the consumer exposure as a function of the biopolyester since the exposure level increases when nanoclays are added in PBSA while it decreases when they are added in PHBV. The incorporation of nano-fillers is far from negligible here, since it induces a change in the exposure level that can exceed 20%. values decrease as a result of reduced consumption. In this specific case, the consumer exposure to additives largely decreased and is now below the tolerated threshold in some cases, especially for adults. Moreover, it is interesting to note that, the addition of nanoclays in the PHBV reduces the exposure level below the acceptable threshold for people aged 11-17 and children.

Considering that the foods discarded from this scenario must be packaged in alternative nonnano packaging, the impact of nanoclays becomes minor here and helps to put into perspective the level of concern that must be raised on this issue. This latter scenario, although referred to "more realistic" perspective, still implies a significantly overestimated level of exposure. Even under these conditions, it is clear that the influence of the nano-fillers does not call into question, on this issue alone, the ways in which bio-nanocomposite materials could be applied in the field of food packaging.

Conclusion

The packaging market is set to undergo major changes and it is still difficult to predict which packaging solution will be favoured in the future to replace plastic materials. Based on the hypothesis of market conversion to bio-nanocomposite packaging, this work provides a first overview on the impact of the incorporation of nanoclays in term of consumer exposure to food packaging additives/contaminants. Without major effect in most cases, the addition of nanoclays can, however, in some cases prove to be harmful and thus slightly increase the level of migration of certain additives or contaminants. This increase is explained by a decrease in the affinity of the molecules for the nanocomposite packaging film, which remains difficult to predict. In view of the results, the influence of nanoclays may seem minor in comparison to the predominant effect of the nature of the food simulant and does not seem to be such as to call into question the food contact properties of the materials but may not be negligible in terms of consumer exposure according to the "worst case" scenario investigated in this current study. It is understood that the exposure levels considered in this study are largely overestimated in view of the still minor growth of nanocomposite materials on the packaging market. As such, the incorporation of nanoclays into packaging materials considered in this study may seem to be without safety concern. Nevertheless, this study highlights all the difficulties in predicting the behaviour of nanocomposite materials, especially when they involve biodegradable polyesters. Packaging is essential to protect foods from deterioration induced by the external environment (Barnes et al., 2007). Combined with modified atmosphere, packaging also helps to prolong the shelf life of a food product and also contributes to improve food safety. Modified atmosphere packaging (MAP) is the removal and/or replacement of the atmosphere in the headspace of the packaging system surrounding the product. Such strategy aims to slow down or totally inhibit microbial development and can also modify the microbial ecology of the product [START_REF] Gill | Active packaging in practice: meat[END_REF][START_REF] Mcmillin | Where is MAP going? A review and future potential of modified atmosphere packaging for meat[END_REF][START_REF] Guillard | Validation of a predictive model coupling gas transfer and microbial growth in fresh food packed under modified atmosphere[END_REF]. The three main gases used in MAP are nitrogen (N2), oxygen (O2), and carbon dioxide (CO2) whose effects on microbial development can be combined. N2 is an inert and tasteless gas used to displace oxygen and prevent package collapse, O2 inhibits the growth of anaerobic microorganisms and CO2 has a bacteriostatic effect, and it slows down the respiration of many products [START_REF] Floros | 10 -Introduction to modified atmosphere packaging[END_REF]. However, the modified atmosphere within the package change over time during storage due to (i) reactions between the food and the gas of the atmosphere and (ii) mass transfer of gases through the packaging. The main objective of the packaging material is thus to maintain as much as possible this atmosphere during food storage.

The massive consumption of plastics in packaging applications [START_REF] Plasticseurope | Plastics -the Facts[END_REF] results in a constant accumulation of plastic wastes in the environment. In face of growing environmental concerns due to the production and consumption of plastics, alternative materials including materials based on bio-sourced and biodegradable polymers appear as promising solutions (Tharanathan, 2003). However, to make them competitive towards conventional polymers, the addition of reinforcing fillers is increasingly being considered to improve their performances especially in term of barrier properties. Due to their availability, low cost and efficiency to significantly enhance barrier properties at very low loadings (1-10 wt%) (Sorrentino et al., 2007) layered silicates are the widest commercial application of reinforcing fillers (Silvestre et al., 2011).

Carbon dioxide permeability

The CO2 permeability of films was determined in triplicate at 23°C and 42 % of relative humidity through an isostatic and dynamic method using gas phase chromatography. The film samples were placed inside sealed stainless steel permeability cells. The lower and upper chambers were each spread by a 5 mL.min -1 flux of permeant gas (CO2) and vector gas (helium), respectively. The flow rates were controlled and monitored by digital flowmeters (Bronkhorst, Germany). The permeability cells were coupled to a gas chromatograph with thermal conductivity detector (GC-TCD, Agilent 7890A, USA), equipped with an automatic multiposition injection valve (Vici, Valco Instruments). This enabled to sequentially analyze online the evolution of CO2 gas concentration in the upper chamber (crossing the film from the inferior chamber). The GC-TCD was equipped with two capillary columns -PoraPlot U (25 m × 0.530 mm id, Agilent) and HP Molesieve (30 m × 0.535 mm id, Agilent) -able to separate CO2, O2 and N2. Data were collected and processed using the ChemStation OpenLab Software (Agilent) and SRA Prochem interface (SRA Instruments). The CO2 permeability, PCO2

(mol.m/(m 2 .s.Pa)), of the material was determined following the Eq. IV.5.

Modelling of gas transfer in the food/packaging system

The mathematical model simulates the evolution of O2 and CO2 concentrations in the headspace of a packaging material composed of a tray and a lid film. The gas exchanges include solubilisation into the food, desorption from the food and permeation through the packaging material. The mathematical model used was previously described [START_REF] Chaix | Mechanistic model coupling gas exchange dynamics and Listeria monocytogenes growth in modified atmosphere packaging of non respiring food[END_REF][START_REF] Guillard | Validation of a predictive model coupling gas transfer and microbial growth in fresh food packed under modified atmosphere[END_REF][START_REF] Guillard | MAP--making in the field of modified atmosphere packaging of fresh non respiring foods[END_REF]. The variations of concentration of gaseous species j (O2, CO2, N2) in the headspace (HS) depends on (i) the mass flow (kg.s -1 ) of species j occurring through the lid film from the surrounding atmosphere to the headspace and on (ii) the mass flow (kg.s -1 ) of species j at the interface between the food sample and the headspace. Assuming that the lid film does not oppose any mechanical resistance to deformation, when the amount of gas species in the headspace varies, the headspace volume VHS changes whereas the total pressure in the headspace PT remains constant. The global mass balance of species j in the headspace can be expressed as followed:

(IV.6)

Where stands for the concentration of gas species j in the headspace (kg.m -3 ).

Chapter IV. Estimation of risks-benefits of adding nanoclays in biodegradable polymer matrices

The gas mixture in the headspace is assumed to obey the ideal gas law and the total pressure in the headspace PT is assumed to remain constant during conservation and equal to atmospheric pressure, which is summarized in the following equation.

(IV.7)

Therefore, headspace volume variation, , can be calculated as the sum of the different aforementioned mass flow and as follows:

(IV.8)

Where R is the ideal gas law constant (=8314 J.mol -1 .K -1 ), T is the temperature (K) and , and are the molar masses (kg.mol -1 ) for O2, CO2 and N2, respectively.

The work of [START_REF] Chaix | Mechanistic model coupling gas exchange dynamics and Listeria monocytogenes growth in modified atmosphere packaging of non respiring food[END_REF] gives more details about the expression of and and about the solubilisation/diffusion of O2 and CO2 within the product.

An oxygen-sensitive product, fresh poultry meat, having short shelf life was selected for this study. It is usually packed in modified atmosphere packaging (MAP) without O2 and with a binary mixture of CO2 and N2 to prolong its short shelf life. A zero O2 concentration is fixed to prevent aerobic microbial growth. In the same way, CO2 is injected in headspace to inhibit the growth of microorganisms. Nitrogen is used as filling gas. Four different materials for the tray were used, PBSA, PHBV and their corresponding nanocomposite containing 5 wt% C-30B. The simulations were also performed using polypropylene (PP), used in commercial food packaging for the formulation of trays, to compare their performances. Moreover, whatever the material used for the tray, the commercial lid film was kept because of its high barrier properties. The input parameters used for the simulations are gathered in Table IV.9.

Table IV.9: Input parameters used for the simulation of the evolution of gas in headspace.

Food case

Fresh poultry Food Mass (g) 225 Thickness (µm) 1 Area (cm²) 200 O2 diffusivity (m².s -1 ) 1.14x10 -9 (20°C) CO2 diffusivity (m².s -1 ) 0.57x10 -9 (20°C) O2 solubility (mol.m -3 .Pa -1 ) 1.33x10 -8 (20°C) CO2 solubility (mol.m -3 .Pa -1 ) 5.70x10 -7 (20°C)

Packaging tray Lenght (cm) 21.5 Width (cm) 13 Height (cm) 2.7 Volume (mL) 600 Thickness (µm) 400

Packaging film Thickness (µm) 30 O2 permeability (mol.m -1 .s -1 .Pa -1 ) 7.99x10 -18 (23°C) CO2 permeability (mol.m -1 .s -1 .Pa -1 ) 1.60x10 -17 (23°C) Headspace % O2 initial 0 % CO2 initial 70 % N2 initial 30

Storage conditions

Temperature (°C) 4 Time (days) 7

Global warming assessment

The packaging system selected as case study is a tray equated to a parallelepiped rectangle whose dimensions are mentioned in Table IV.9, and for which the volume of material is of 18.6 cm 3 . The volume of the material was thereby kept equal for all materials. Due to the intrinsic density of the considered materials, the final weight of the trays varies according to the nature and the proportion of each constituent (Table IV.10).

The scenarios included in this study were trays made of PP (reference material), neat PHBV and PHBV reinforced with 5 wt% of C-30B. PBSA and PBSA NCP were not considered for this study due to their non-competitiveness regarding gas transfers, compared to reference material.

The main properties of the raw materials are presented in Table IV.10 corresponding respectively to PP (PPH9020 from Total Petrochemical), PHBV and C-30B (as described in Section 2.1). [START_REF] Yates | Life cycle assessments of biodegradable, commercial biopolymers -A critical review[END_REF]. For this study, global warming impact value for PHBV was found in the work of David et al., (2019) who used inventory for PHBV made from sugar cane obtained from the work of [START_REF] Harding | Environmental analysis of plastic production processes: Comparing petroleum-based polypropylene and polyethylene with biologically-based poly-Bhydroxybutyric acid using life cycle analysis[END_REF]. The global warming impact related to poultry was recovered from Agribalyse database provided by the French Environment and Energy Management Agency (ADEME) and calculated according to the ReCiPe method. The reported value is weighted average including organic sector and conventional industry. The benefits, at the level of global warming impact, due to substitution of PP with PHBV and to the addition of nanoclays in PHBV were estimated by (i) a reduction in food losses (fresh poultry) at the distributor or consumer level and (ii) a reduction of the amount of material required to fulfil the function.

Results and discussion

Mass transfer characterization

Oxygen and carbon dioxide permeabilities, needful for the modelling of gas transfers in the food/packaging system, were determined for neat and nanocomposite PBSA and PHBV based materials and are summarized in Table IV.11. As expected, the addition of nanoclay decreases the gas permeability, except in the case of CO2 for PHBV for which no significant difference was measured.

The results of O2 and CO2 permeabilities obtained for PBSA and PBSA NCP are in good agreement with that found in another study conducted on the same material (Charlon et al., 2015). For PHBV and PHBV NCP, the permeability values are comparable to those found in the literature but the CO2/O2 selectivity is half as high (Crétois et al., 2014).

Concerning the impact of the addition of nanoclay, the decrease of the O2 permeability of 27.3% for PBSA and 76.9% for PHBV are similar to the results reported in previous studies.

For PBSA reinforced with 5 wt% hectorite, Dean et al., (2009) found a decrease of the O2 permeability from 38% to 55% depending on the organo-modified nanoclay used. Charlon et al., (2015) observed a decrease of O2 permeability from 20% to 51% depending on the nanoclay type and the processing method used for the production of films. For PHBV, a decrease of 32% was evidenced with the addition of 5 wt% of nanoclays [START_REF] Sanchez-Garcia | Novel clay-based nanobiocomposites of biopolyesters with synergistic barrier to UV light, gas, and vapour[END_REF].

For CO2 permeability, the decrease of 41.1% for PBSA is in agreement with the improvements of 39% and 52% previously obtained for PBSA/C-30B nanocomposite depending of the processing method (melt mixing or melt mixing coupled to water injection respectively) (Charlon et al., 2015). On the other hand, the absence of any effect induced by the presence of nanoclays on CO2 permeability of PHBV material is consistent with a previous study conducted on the same material demonstrating also a non-conventional behavior of the addition of nanoclays with an increase of the permeability following an increase of the loading rate (Crétois et al., 2014). This behavior, which is sometimes observed, can be due to the dispersion and orientation of the nanoclay platelets in the polymer matrix and to the quality of the polymer/nanoclays interface.

The difference in the impacts produced by the addition of nanoclay in the two materials suggests a difference in the level of their dispersion/exfoliation in the two polymeric matrices but can also be explained by a different impact on the level of crystallinity of the polyesters [START_REF] Yano | Synthesis and properties of polyimide-clay hybrid[END_REF]. PBSA NCP 1.18×10 -16 ± 0.01×10 -16 1.55×10 -15 ± 0.17×10 -15 PHBV 5.73×10 -17 ± 2.06×10 -17 6.93×10 -17 ± 0.89×10 -17 PHBV NCP 1.32×10 -17 ± 0.57×10 -17 7.21×10 -17 ± 0.67×10 -17 PP 7.49×10 -17 1.09×10 -16

Analysis of model simulation

The evolution of O2 and CO2 in the headspace of a tray containing fresh poultry meat and made with different materials was studied over storage time, knowing that the O2 recovery must be avoided and CO2 concentration must remain high enough.

Chapter IV. Estimation of risks-benefits of adding nanoclays in biodegradable polymer matrices Poultry meat is known to be a fast perishable product, even preserved at low temperature [START_REF] Jiménez | Spoilage microflora in fresh chicken breast stored at 4°C: influence of packaging methods[END_REF] and MAP technology is often used at industrial scale to prolong the shelf life. Typically, poultry meat is packed in CO2/N2 atmosphere with low residual O2 concentration [START_REF] Höll | Identification and growth dynamics of meat spoilage microorganisms in modified atmosphere packaged poultry meat by MALDI-TOF MS[END_REF]. Moreover, for a longer shelf life extension, the CO2 concentration should be higher than 50% [START_REF] Sante | Effect of modified atmosphere packaging on color stability and on microbiology of Turkey breast meat[END_REF]. On this basis, the gas composition of the headspace is initially of 0 % O2, 70 % CO2 and 30 % N2 (Table IV.9).

The evolution of the gaseous composition in the headspace of the packaging is reported in The initial O2 and CO2 atmosphere conditions are rapidly modified during the storage, whatever the tray material used. The percentage of O2 increases rapidly to 0.8% during the first stage (1.25 days) for all materials and reaches after 7 days between 1.14% and 1.79% for the most barrier material (PHBV NCP) and the less permeable material (PBSA), respectively. The percentage of CO2 decreases rapidly from 70% to 45% during the first two days and reaches after 7 days between 43.3% and 41.8% for PHBV NCP and PBSA, respectively. These results can be explained by two concomitant phenomena i.e. (i) the sorption of CO2 and desorption of O2 at food-headspace interface and (ii) the gas permeation through the packaging.

Pseudomonas fluorescens is an aerobe microorganism that can grow in poultry meat. Its minimal concentration required for microbial growth, (% O2 min) is equal to 0.25% (expressed in % equivalent in headspace) and it is very sensitive to CO2 with a maximal inhibitory concentration (% CO2 max) equal to 40%. In the simulation, the % O2 min is reached during the first half-day regardless of the material used since the evolution of O2 is at this stage, mainly due to the desorption from the poultry into the headspace. Considering the reference material (PP), the O2 concentration in the headspace after 7 days is 1.36%, this value being considered as the oxygen content threshold reached at the expiry of the use-by date. On this basis, the most suitable tray is the one made with PHBV NCP since it maintains the lower O2 concentration. This percentage of 1.36% is reached after 9.9 days and 11.4 days of storage by using PHBV or PHBV NCP as tray material respectively. Assuming that product loss is proportional to its shelf life (this hypothesis having never being specifically studied on the fresh poultry), the use of PHBV allows to increase the food shelf life of 2.9 days compared to PP and the addition of nanoclays in PHBV allows to prolong the shelf life of 1.5 days more. In comparison, the performances of neat and nanocomposite PBSA in term of O2 are lower than PP material, reaching 1.60% and 1.79% respectively. With respect to the threshold of O2 content, the the losses of 4.1% and avoid the waste of 4.6 trays/person/year. The global warming impact decreases at 124.90 kg CO2eq/person/year.

Substitution of PHBV with PHBV NCP-based packaging

The addition of 5 wt% C-30B in PHBV allows to increase the shelf life of fresh poultry by 1.5 days as reported in Section 3.2, which represents an increase of 15% compared to neat PHBV. At the distributor level, as for the substitution of PP with PHBV, the waste of 4.2 trays/person/year is avoided, which corresponds to a global warming impact of 125.49 kg CO2eq/person/year. At the consumer level, it allows to waste 1.5 trays less than with the PHBV, the global warming impact decreases at 123.02 kg CO2eq/person/year. Table IV.12 summarizes the global warming impact obtained for each case studies. The use of PHBV NCP could decrease the impact to a greater extent compared to PHBV, except in the scenario of a reduction of losses at the distributor level since the impact of a tray of PHBV NCP is slightly higher compared to that of PHBV due to its higher weight. Moreover, it seems that the reduction of the losses at the consumer level is the best option to decrease the CO2 footprint. In fact, a decrease of 2.8% is achieved by replacing PP with PHBV NCP. Despite the low contribution of packaging on the overall carbon footprint, the material savings are also an effective way to decrease the global warming impact of the food-packaging system. However, it is important to keep in mind that this scenario must be validated on the basis of the packaging specifications, which are also based on technical considerations (machinability, production rate, mechanical resistance to use, etc.). The substitution of conventional non-degradable polymers by bio-sourced and biodegradable polymers, such as PHBV, proved to be an interesting alternative for the packaging of oxygen-sensitive products such as fresh poultry meat. The addition of reinforcing nanofillers in this matrix further increases the performances of PHBV. However, this biopolyester matrix which is supposed to be eco-friendly, displayed higher environmental impacts than petrochemical PP because of its higher density and lower level of technological development. Due to the higher barrier properties of PHBV, two distinct alternatives may be considered to reduce their impact. Among the possible scenario, the most efficient way to scale back the global warming impact of packaging trays containing fresh poultry is the reduction of the losses and waste at the consumer level resulting from the increase of the product shelf life.

Moreover, as the permeability of PHBV is largely lower compared to PP, the reduction of raw material used to formulate the packaging allows a material economy resulting in 1.7% decrease of the CO2 footprint, this option being subject to validation of its applicability on an industrial scale. The addition of nanoclay in the biodegradable matrix reinforces the decrease of the impact. These preliminary results must be analyzed with regard to their limit of application especially since LCA tools cannot, up to now, anticipate the long-term effects of plastic accumulation in the environment, which represents a major environmental benefit of biodegradable materials for which the process of production is going to positively evolve in the foreseeable future. In addition, the approach proposed in this study leads to considering the addition of nanoclay as an environmentally beneficial strategy. However, it is well known that the incorporation of nanoparticles into packaging raises many questions, in particular about their influence on the biodegradability of the material, their fate and persistence in the environment after the degradation process and their subsequent impact on ecosystems. These questions will inevitably have to be placed in perspective for future more comprehensive studies on this issue.

Conclusion

Le marché de l'emballage alimentaire subit des changements majeurs avec notamment le remplacement progressif des matériaux plastiques conventionnels par des matériaux biodégradables entrainant l'incorporation de renforts dans ces matrices.

Basé sur une conversion du marché vers des emballages alimentaires bio-nanocomposites, ce travail fournit un aperçu de l'impact de l'incorporation de nano-argiles en terme d'exposition du consommateur à un panel d'additifs. Il s'agit de molécules modèles représentatives de celles utilisées dans les emballages alimentaires mais qui pourraient être potentiellement remplacées par des molécules avec une toxicité plus faible. L'ajout des nano-argiles n'induit pas d'effet majeur dans la plupart des cas mais il faut cependant être prudent sur les quantités d'additifs utilisées pour ne pas dépasser la dose journalière tolérée. Une évaluation des risques au cas par cas reste donc recommandée jusqu'à ce que plus d'informations soient disponibles.

Concernant l'impact de ces renforts sur les propriétés barrières des matériaux, une diminution des perméabilités à l'oxygène et au dioxyde de carbone a été mesurée. Afin de quantifier ce bénéfice, une augmentation de durée de vie dans un cas d'étude réel (poulet) a été calculée. Les matériaux de PBSA pur et nanocomposite se sont montrés inadaptés à l'emballage de ce type de produits sensibles à l'oxygène, par rapport au matériau de référence. En revanche, le PHBV et d'autant plus le PHBV nanocomposite, dans lequel l'ajout de nano-argiles a permis de diminuer la perméabilité à l'oxygène de 77% se sont révélés concurrentiels vis-à-vis de leur benchmark. Un gain de durée de vie de 2.9 jours en utilisant le PHBV et de 1.5 jours supplémentaires en utilisant le PHBV NCP a été obtenu, permettant d'envisager une réduction des pertes alimentaires. La diminution des perméabilités aux gaz peut également conduire à diminuer la quantité de matière nécessaire pour formuler le matériau et atteindre la perméabilité du matériau pur ou du benchmark. L'étude d'impact environnemental de l'ensemble des cas de figure envisagés a montré qu'une diminution des pertes au niveau du consommateur permet d'obtenir le gain le plus important suivi de près par la diminution de la quantité de matériau, qui permet également de faire diminuer le prix d'une barquette. Cependant, pour ce dernier scénario, il est important de s'assurer de l'usinabilité et de la résistance mécanique de telles barquettes avec de faibles épaisseurs. Since their first appearance in the 1950 , the production of plastic materials continues to grow, the packaging application being the largest plastic market with nearly 40% of the demand in 2017 in Europe (PlasticsEurope, 2018). This growing plastic consumption leads to an exponential accumulation of these non-biodegradable materials in the environment and the packaging sector has become the primary waste producer with more than half of plastic wastes (Geyer et al., 2017). To face the environmental issues, alternatives are studied to substitute these materials with biodegradable plastics potentially derived from renewable feedstocks. In 2018, 0.91 Mt of biodegradable polymers were produced which represents only 0.3% of the total worldwide plastics production. However, the demand for more sophisticated biopolymers, applications, and products leads the market to rise continuously (Source: European Bioplastics 2018).

Among the biodegradable available plastics, aliphatic polyesters such as poly(butylene succinate-co-butylene adipate) (PBSA) and poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) appear as promising polymers to replace conventional polymers for food packaging applications because of their biodegradability in accordance with the standard in force NF EN 13432 dedicated to biodegradation in compost medium. However, after use, a large quantity of plastic materials is not collected to be treated and the study of their biodegradability in soil medium is of great interest. The biodegradability of plastic material in soil is evaluated according to NF EN 17033: 2018 standard. The global production of biodegradable polymers now represents 10.6% for PBSs and 3.2% for PHAs of the production of all biodegradable polymers (Source: European Bioplastics 2018).

In addition to the fact that the thermal, mechanical or optical properties of these two polymers do not always meet the requirements of packaging applications, their barrier properties are still weak compared to synthetic polymers and their high sensitivity to water acts as a brake on their emergence on the market. Among the strategies deployed to improve the performance of such materials and to make them more competitive, their blend with nanomaterials like nanoclays is an effective alternative. Many authors have studied the improvement of barrier properties due to the strengthening of polymers by nanoclays. Different biodegradable polymers have been studied with this objective, starch [START_REF] Park | Preparation and properties of biodegradable thermoplastic starch/clay hybrids[END_REF]Tang et al., 2008;Boonprasith et al., 2013;Mohan et al., 2016), PLA [START_REF] Chang | Poly(lactic acid) nanocomposites with various organoclays. I. Thermomechanical properties, morphology, and gas permeability[END_REF]Koh et al., 2008;Tenn et al., 2013;[START_REF] Ortenzi | Evaluation of polymerization of L -lactide with silane-modified nanosilica and montmorillonite[END_REF]Bartel et al., 2017), PCL (Tortora et al., 2002;Gain et al., 2004;Luduena et al., 2013), PBS/PBSA [START_REF] Okamoto | New poly(butylene succinate)/layered silicate nanocomposites. II. Effect of organically modified layered silicates on structure, properties, melt rheology, and biodegradability[END_REF]Dean et al., 2009;Charlon et al., 2015), PHAs (Crétois et al., 2014;Farmahini-Farahani et al., 2017;Akin and Tihminlioglu, 2018).

These expected benefits promote the development of bio-nanocomposite materials, which is seen as a promising way, combining profit in terms of environmental impact and maintaining the functional quality of packaging. However, the environmental impacts of incorporating inorganic nanoparticles (NPs) into a biodegradable packaging matrix are difficult to assess. The biodegradability of bio-nanocomposite packaging therefore raises two fundamental questions, i.e. (1) what is the influence of the NPs on the stability of biomaterials and consequently on their degree and rate of degradation?; (2) what is the fate of NPs resulting from these materials in the natural environment and how they impact the soil? The first question has been a concern in the packaging community for several years and the few works investigating the influence of NPs on material biodegradability have highlighted complex and contrasted effects which involve two main mechanisms (Maiti et al., 2003;Wang et al., 2005b;Steeves et al., 2007;Iggui et al., 2015):

(1) The nucleating effect of inorganic nanomaterials could result in crystallinity variation of the polymer (Luduena et al., 2013) with consequences on the capacity and the kinetics of water sorption in materials and subsequent hydrolysis of polymers [START_REF] Massardier-Nageotte | Aerobic and anaerobic biodegradability of polymer films and physico-chemical characterization[END_REF].

(2) The presence of such non permeable reinforcement can hinder the access of the microorganisms to attack the chemical bonds of the polymer (Fukushima et al., 2010).

These combined effects are expected to have a significant impact on the inertia of the materials and consequently on their degradability. A good understanding of these phenomena is therefore, once again, a key step in the design of nanomaterials that would be safe for the consumer and the environment. The aim of the present work was to evaluate the influence of the addition of organo-modified montmorillonite, Cloisite 30B (C-30B) on the end-of-life of two distinct biodegradable polymers: PBSA and PHBV. Considering that after use, a part of these materials will be composted in industrial conditions and the other part will be abandoned in the environment, two distinct scenarios were investigated. In this way, (i) biodegradation tests in soil were performed based on methodology described in ISO/FDIS 17556: 2012 with, in parallel, (ii) disintegration tests under composting conditions following the ISO 20200: 2015 (disintegration at lab-scale) and ISO 16929: 2013 (disintegration at pilot-scale). The composts resulting from the disintegration of both materials were then used to amend a soil in which winter forage rapeseed were sown. The ecotoxic effect which was poorly investigated on these two biodegradable polymers (Steeves et al., 2007;Balaguer et al., 2016) was evaluated on the plant, soil and microbiota as proxy.

Materials and Methods

Materials

Two different biodegradable polymers, the poly(butylene succinate-co-butylene adipate) (PBSA, PBE 001, density 1.24 g.cm -3 ) and the poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV, PHI 002, density 1.23 g.cm -3 ) were commercially procured from Natureplast, France.

The selected clay was a commercial organo-modified montmorillonite, the Cloisite 30B (C-30B) supplied by BYK additives & instruments, Germany.

Preparation and characterization of bio-nanocomposite materials

Materials in form of pellets and films with 5 wt% C-30B were produced by a melt extrusion process using an EuroLab 16XL co-rotating twin-TM , Germany) with a L/D ratio of 40 and a screw diameter of 16 mm as previously described (Lajarrige et al., 2019).

The film thickness was determined using a digimatic micrometer 0-25 mm (Mitutoyo, France).

The percentage of nanoclays in PBSA and PHBV was determined using Mettler TGA2 apparatus. Specimens of around 10 mg weighed using a microbalance (Sartorius micro PRO 11) were heated from 25°C to 800°C at a rate of 10°C.min -1 under air flow of 50 mL.min -1 . The percentage of nanoclay present in the film was calculated using the remaining residue (R) obtained at the end of the thermal cycle, for neat polymer, nanocomposite polymer and nanoclays according to the following equation:

(V.1)

The carbon content of neat PBSA and PHBV and their corresponding nanocomposite was determined using an Elementar Vario Micro Cube analyzer.

Evaluation of the biodegradability in soil

The aerobic biodegradability of PBSA, PHBV and their corresponding nanocomposites was evaluated under controlled soil conditions by respirometric tests according to ISO/FDIS 17556: 2012 as methodological standard and NF EN 17033: 2018 as specification standard.

The method measures the conversion of organic carbon in CO2 over time, this later being proportional to the percentage of biodegraded substrate. Microorganisms assimilate polymer chains and ultimately convert them aerobically into biomass, water and CO2.

Medium and chemical reagents

The soil used was a vegetal soil displaying the following characteristics: pH 6.8 (H2O), 2.3 wt% of organic matter, 16.85 wt% of clay, 26.85 wt% of lime and 56.3 wt% of sand. Barium chloride-dihydrat, sodium hydroxide, hydrochloric acid (Grosseron, France), thymolphtalein (Alfa Aesar, Germany), and microcrystalline cellulose (Merck, Germany) were used for biodegradation tests.

Start-up of the experiment

After manually removal of large inert objects, the soil was dried and sieved discarding 2 mm to obtain homogeneous medium.

The materials in form of pellets were cooled down in liquid nitrogen before being grinded into a powder of 500 nm using an Ultra Centrifugal Mill Retsch ZM-200.

The respirometric equipment consisted of a hermetic 1L glass reactor containing three flasks as previously described (Chevillard et al., 2012;Lammi et al., 2019). One of the flasks contained 25 g of dried soil mixed with 50 mg of equivalent carbon of the grounded samples.

The second flask contained distilled water to maintain a constant relative humidity of 100%.

The last flask contained a solution of 0.2 N hydroxide sodium (NaOH), which allows capturing the CO2 released by the soil microorganisms when degrading the material. The reactors were incubated in the dark at 28°C ± 2°C. Experiments included blank sample, which contained soil without material and control sample, which contained soil mixed with cellulose as positive reference material. According to ISO/FDIS 17556 methodological standard, the two following criteria should have been fulfilled to consider the test as valid: i) at least 60% biodegradation reached in less than 2 years for the positive reference and ii) the amount of carbon dioxide evolving from the three blank samples should be within 20 % of the mean at the plateau phase or at the end of the test. According to NF EN 17033: 2018 specification standard, the material is considered as biodegradable if its degree of biodegradation is at least equal to 90% in absolute, or relative to that of the positive reference, after less than 2 years of incubation. All samples were run in triplicate.

To evaluate the biodegradation, titrimetric assays of the NaOH solutions were performed over time to determine the CO2 released by microorganisms. For that purpose, 5 mL of a solution of barium chloride (20% w/v in distilled water) was added in the NaOH solutions before titration to ensure that all the Na2CO3 formed during incubation was precipitated. The excess of NaOH was then titrated back with 0.1N hydrochloric acid using thymolphtalein (0.1% prepared in 95% ethanol) as color indicator.

The percentage of biodegradation (%B) is calculated from the ratio of the cumulative amount of CO2 produced by the sample related to the maximal theoretical amount of CO2 that could be released (Eq.V.2).

(V.2)

With:

(V.3)
Where C, is the amount of carbon (mg) of the sample introduced in the soil, and , the molecular weight of carbon dioxide (44 g.mol -1 ) and carbon (12 g.mol -1 ), respectively.

After each dosage, a new NaOH solution (0.2 N) was placed in the hermetic glass reactor.

The flasks containing soil were weighted and the water content was adjusted to keep the soil at 80% of its water-holding capacity.

Finally, the experimental degradation data were modelled using Hill (for cellulose) and Boltzmann (for the tested materials) equations (Deroiné et al., 2015).

Evaluation of the ecotoxic effects

The ecotoxicity tests determined the effects on plants, soil and microbiota after exposition to composts in which PBSA, PHBV and their corresponding nanocomposite were degraded.

The materials were degraded under composting conditions following the ISO 20200: 2015 (disintegration at lab-scale) and ISO 16929: 2013 (disintegration at pilot-scale). The resulting composts were then used to amend a soil for the culture of a field crop plant. The biocompatibility of the residual nanoparticles was thus investigated using the response of the plant, soil and microbiota as proxy.

Reagents

Mature green compost was collected in the waste management center of Aspiran (France)

and sieved through 5 mm meshes.

Disintegration test

The method was already described by (Balaguer et al., 2016). 1 kg of the solid synthetic waste was introduced in polypropylene reactors, which were perforated to facilitate the gas transfer. 100 g of polymer, 90% (w/w) in powder form with a particle size of 500 µm and 10%

(w/w) in the form of 25 mm × 25 mm films were mixed with the solid synthetic waste. The reactors were closed and incubated in the dark at 58 ± 2°C during 90 days. Samples were run in triplicate. Over specific periods of time, water was added to maintain adequate moisture conditions and solid synthetic waste was carefully mixed to maintain aerobic conditions. After 90 days, the compost resulting was dried in open air and sieved through 5 mm and 2 mm sieves.

Residual film samples were recovered, cleaned to eliminate stuck compost and dried at 40°C until constant weight was reached. Then, the degree of disintegration, D was calculated (Eq.

V.4).

(V.4)

Where is the weight of dry material in pieces of 25 mm × 25 mm introduced initially and is the weight of the clean and dry fraction of films recovered after sieving.

Ecotoxicity test

This compost was then used to amend a low-carbon calcareous soil (25% compost) for the culture of a winter forage rapeseed. The biocompatibility of the residual nanoparticles was assessed using the response of the plant, soil and microbiota as proxy. On the plant side, the tests measured the immediate toxicity of the compost (germination) or the latent toxicity (growth inhibition), the shoot biomass, the number of leaves by plant and the pigment content (chlorophyll and carotenoids) in the shoots [START_REF] Wellburn | The spectral determination of chlorophylls a and b, as well as total carotenoids, using various solvents with spectrophotometers of different resolution[END_REF][START_REF] Khan | Effect of ellagic acid on growth and physiology of canola (Brassica napus L.) under saline conditions[END_REF][START_REF] Zhu | Physiological, biochemical, and molecular differences in chloroplast synthesis between leaf and corolla of cabbage (Brassica rapa L. var. chinensis) and rapeseed (Brassica napus L.)[END_REF]. On the soil side, the tests monitored the enzyme activity and the microbiota composition [START_REF] Hamidat | Design defines the effects of nanoceria at a low dose on soil microbiota and the potentiation of impacts by the Canola plant[END_REF].

Statistical analysis

Statistical analyses and data visualization were performed in XLSTAT (Addinsoft, 2019).

The variables were checked for normality (Shapiro-Wilk test) and homoscedasticity (Levene test). The significance of results was examined by one-way analysis of variance (ANOVA) followed by posthoc tests (Student-Newman-Keul test). For data that were not normally distributed, Kruskal-Wallis test and Conover-Iman post-hoc tests were performed.

The significance was set at p < 0.05. In the graphs, error bars are standard deviations.

Principal component analysis (PCA) was used to visualize the data set and to reduce the dimensionality by projecting each sample onto the two first principal components [START_REF] Ringnér | What is principal component analysis?[END_REF]. PCA was performed on 100 observations, each corresponding to an individual experiment. The factors (levels) were related to the nature of the biodegradable polymer (PBSA or PHBV), and the nanocomposite (presence or absence). The variables of interest were C/N ratio, total dry solids (DS), total volatile solids (VS), plant mass shoot, plant number of leaves, and soil and rhizosphere enzymatic activities. As the variables were measured in different units, they were centered and scaled to unit variance. Differences among groups with multiple response variables were assessed using permutation based multivariate analysis of variance (PERMANOVA, 1000 permutations)

3 Results and discussion

Materials characterization

Biodegradability of neat and nanocomposite polymers

The degree and kinetic of biodegradation in soil of neat and nanocomposite materials were The biodegradability experiments were validated since the degree of biodegradation of cellulose is higher than the 60% set by the standard (NF EN 17033: 2018). This threshold was obtained after only 13 days of biodegradation showing the high activity of the soil used.

The biodegradation kinetic curves obtained for each material are different from that of the reference material (cellulose). The biodegradation of cellulose increased rapidly until day 20 and continued to increase at a much slower rate until day 100 before reaching a plateau. As regards the four tested materials, a succession of two phases of increase followed by a slowdown is observed. This difference in degradation kinetics results in two different types of modelling: a single sigmoidal shape curve according to Hill equation for cellulose and a double sigmoidal shape curve fitting with Boltzmann equation for the materials under study.

The tested materials degraded well and even exceeded the cellulose. Thus, according to the standard (NF EN 17033: 2018) that mentions that materials have to reach 90% of biodegradation related to the reference material to be considered as biodegradable, the tested materials are biodegradable. By considering the degree of biodegradation at the end of the experiment, the materials ranking as follow: PBSA < PHBV NCP < PHBV < PBSA NCP and reaching a degree of biodegradation greater than 100%. This phenomenon was observed for the first time by [START_REF] Löhnis | Nitrogen availability of green manures[END_REF][START_REF] Bingeman | The effect of the addition of organic materials on the decomposition of an organic soil[END_REF]. It occurs when there is an increased release of the soil-derived carbon as CO2 compared to the mineralization in the soil without any additions [START_REF] Kuzyakov | Review of mechanisms and quantification of priming effects[END_REF]. This peculiar behavior results from a stimulation of the organic matter mineralization due to its easy decomposition. It was already observed for films made of PLA [START_REF] Tuominen | Biodegradation of lactic acid based polymers under controlled composting conditions and evaluation of the ecotoxicological impact[END_REF]Balaguer et al., 2016), starch-polystyrene foams [START_REF] Pushpadass | Biodegradation characteristics of starch polystyrene loose-fill foams in a composting medium[END_REF], PHBV (Lammi et al., 2019).

The results show that during the first stage (0 30 days), PBSA appeared to biodegrade faster than PHBV. This result is quite surprising since an opposite trend was observed in another study undertaken on the same materials but in a different soil (results not shown) and also in a comparative study of both PBSA and PHBV polymers by respirometric test and mass loss measurement in composting conditions (Salomez et al., 2019). After 20 days of incubation, they obtained a percentage of biodegradation of 15% for PBSA against 53% for PHBV and a mass loss five times higher for PHBV compared to PBSA. Moreover, [START_REF] Mercier | Fate of eight different polymers under uncontrolled composting conditions: relationships between deterioration, biofilm formation, and the material surface properties[END_REF] observed on their study conducted on eight different polymers, a most significant weight loss with PHA compared to other tested polymers, and among them PBS. They also reported that many microorganisms isolated from various environments are able to intracellularly hydrolyze PHA in a wide range of hydroxyalkanoic acids.

The addition of nanoclays in a biodegradable polymer matrix leads to contrasted effects depending on the matrix, PHBV or PBSA. The influence of the addition of nanoclays on the biodegradation of polymers has been extensively studied. Whatever the effect observed it can be ascribed to (i) a chemical/catalytic effect in link with the hydroxyl groups located at the edges of the nanoclays platelets [START_REF] Sabet | Interfacially compatibilized poly(lactic acid) and poly(lactic acid)/polycaprolactone/organoclay nanocomposites with improved biodegradability and barrier properties: Effects of the compatibilizer structural parameters and feeding route[END_REF][START_REF] Chevillard | How the biodegradability of wheat gluten-based agromaterial can be modulated by adding nanoclays[END_REF] or (ii) a structural effect in relation with the degree of dispersion and exfoliation of the nanoclay platelets preventing both water and oxygen diffusion and/or (iii) a nucleating effect influencing the crystallinity phase.

In PHBV matrix no significant impact neither on the degree of biodegradation nor on the kinetic of biodegradation was evidenced. Thus, the mechanism of degradation of PHBV does not seem to be impacted by the presence of nanoclays and/or this type of nanoclays. Several studies have investigated the influence of the addition of nanoclays on the biodegradation of PHAs with contrasting conclusions. Sometimes, the biodegradation is enhanced by the presence of nanoclays due to the nucleating effect of nanoclays (Maiti et al., 2007). They induce changes in crystallinity such as smaller crystallite size and therefore larger amounts of interspherulitic area (amorphous regions), which can be degraded by microorganisms. Other times it is slowed down (Maiti et al., 2003;Wang et al., 2005a;Iggui et al., 2015). This decrease of the biodegradability was attributed to the barrier effect of dispersed nanoclays that induce a more Chapter V. Impact of nanoclays on the end-of-life of biodegradable polymer matrices tortuous path reducing the diffusion of water and microorganisms from the surface into the bulk of the material.

On the other hand, in PBSA matrix, an impact on both degree and kinetic of biodegradation was observed. At the beginning of the experiment, PBSA degraded quickly, faster than all other materials before slowing down after 30 days. Conversely, when nanoclays are added, the biodegradation started later after a period of latency and at a slower rate. Finally, after 65 days, the degree of biodegradation of PBSA NCP becomes higher than that of neat PBSA. In this case, the nanoclays slow down the kinetic of biodegradation but have a positive impact on the degree of biodegradation with a plateau reached at higher values. Steeves et al. (2007) These results show that the impact of nanoclays on the biodegradation is polymer dependent. This may be due to the degradation mechanism inherent to each polymer. Indeed, as it has been demonstrated in previous work (Salomez et al., 2019), the degradation of PHBV leads to the formation of holes whereas PBSA fragments. Such difference in degradation was mainly attributed to a different morphology and structural organization of the crystalline phase in each polymer. As consequence, the porous structure generating diffusion channels allows the microorganisms to progress more rapidly into the core of the PHBV-based material without any hindrance induced by nanoclays. On the other hand, the degradation of PBSA-based materials appears more frontal. In this case, dispersed nanoclays act as obstacles to the advancement of microorganisms and slow down the biodegradation of the material.

Chapter V. Impact of nanoclays on the end-of-life of biodegradable polymer matrices

Disintegration of neat and nanocomposite polymers

The results of disintegration are shown in Figure V.2 and the degree of disintegration obtained after 90 days of composting is reported in Table V.2. V.2). The results show that the incorporation of nanoclays did not hinder the disintegration of polymer matrices. Moreover, in the case of PHBV, it seems that they enhance the disintegration process. The lower degree of disintegration obtained for PHBV can be ascribed to the higher degree of crystallinity evidenced in a previous study (Lajarrige et al., 2019). The impact of bioresidues on plant growth was evaluated by germination, shoot biomass and number of leaves.

Conclusion

Les tests de biodégradation en sol réalisés sur les matériaux de PBSA et PHBV purs et nanocomposites ont révélé des comportements différents en fonction de la matrice polymère act sur la biodégradabilité des matériaux alors que dans le cas du PBSA, un retard de biodégradation est observé en présence de nano-argiles puis après 60 jours, le taux de biodégradation du matériau nanocomposite devient supérieur à celui du PBSA pur. La dégradation du PHBV entraîne la formation de trous alors que le PBSA se fragmente. Ces comportements de dégradation différents peuvent expliquer les impacts variables de la présence de nano-argiles. Cependant, la biodégradabilité de tous les matériaux étudiés a été confirmée.

Les tests de désintégration réalisés en compost ont montré un impact négligeable de la présence de nano-argiles sur la perte de masse du matériau et même un effet profitable dans le u degré de cristallinité en présence de nano-argiles.

-argiles résiduelles étudié sur les plantes, le sol et la les propriétés thermiques, etc. pourraient y être ajoutés.

De plus, les résultats de cette balance bénéfices-risques pourraient être utilisés pour initier le développement de de, alcoolique, gras ou encore contenant des microorganismes) en fonction des exigences (emballage biodégradable, transparent, coût raisonnable, etc.) des différentes parties impliquées re, consommateurs, etc.).

Des outils de ce genre ont déjà été développés notamment dans le cadre du projet EcoBioCAP Exploration des risques et des bénéfices liés à l'utilisation de nano-argiles dans des matériaux biodégradables destinés au contact alimentaire La substitution des matériaux plastiques conventionnels par des matériaux biodégradables et biosourcés est une alternative intéressante pour faire face aux problèmes environnementaux liés à leur matériaux nanocomposites, emballage alimentaire, Les bionanocomposites sont produit par extrusion en utilisant comme matrice deux polyesters (PBSA ou PHBV), choisis pour leur différence de propriétés de transfert, et comme renfort des nano-argiles lamellaires. La thèse se focalise sur deux questions scientifiques majeures : (i) comprendre -argiles sur les propriétés du matériau à tous les stades de son cycle de vie : après production, au cours de son usage au contact alimentaire et lors de sa fin de vie ; (ii) étudier la balance bénéfices-risques liée -argiles dans des matériaux biodégradables dédiés de s nanoleur aptitude au contact alimentaire.

sai définies dans le règlement européen N° 10/2011 dédié aux matériaux nonméthodes plus adaptées à ce type de matériaux ont été développées. Par la suite, les bénéfices en terme s. Pour finir, une étude de -argiles sur la fin de vie de ces matériaux a été menée par des tests de Mots-clés : bio-nanocomposite, nano-argile, aptitude au contact alimentaire, propriétés de transfert de matière, fin de vie Exploring of risks and benefits related to the use of nanoclays in biodegradable materials for food contact application

The substitution of conventional plastic materials with biodegradable and bio-based materials is an interesting alternative for dealing with environmental problems related to their wide use. The aim of this thesis is to develop new nanocomposite materials, dedicated to food packaging, safe for the consumer and the environment. The bio-nanocomposites are produced by extrusion using two biopolyesters as matrix (PBSA or PHBV), chosen for their differing transfer properties, and lamellar nanoclays as reinforcement. The investigations were focused on two major scientific issues: (i) to understand the impact of the introduction of nanoclays on the material properties at all stages of its life cycle: after production, during its use in food contact and during its end-of-life; (ii) to study the benefits-risks balance related to the addition of nanoclays in biodegradable materials dedicated to food packaging. The first part of this work aims to evaluate the impact of the addition of nanoclays on the properties of transfer and inertia of these materials and consequently of their suitability for food contact. In this way, the conditions defined in the European regulation No 10/2011 dedicated to nondegradable materials were first used, then methods more adapted to this type of materials have been developed. Subsequently, the benefits in term of barrier properties and the risks related to consumer exposure to additives present in the materials were quantified using different case studies. Finally, a study of the impact of the nanoclays on the end-of-life of the materials was conducted by biodegradation tests in soil and ecotoxicity tests on plants and soil. Keywords: bio-nanocomposite, nanoclay, suitability for food contact, mass transfer properties, endof-life

Figure 3 :

 3 Figure 3 : Diagramme représentant les différentes tâches étudiées.

Figure I. 4 :

 4 Figure I.4: The structure of 2:1 layered silicate (Pavlidou and Papaspyrides, 2008)

Figure I. 5 :

 5 Figure I.5: The different structures and morphologies of polymer/clay materials according to the interaction between layered silicate and polymer.

Figure I. 6 :

 6 Figure I.6: Tortuosity path of permeate molecules in (i) microcomposite or (ii) nanocomposite material.

  60 mg/kg. Recently, the EFSA Panel on Food Contact Materials, Enzymes and Processing Aids (CEP) concluded that the poly((R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate) (PHBH) (FCM No 1059) is not of safety concern for the consumer if it is used either alone or blended with other polymers in contact with all kinds of food, under contact conditions of more than 6 months at room temperature or below, including hot-fill or a short heating up phase (EFSA CEP Panel, 2019).

Figure I. 8 :

 8 Figure I.8: Oxygen (1), carbon dioxide (2) and water vapor (3) permeabilities reduction or augmentation according to nanoclay content.

  Figure I.10: Elastic modulus (1), tensile strength (2) and elongation at break (3) reduction or augmentation according to nanoclay content. (A) full scale, positive axis (B) and (C) negative axis.

Figure I. 11 :

 11 Figure I.11: Evolution of the biodegradability according to nanoclay content.

  who evaluated the degradation by weight loss of PCL filled with 5 wt% Cloisite 30B or Nanofil 804. The neat PCL shows a full degradation after eight weeks while the weight loss is of 93% and 90% for PCL reinforced with 5 wt% of Cloisite 30B and Nanofil 804, respectively. Guaras et al. (2016) reached similar conclusion on a blend made of PCL and thermoplastic starch (TPS) with and without compatibilizer. The effect of the nature of intercalating agent was investigated by Luduena et al. (2013) on unmodified (C-Na + ) and modified (C-30B and C-20A) MMTs. The results of this work show a very contrasted effect of the intercalant agent with, in particular, a reduction in the degradation rate of PCL in the case of C-30B, an augmentation in presence of C-NA + and no effect in presence of C-20B. This result is in contradiction with the conclusion made by Wu et al. (

  tendency was found by[START_REF] Shayan | Effect of modified starch and nanoclay particles on biodegradability and mechanical properties of cross-linked poly lactic acid[END_REF] on a blend of PLA/TPS reinforced with Cloisite 30B.[START_REF] Fukushima | Biodegradation of poly(lactic acid) and its nanocomposites[END_REF], studied the biodegradation of PLA nanocomposites based on two different modified montmorillonites: Cloisite 30B and Nanofil 804, at 5 wt% loading prepared by melt blending in compost and in a medium containing microorganisms isolated from the compost. The results show no significant decrease of the mass loss up to 39 weeks. However, in composting conditions, SEC and DSC experiments indicate that the degradation of PLA is greater when nanoclays are added, especially with C-30B. The better C-30B dispersion can influence the level of hydrolytic degradation of the polymer matrix, which seems to start from the interface between polymer and fillers. In microbial medium, the addition of Nanofil 804 does not affect the degradation of PLA, while the Cloisite 30, leads to a rejection response towards B. licheniformis preventing its growth close to the organoclay.The biodegradability of polyhydroxyalkanoates (PHAs) nanocomposite has also been the subject of several investigations. The weight loss of PHB and corresponding nanocomposites, PHB-fluoromica and PHB-MMT was determined as a function of compost time byMaiti et al.

  studied the potential toxicity on algal population growth (Chlamydomonas reinhardtii) of three different nanoclays: a natural nanoclay (MMT-Na + ) and two modified nanoclays (Cloisite 30B and Novaclay TM ). The results evidenced that neither natural nanoclay nor the modified nanoclay (Novaclay) adversely affected algal population growth at concentrations up to 100 mg/L while Cloisite 30B caused a severe reduction in algal population growth even when the concentration of the nanoclay was rather low. In the other study, Balaguer et al. (2016) study the ecotoxicity on garden cress (L. sativum) of PLA film reinforced with three different nanoclays: MMT modified with (1) an ammonium quaternary salt, (2) calcium carbonate and (3) silicon dioxide. The authors observed no phytotoxicity (chlorosis, necrosis, wilting, leaf and stem deformation) or mortality with the addition of nanoclays in PLA matrix, despite being L. sativum one of the most sensitive species in ecotoxicity tests on plants.

  Figure II.1: Formula of (A) PBSA and (B) PHBV.

Figure

  Figure II.2: Twin-screw profile. Table II.4: Temperature profiles of the extruder from the feeder to the die. PBSA PHBV Masterbatch preparation 120 120 130 130 140 140 150 150 160 160 140 150 160 160 160 170 175 180 180 180 Dilution of masterbatch 120 140 140 150 150 150 160 160 180 180 140 150 160 160 160 170 175 180 180 180 Production of films 100 100 100 125 130 130 130 135 135 135

Figure II. 3 :

 3 Figure II.3: Schematic description of the test chamber for optical permeation measurement of films.

  paper. The concentration of additives remaining in films was evaluated by extraction in Chapter II. Materials and Methods 83 20 mL of n-hexane at 23°C during 24h under magnetic agitation. The solution was analyzed by GC-FID.

Figure

  Figure II.4 represents the experimental protocol used for the migration tests using microbial solid food simulants.

Figure II. 4 :

 4 Figure II.4: Experimental protocol used for the migration tests using microbial solid food

  , polyamides (Pereira deAbreu et al., 2010), and polyolefins[START_REF] Otero-Pazos | Determination of partition coefficients of selected model migrants between polyethylene and polypropylene and nanocomposite polypropylene[END_REF] Nasiri et al. 2016; Nasiri et al. 2017), conclude that nanoparticles decrease the diffusivity of low molar mass substances. Such results suggest the applicability of the Piringer model in a migration prediction approach based on an overestimated value of diffusivity.

  degree of exfoliation of nanoclay platelets in biopolymer matrices was measured by XRD (Fig. III.1 A) and TEM (Fig. III.1 B). As expected, the analysis of the C-30B powder by

3 :

 3 Fig. III.3: Comparison of Dapp of additives in neat (grey bars) and nanocomposite (black bars) PBSA (Fig. A and B) and PHBV (Fig. C and D) films in contact with food simulants: 95% ethanol (Fig. A and C) and isooctane (Fig. B and D).

Fig

  Fig. III.4, below, shows the Dapp of additives in function of their molar mass in neat and nanocomposite PBSA (Fig. III.4 A) and PHBV (Fig. III.4 B) in contact with 95% ethanol and isooctane food simulants. The linear dependence of Log (D) = f (M) is clearer for PBSA thanPHBV. The dependence of the diffusion coefficient on size has already been demonstrated in natural rubber and in a glassy synthetic polymer[START_REF] Chern | Material selection for membrane-based gas separations[END_REF] Reynier et al. 2001;

  3. The sorption of ethanol by both tested packaging films could explain the high Dapp values measured during the contact with this food simulant (Fig. III.3).

for 5

 5 min, cooled to -30°C and finally reheated to 200°C. Temperatures were adjusted at a rate of 10°C.min -1 . The crystallization temperature (Tc) determined from cooling ramp, melting temperature (Tm) and melting determined from the DSC curves using the Universal Analysis 2000 software by TA Instruments. The crystallinity degree (XC) of neat and nanocomposite polymers was determined by the equation: (III.10)

Figure

  Figure III.7: Overall migration of PHBV, PHBV NCP and the corresponding aged materials

  Figure III.8 and III.9 for PHBV, PHBV NCP (Figure III.8), PBSA, PBSA NCP (Figure III.9) and the corresponding UV treated materials.

  (PHBV) and Figure III.12 (PBSA), showing the respective impacts of: polymer type, food simulant, nanoparticle incorporation and accelerated aging.

  Figure III.15: Remaining film mass of neat (empty symbols) and nanocomposite (full symbols) PBSA films in contact with P. Candidum (A) or L. Bulgaricus (B) (circle) or not (control) (diamond).

Figure

  Figure III.16 B, C and D. The SEM image B, showing the surface of PBSA after 10 days of contact with agar plate inoculated with P. Candidum evidenced the occurrence of pores and interstices, which can be attributed to areas degraded by the microorganism. The same observation can be made from the image C corresponding to the surface of PBSA after 10 days of contact with agar plate inoculated with L. Bulgaricus. On the other hand, nanocomposite material in contact with agar plate inoculated with L. Bulgaricus (image D) does not exhibit any superficial damaging. These results, in accordance with those of the mass loss (Figure III.15), suggest that nanoclays prevent the degradation of the material by hindering the access and the progression some microorganisms into the polymeric matrix.

Figure III. 17 :

 17 Figure III.17: Example for PBSA NCP in contact with P. Candidum of kinetic curves of the mass of additives remaining in film samples (A) and having migrated into the agar plate (B).

Figure

  Figure III.18: Material balance of additives between the film and the agar plate after 10 days of contact with PDA agar plate inoculated or not with P. Candidum.

  Figure III.19: Desorption rate of additives from the films (A) PBSA in contact with uninoculated agar plate, (B) PBSA NCP in contact with uninoculated agar plate, (C) PBSA in contact with agar plate inoculated with P. Candidum and (D) PBSA NCP in contact with agar plate inoculated with P. Candidum.

Figure

  Figure III.20: Impact of the microorganism on the migration of additives from films to agar.

  plus évident et dépend de la matrice et du simulant alimentaire utilisés. En revanche, le traitement a révélé avoir un effet positif en limitant la migration globale et la migration des additifs dans les simulants alimentaires étudiés. Cependant, ce traitement impacte les propriétés mécaniques des matériaux qui deviennent fragiles et cassants, il sera donc important de prendre . tement peut expliquer ces résultats. Ce travail a également permis de démontrer que les conditions définies pour les matériaux plastiques conventionnels sont potentiellement applicables au cas particulier des matériaux biodégradables. Lorsque les matériaux sont mis au contact de simulants alimentaires microbiens, un impact positif de la présence des nano-argiles est mis en évidence avec une diminution des vitesses de migration et une rétention plus élevée des additifs dans les matériaux. Cependant, , tels que les produits laitiers, semble être délicate dégradent les matériaux entraînant des pertes de masse non CHAPTER IV. ESTIMATION OF RISKS-BENEFITS OF ADDING NANOCLAYS IN BIODEGRADABLE POLYMER MATRICES Chapter IV. Estimation of risks-benefits of adding nanoclays in biodegradable polymer matrices Un des paramètres principaux à prendre en compte dans la formulation d'un emballage alimentaire est son aptitude au contact alimentaire. Elle tient compte de la mesure de migration globale qui ne doit pas excéder 10 mg de substances par dm² de surface de matériau et des mesures de migration spécifique dont les limites sont fixées pour chaque substance (référencées dans la liste positive des substances autorisées dans la formulation des emballages) en fonction de leur toxicité potentielle. Dans la première partie (Article 4), une évaluation de l'exposition du consommateur à un panel d'additifs autorisé au contact alimentaire, est menée en considérant la substitution de nos emballages actuels par deux matériaux biodégradables et incorporant ou non des nano-argiles (PBSA, PBSA NCP, PHBV et PHBV NCP). L'étude bibliographique a montré que de nombreux travaux s'intéressent au renforcement des propriétés des matériaux par des argiles lamellaires. Elles permettent notamment d'améliorer les propriétés barrières des matériaux en diminuant leur perméabilité aux gaz (vapeur d'eau, oxygène et dioxyde de carbone). Ainsi, dans une seconde partie (Article 5), les bénéfices de leur ajout sur ces propriétés barrières sont étudiés. Afin de quantifier ces bénéfices, les perméabilités à l'oxygène et au dioxyde de carbone des matériaux vierges et nanocomposites sont utilisées pour déterminer (i) le gain en terme de durée de vie d'un aliment sensible à l'oxygène pris comme exemple et (ii) le gain en terme de matériau utilisé pour formuler l'emballage alimentaire. Ces différents scénarios sont ensuite utilisés pour estimer le gain en terme d'impact environnemental et déterminer quelle est la meilleure option à suivre pour limiter cet impact. Chapter IV. Estimation of risks-benefits of adding nanoclays in biodegradable polymer matrices Evaluation of the consumer exposure to additives related to the packaging of foods in bio-nanocomposite materials (Article 4) Anaïs Lajarrige a , Nathalie Gontard a and Stéphane Peyron a a JRU IATE 1208, CIRAD, INRA, Montpellier SupAgro, University of Montpellier, 2 place Pierre Viala, F-34060 Montpellier 01, France

  Figure IV.2: Comparison of additives relative migration from neat PBSA, PHBV, PBSA NCP and PHBV NCP films into food simulants: 3% acetic acid (Fig. A), 10% ethanol (Fig. B), 20% ethanol (Fig. C), 50% ethanol (Fig. D), 95% ethanol (Fig. E), isooctane (Fig. F) and Tenax (Fig. G).

Figure IV. 3

 3 Figure IV.3 in which are compared the four different materials (PBSA, PBSA NCP, PHBV and PHBV NCP) and the PP as reference material.

  Impact of nanoclays on the end-of-life of biodegradable polymer matrices Un autre paramètre important à prendre en compte lors du développement de nouveaux emballages alimentaires est leur traitement de fin de vie. Le PBSA et le PHBV étant des matériaux biodégradables et une grande partie des emballages alimentaires finissant dans après usage, leur biodégradabilité en sol est étudiée afin de déterminer -argiles dans leur formulation sur ce paramètre. A ce sujet, la synthèse bibliographique a montré des résultats contrastés en fonction du type de polymère, du type de nano-argile, de leur compatibilité et du milieu utilisé pour la dégradation. Ces matériaux ont également été dégradés en compost pendant 90 jours e compost résiduelles sur les plantes, le sol et le microbiote. L écotoxicité a été réalisée en collaboration avec le CEA de Cadarache.

  evaluated using respirometric tests. The biodegradation kinetic of each tested material are presented in Figure V.1.

  , studied the impact of the incorporation of three different modified montmorillonite layered silicates ((a) MLS-1330 12-aminolauric acid; (b) MLS-1855 stearylbis(2-hydroxyethyl)methyl quaternary amine chloride; (c) MLS-1856: stearyl bis(2-hydroxyethyl)amino hydrochloride) in PBSA and found the same tendency, that is total amounts of net mineralization greater for the nanocomposite than for the neat PBSA.

Figure V. 1 :

 1 Figure V.1: Biodegradation kinetic curves of PBSA, PBSA NCP, PHBV, PHBV NCP and cellulose (reference material).

Figure V. 2

 2 Figure V.2 shows the physical changes in the structure of the four films, PBSA, PHBV and their corresponding nanocomposite. All the materials visually disintegrated over time under composting conditions. The degree of disintegration of the materials is calculated after 90 days of composting (TableV.2). The results show that the incorporation of nanoclays did not hinder

  Figure V.2: Evolution of disintegration of neat and nanocomposite PBSA and PHBV under simulated composting conditions. Table V.2: Degree of disintegration of neat and nanocomposite PBSA and PHBV after 3 months in simulated composting conditions.

  rhizosphère a montré un impact variable en fonction des différents paramètres étudiés. Une augmentation de la biom -argiles dans du nombre de feuilles par plante, quelle que soit la matrice polymère, une augmentation est observée. Trois enzymes : -glucosidase, phosphatase et -N-acetylglucosaminidase (NAGase) sont obtenus ne -argiles sur ces paramètres. Des tests es résultats ont montré un impact significatif des nano-argiles dépendant du polymère utilisé. de la construction par exemple, est devenu une nécessité. Le marché de era privilégiée pour remplacer les matériaux actuels.technologie dans ce secteur est due à plusieurs perspectives de développement : (i) amélioration des propriétés des matériaux, (ii) emballages actifs destinés à étendre la durée de conservation matériaux biodégradables issus de ressources renouvelables. Certains de ces emballages incorporant des nanomatériaux sont déjà sur le marché tels que des bouteilles en PET contenant des nanocharges lamellaires ou encore des emballages en polypropylène contenant des nanoparti En biodégradables et/ou bio-sourcés, occupent une part de marché encore mineure en Europe. Leur essor commercial ne pourra la condition que leur aptitude au contact alimentaire soit approuvée. es matériaux a pour but de les rendre plus compétitifs vis-à-vis des matériaux conventionnels utilisés hui. Cependant, la conception de matériaux bio-nanocomposites fait face à plusieurs verrous. En glementation spécifique définie pour ce type de matériaux, la question est de savoir comment évaluer leur aptitude au contact alimentaire. De plus, il y a peu de connaissances sur le devenir de ces nanoparticules résiduelles après usage de ces matériaux. était de produire des matériaux nanocomposites biodégradables et bio-sourcés imentaire, à partir de deux polyesters différents (PBSA et PHBV) et de nano-argiles lamellaires (Cloisite 30B) utilisées en tant que renfort des propriétés barrières. L objectif scientifique était la compréhension de ces systèmes priétés barrières des matériaux présente différents avantages i.e. (1) améliorer leurs performances et compétitivité vis-à-vis des matériaux conventionnels, (2) augmenter la durée de conservation des aliments emballés, notamment pour les produits Concernant le deuxième point, les aliments 2 sont habituellement emballés dans des emballages sous atmosphère modifiée (MAP) sans oxygène et avec un mélange de gaz CO2/N2 pour prolonger leur durée de vie. Une 2 et au CO2 bénéfice des diminutions de perméabi , des filets de poulet qui ont une faible durée de conservation, a été étudié (Article 5). Pour cela, le logiciel MAPproduits alimentaires frais réfrigérés et non-respirant, a été utilisé. Il prend en compte la 2 et du CO2 ainsi que leur désorption et dissolution dans . Le PHBV, qui a de meilleures propriétés barrières que le PBSA et plus proches de son « benchmark » le polypropylène (PP) emballage de ce type -argiles a montré une augmentation de la durée de vie de 1.5 jours par rapport au PHBV pur. Il limite, dans une plus grande mesure que les autres matériaux, , ce qui entraîne (i) une reprise en O2 (ii) un transfert de CO2 rieur plus faible également, conservant les concentrations initiales plus longtemps. L mélioration des propriétés barrières peut se trouver ailleurs il est utilisée pour fabriquer proportionnel au pourcentage de diminution de la perméabilité peut être envisagé. la quantité de matériau contribuent à limiter leur impact environnemental. En se focalisant sur La réduction des pertes au niveau du -argiles ayant été démontré, leur impact sur les propriétés a ensuite été étudié. General discussion, conclusions and prospective des matériaux Une seconde partie de ce travail a consisté à tie de matrices polymères biodégradables et donc sur leur aptitude au contact alimentaire. migration globale et spécifique définis dans le cas des matériaux plastiques destinés à entrer en contact avec des denrées alimentaires, selon le règlement (CE) N° 10/2011. Il définit les ces matériaux et les limites de migration à ne pas dépasser pour chacune de ces substances. Les propriétés de transfert peuvent être étudiées par ces tests de migration spécifique, notamment leur diffusivité dans le matériau et leur coefficient de partition entre le matériau et le simulant alimentaire. représentatif des possibles substances utilisées dans la formulation des emballages alimentaires (Article 1). La diffusivité est un paramètre important pour les applications industrielles car la réglementation (CE) N° 10/2011 niveaux de migration. des données expérimentales reposant sur la mise en de test complexes, coûteux et chronophages. Pour déterminer le coefficient de diffusion de ces additifs, des cinétiques de désorption ont été réalisées dans différents simulants alimentaires (éthanol 95% et isooctane) argiles sur ce paramètre a été étudié en comparant les coefficients de diffusion obtenus en présence et en absence de nano-argiles. Contrairement à ce qui était observé dans le cas des matériaux plastiques conventionnels tels que le polyéthylène (Nasiri et al., 2016), une augmentation du coefficient de diffusion en présence de nano-argiles a été obtenue dans la plupart des cas. Ce résultat soulève donc de nouvelles questions liée et différents facteurs pouvant influencer la diffusivité des molécules ont été étudiés : -La dispersion et exfoliation des nano-: une mauvaise dispersion et exfoliation peut conduire à la création de chemins préférentiels et ainsi augmenter la diffusivité des additifs. Les observations en microscopie électronique à transmission et les résultats de diffraction des rayons X ont révélés une bonne dispersion des nano-s avec un mélange de structures intercalées et exfoliées. General discussion, conclusions and prospective -Le contact entre le polymère et le solvant peut modifier les propriétés de transfert du matériau et les nano-argiles sont susceptibles de modifier ces interactions. La sorption de solvant par le matériau qui induit souvent un gonflement du matériau peut accélérer la migration des additifs et augmenter leur coefficient de diffusion. éthanol les différences importantes de diffusivité. Par exemple, le même pourcentage de sorption coefficients de diffusion est obtenu. -Le degré de cristallinité influence également les propriétés de transfert des matériaux puisque l lieu que dans les zones amorphes. La -argiles va dans le sens de Cependant, les diminutions de degré de cristallinité ne peuvent expliquer, à elles seules, les grandes différences de diffusivité obtenues entre les différents matériaux et simulants alimentaires au contact retenus dans cette étude. expériences complémentaires en étudiant la cristallinité de manière plus approfondie et pas Des mesures en flash-DSC pour étudier plus en détail les zones amorphes et déterminer notamment le pourcentage de fraction amorphe rigide et de fraction amorphe mobile de ces zones amorphes serait intéressant. Un autre paramètre important à prendre en compte est la nature, dimension et niveau interaction interface entre la matrice et les nano-argiles. Une incompatibilité entre les constituants du matériau nanocomp dans certain cas à une augmentation de la diffusivité des molécules dans la matrice. Cependant reste difficile à mener. ct du vieillissement du matériau sur ces propriétés de t ). La structure de ces matériaux étant instable dans le temps du fait de leur caractère biodégradable, contrairement aux polymères conventionnels, il est important de d sont suffisamment stables pour être utilisés au contact alimentaire ou si des mesures particulières doivent être prises. Les résultats ont montré une modification de leur structure avec notamment une augmentation de leur degré de General discussion, conclusions and prospective cristallinité qui conduit à de meilleure -à-dire à une diminution des valeurs de migration globale. Une diminution des migrations spécifiques est également obtenue dans la plupart des cas, e à une diminution de la diffusivité des additifs, qui augmente dans la majorité des cas. Ce travail a permis de démontrer que les recommandations fournies dans le règlement (CE) N° 10/2011 peuvent être appliquées au cas particulier des polymères biodégradables et que ces matériaux vieillis restent aptes au contact alimentaire. En revanche, le traitement appliqué à ces matériaux visant à simuler plusieurs mois de agressif, conduisant à des matériaux très fragiles et cassant. Pour compléter cette étude, il serait intéressant de réaliser des cinétiques de vieillissement et de mesurer en parallèle les propriétés mécaniques de ces matériaux. En se fixant des propriétés mécaniqu E nanocomposites, des essais ont été mis en place en utilisant des simulants alimentaires que les fromages et les yaourts (Article 3). Ces matériaux étant biodégradables et donc sensibles , vant conduire à de nouveaux risques pour la santé du consommateur. Une faible migration des additifs vers le simulant alimentaire a été observée mais les résultats ont montré que les microorganismes peuvent métaboliser certains additifs. De plus, la structure des matériaux est impactée par la présence des microorganismes. Une perte de masse du matériau comprise entre 0.5 et 1% a été mesurée après 10 jours de contact avec les simulants microbiens. Ainsi, au vue de ce contenant des microorganismes. En ce qui concerne, l ajout de nano-argiles dans des matrices biodégradables au contact de ce type de simulants alimentaires, un effet positif a été observé. au cours de ce travail. Cela inclut les simulants alimentaires un impact non négligeable sur la dégradation des matériaux après 10 jours de contact. Il a été montré que les nanoparticules incluses dans la matrice polymère et don ne peuvent (Störmer et al., 2017). Dans le cas des polymères biodégradables, des nt intercalant, utilisé pour modifier les nano-argiles et leur permettre une meilleure dispersion et exfoliation dans la matrice, doit également être étudiée. Cet agent intercalant contient un ammonium quaternaire polymère. Pour cette raison la substitution des nano-argiles modifiées par des nanotalcs, qui ne nécessitent pas de traitement pour permettre une bonne dispersion, se présente comme une alternative prometteuse. (CE) N° 10/2011 dans la formulation des emballages alimentaires (Article 4). Les migrations spécifiques de cinq additifs (méthyl salicylate, benzophénone, DEHA, uvitex OB et irganox 1076) ont été déterminées dans les différents simulants -à-: aqueux, acide, gras, alcoolique et sec et pour les quatre matériaux envisagés (PBSA, PBSA NCP, PHBV et PHBV NCP). Par la suite et en utilisant les consommations alimentaires quotidiennes, déterminées ANSES (2017), auprès de trois classes de population (0-11 ans, 12-17 ans et 18-79 ans) et en considérant que ces aliments sont calculée et comparée à la limite tolérée par la réglementation. Trois scénarios ont été envisagés en partant du « worst case », dans lequel toutes les boissons et aliments sont conditionnés dans ces matériaux, vers un cas plus réaliste mais qui surestime cependant consommateurs. Un exemple de résultat est présenté en Figure 2 consommateur (pour les 18-79 ans) à trois additifs est représentée en considérant que les aliments sont emballés soit dans du PHBV soit dans du PHBV NCP, selon les trois scénarios General discussion, conclusions and prospective envisagés. , fixées par la réglementation, sont également représentées mais seulement celle de la benzophénone est visible, celles du méthyl salicylate et s (88.24 mgadditif.j -1 et 17.65 mgadditif.j -1 , respectivement).osition du consommateur en se bien inférieure à la limite fixée par la ré la faible dose journalière tolérée (DJA) définie pour cet additif. Dans ces cas critiques, une diminution de la concentration initiale en additif peut être envisagée. --dans la consommateur dans quelques cas. Cependant, ce travail a été effectué en utilisant le cas le plus plus, certains des additifs utilisés dans cette étude, tels que la benzophénone, ont des DJA que des additifs plus sûrs seraient incorporés dans ces matériaux biodégradables.

Figure 2 :

 2 Figure 2 : Exposition quotidienne du consommateur à trois additifs en considérant que les aliments, selon trois scénarios, sont emballés dans du PHBV ou PHBV NCP.

Table I .

 I 1: Commercially available clay minerals used in food packaging application.

	Commercial name	Chemical structure of organic modifier	Modifier concentration	d001 spacing (Å)
	Cloisite ® Na +	Unmodified clay	CEC = 92.6	11.7
	Cloisite ® 6A	Dimethyl dihydrogenated tallow ammonium	140 meq/100 g of clay 33.4
	Cloisite ® 10A	Dimethyl benzyl hydrogenated tallow quaternary ammonium	125 meq/100 g of clay 19.2
	Cloisite ® 15A	Dimethyl dihydrogenated tallow quaternary ammonium	125 meq/100 g of clay 31.5
	Cloisite ® 20A	Dimethyl dihydrogenated tallow quaternary ammonium	95 meq/100 g of clay 24.2
	Cloisite ® 25A	Dimethyl, dihydrogenated tallow, 2-ethylhexyl quaternary	95 meq/100 g of clay 18.6
		ammonium
	Cloisite ® 30B	Methyl tallow bis-2-hydroxyethyl quaternary ammonium	90 meq/100 g of clay 18.5
	Cloisite ® 93A	Methyl dehydrogenated tallow ammonium	90 meq/100 g of clay 23.6
	Nanocor ® I.30P Octadecyl ammonium	28-32 wt%	20.8
	Nanocor ® I.34TCN	Methyl octadecyl di-2-ammonium hydroxyethyl quaternary	25-30 wt. %	20
	Nanocor ® I.44P Dimethyl dialkyl tallow amine	25-30 wt%	26
	MAE	Dimethyl dialkyl (tallow) ammonium	120 meq/100 g of clay 30
	MEE	Methyl bis-2-hydroxyethyl coco quaternary ammonium	120 meq/100 g of clay 23

  particles, in an unbound state or as an aggregate and where, for 50% or more of the particles in the number size distribution, one or more external

	dimensions is in the size range 1 nm -	(The European Commission, 2011). The
	European Food Safety Authority (EFSA) proposes a suitable risk assessment approach taking
	f the ENM in addition to those common to the equivalent
	non-
	technical guidance that mention nanomaterials. The Regulation (EC) No 10/2011, specifically
	states that new technologies that engineer substances in particle size that exhibit chemical and
	physical properties that significantly differ from those at a larger scale, for example,
	nanoparticles, should be assessed on a case-by-case basis as regards their risk until more
	information is known about such new technology. Up today, only few substances have been

The Food and Drug Administration (FDA) has not established admitting that these terms are commonly used in relation to the engineering (i.e., deliberate manipulation, manufacture or selection) of materials that have at least one dimension in the size range of approximately 1 nanometer (nm) to 100 nm (U. S. Food and Drug Administration, 2014). The European Commission defines a nanomaterial as "a natural, incidental, or manufactured material containing in the positive list of the Regulation (EC) No 10/2011 and authorized to be used in PET bottles without any specific migration limit. Two other substances are authorized in this list but they are not listed as nanomaterials: carbon black (FCM No 411) and silicon dioxide (FCM No 504).

Table I .

 I 2: Analysis of the publications related to the study of barrier properties of biodegradable polymers reinforced with 2:1 phyllosilicates.

	(Crétois et al., 2014) (Masclaux et al., 2010) (Strawhecker and Manias, 2000)	PHBV Native potato starch ± glycerol Poly(vinyl alcohol)	Cloisite 30B Nanofil® 757 (MMT) Cloisite Na +	0, 2.5, 5, 7.5, 10 0, 2.5, 7.5 Solution blending Melt blending and compression molding 0, 2, 4, 6 Solution blending	Oxygen, carbon dioxide and water vapor Oxygen Water vapor
	Reference (Abdollahi et al., 2013) (Akin and Tihminlioglu, 2018) (Alboofetileh et al., 2013) (Alves et al., 2011) (Bae et al., 2009) (Bartel et al., 2017) (Boonprasith et al., 2013) (Chang et al., 2002) (Charlon et al. 2015) (Chen and Yang, 2015) (Chien and Lin, 2007) (Chivrac et al., 2010) (Corrêa et al., 2012) (Dean et al., 2009) (Di Maio et al., 2014) (Duan et al., 2013) (Memis et al., 2017) (Swain et al., 2014) (Mohan et al., 2016) (Tang et al., 2008) (Mondal et al., 2014) (Echeverría et al., 2014) (Falcao et al., 2017) (Farmahini-Farahani et al., 2017) (Gain et al., 2004) (Hassannia-Kolaee et al., 2016) (Issa et al., 2018) (Kasirga et al., 2012) (Koh et al., 2008) (Kumar et al., 2010a) (Kumar et al., 2010b) (Lavorgna et al., 2010) (Lee and Kim, 2010) (Luduena et al., 2013) (Luecha et al., 2010) (Martino et al., 2010) (Sothornvit et al., 2010) (Sothornvit et al., 2009) (Slavutsky et al., 2014) al., 2003) (Sinha Ray et (S. Singh et al., 2010) (Scarfato et al., 2017) (Müller et al., 2012) (Tang et al., 2018) (Okamoto et al., 2003) (Ortenzi et al., 2015) (Tenn et al., 2013) (Park et al., 2002) (Thellen et al., 2005) (Picard et al., 2011) (Tortora et al., 2002) (Rhim et al., 2006a) (Rhim et al., 2009) (Tunc et al., 2007) (Rhim, 2011) Agar Polymeric matrix Sodium alginate PHB, PHBV Cloisite 10A Type of nanoclay MMT-Na + Sodium alginate (1% et 1.5%) MMT-Na + Pectin (33%) + k-carrageenan (67%) Nanomer 1.34 TCN (OMMT) Gelatin 200 bloom-fish-2360µm Cloisite Na + PLA4043D Cloisite 30B PBS/Cassava starch (TPS) (75/25% w/w) Cloisite Na + , 30B PBS/TPS (25/75) Cloisite Na + , 30B PLA C16-MMT and DTA-MMT Cloisite 25A PBS and PBSA Cloisite-Na + , 30B PBAT and MA-grafted-PBAT MMT, ODA-MMT, DHA-MMT Poly(vinyl acetate) (PVAc) MMT Wheat starch MMT-Na, OMMT-CS plasticizer PHBV, PHBV + 10% Cloisite 30B PBSA Modified hectorite-Na + (Na-FHT) PLA 4032D Cloisite 30B PLA 4060D amorphous and PLA 4032D Cloisite 30B Cloisite Na + and Starch rice Cloisite 30B Fenugreek seed Nanomer® MMT, I.44 P Corn starch Nanomer I30E (OMMT) Corn starch (S8250) Cloisite 30B (OMMT) Wheat starch and potato MMT PBAT MMT-Na + starch Cloisite Na + , crystallisable Soy protein isolate Cloisite-Na + PBAT Cloisite 20A PHBV Cloisite® 30B PCL MMT-Na and MMT-(OH)2 MMT-(OH)2 MMT-(OH)0.5, MMT-(OH)0.75, MMT-(OH)1 and MMT-(OH)2 Whey protein isolate MMT-Na + Sweet potato starch MMT Chitosan MMTK-10 PLA Cloisite 15A, 20A, 30B Soy protein isolate Cloisite Na + Soy protein isolate Cloisite 20A, 30B molecular Soy protein isolate MMT-Na PCL Cloiste Na + , 30B, 20A Zein MMT Nanomer I.34 TCN PLA + 15% or 20% plasticizer Cloisite 30B isolate Cloisite 30B Whey protein Whey protein isolate 20A, 30B Cloisite Na + , (BG) MMT-Na + Brea gum (OMSFM) fluorine mica PLA synthetic modified chitosan (CS) Oragnically weight MMT-Na + PLLA 4032D Cloisite 30B High and PLA 4060D Cloisite 30B PLA 4032D Cassava starch Cloiste Na + and 30B PLA 6752D 10A, 15A, 20A, 25A, 93A PBS C18-MMT Cloisite 30B PLA Cloisite 15A and modified MMT PLA Cloisite 30B as received and dried Thermoplastic potato starch (TPS) Cloiste Na+, 30B, 10A, 6A PLA Cloisite 25A PLA 2002D Nanofil 804 (OMMT) PCL MMT-Na + Chitosan Cloisite-Na + , 30B PLLA Wheat gluten OMMT Cloisite Na + , 20A, 30B Cloisite Na + (Yahiaoui et al., PCL OMMT 2014) PLA coated (Rodríguez-Marín et al., 2013) Banana flour and rice flour Cloisite-Na + , (Weizman et al., 2016) with 8wt% of whey protein Cloisite 30B, 15A OMMT (Sanchez-Garcia et al., 2010) PLA, PHBV, PHB, PCL MMT isolate (Sanchez-Garcia and Lagaron, 2010) PLA, PHBV and PCL NanoBioTer AC11 mica (Zenkiewicz and Richert, 2008) PLA 2002D Cloisite 30B, Nanofil 2	Nanoclay content (wt%) 0, 1, 3, 5 Solution blending Processing method Diffusing molecule Water vapor 0, 1, 2, 3 Solution blending Water vapor 0, 1, 3, 5 Solution blending Water vapor 0, 1, 5, 10 Solution blending Water vapor and carbon dioxyde 0, 1, 3, 5, 7, 9 Solution blending Oxygen and water vapor 0, 2, 4, 6 Melt blending and flat film extrusion Oxygen and water vapor 0, 5 Melt blending and compression molding Oxygen and water vapor 0, 5 0, 4, 6, 10 Solution blending Oxygen 0, 6, 10 0, 5 Melt blending with or not water injection Oxygen, carbon dioxide and water vapor 0, 3 Melt blending Water vapor 0, 1, 3, 5, 10 In-situ polymerization and casting Water vapor 0, 3, 6 Melt blending and compression molding Water vapor 0, 3 Melt blending Oxygen 0, 5 Melt blending and injection molding Oxygen 0, 3, 6 Melt blending Oxygen Melt blending and 0, 2, 5, 8, 10 Solution blending Oxygen 0, 2.5, 5, 7.5 Solution blending Water vapor 0, 3, 6, 9 0, 1, 2, 3, 5, 7, 10 Solution blending Melt blending and solution blending Water vapor Oxygen 0, 3, 6, 9 0, 1, 2, 3, 4, 5, 6 compression molding Water vapor 0, 2, 4, 8 Solution blending Water vapor 0, 2.5, 5, 7.5, 10 Solution blending Water vapor 0, 1, 3, 5 Melt blending Oxygen and carbon dioxide 0, 1, 3, 5, 10, 20, 30, 40 Solution blending Water vapor 0, 3 Melt blending Carbon dioxide 0, 3 Two-step 0, 3 In-situ polymerization 0, 1, 3, 5 Solution blending Water vapor 0, 3 Solution blending Water vapor 0, 0.5, 1, 2.5, 5 Solution blending Oxygen and water vapor 0, 0.1, 0.2, 0.4, 0.8 Solution blending Oxygen and carbon dioxide 0, 5, 10, 15 Melt blending and 0, 5, 10, 15 Melt blending and solution blending Water vapor 0, 3, 6, 9, 12, 15 Solution blending Oxygen and water vapor 0, 5 compression molding 0, 1, 3, 5, 10 Solution blending Water vapor 0, 1, 3 and blown extrusion 0, 1, 3, 5 compression molding Oxygen 0, 5, 10, 20 Solution blending Water vapor Melt blending and 0, 5 Solution blending Water vapor Solution blending Solution blending dioxide 0, 5 Oxygen and carbon 0, 1, 3, 5 Water vapor Water vapor Melt blending and 0, 4, 7, 10 Melt blending Oxygen 0, 3, 10 Solution blending Water vapor vapor 0, 1, 3, 5 Melt blending Oxygen and water vapor 0, 4 Melt blending Oxygen and water solution blending Water vapor 0, 3, 5 Melt blending and compression molding Water vapor 0, 5 Melt blending and compression Water vapor molding 0, 3.6 Melt blending 0, 1, 3, 5, 10 Oxygen 0, 1 Solution blending Oxygen, carbon dioxide and water 0, 5, 10, 15, 20 Melt blending and Oxygen and water compression vapor molding vapor 0, 5 Melt blending and Melt blending and Oxygen and water 0, 5 blowing vapor Water vapor In situ injection molding 0, 3.8, 7.9 Melt blending and compression 0, 6, 16, 30, 44 polymerization and Water vapor compression Oxygen molding molding 0, 5 Solution blending Water vapor Oxygen, carbon 0, 1, 2.5, 5, Solution blending dioxide and water 7.5, 10 vapor 0, 5 Solvent blending Water vapor 0, 2.5, 5, 10, 15, 20 Solution blending Melt blending and 0, 3 compression Water vapor molding Water vapor 0, 5 Solution blending Water vapor Solution blending Oxygen and water 0, 0.5, 1 and coating vapor 0, 5 Melt blending or solution blending Oxygen and water vapor vapor 0, 1, 5, 10 Solution blending Oxygen and water Oxygen, carbon 0, 5 Melt blending dioxide and water vapor

  .3.

	Table I.3: Analysis of the publications related to the study of mechanical properties of biodegradable polymers reinforced with 2:1 phyllosilicates. Reference Polymer matrix Type of nanoclay Nanoclay content (wt%) Processing method Mechanical property (Abdollahi et al., 2013) Sodium alginate MMT-Na + 0, 1, 3, 5 Solution casting E, TS, EB (Akin and Tihminlioglu, 2018) PHB, PHBV Cloisite 10A 0, 1, 2, 3 Solution casting E, TS, EB 2013) Sodium alginate MMT-Na + 0, 1, 3, 5 Solution casting TS, EB (Di Maio et al., 2014) PLA Cloisite 30B 0, 3, 6 Melt compounding E, EB (Scarfato et al., 2017) PLA Cloisite 30B 0, 4 Melt compounding E, TS, EB (Alboofetileh et al., (Avella et al., 2005) Potato starch MMT 0, 4 Solution casting E, TS, EB (Bae et al., 2009) Fish gelatin MMT-Na + 0, 1, 3, 5, 7, 9 Solution casting TS, EB (Bartel et al., 2017) PLA Cloisite 30B 0, 2, 4, 6 (Guaras et al., 2016) PCL/TPS MMT-Na + , Cloisite 30B 0, 1, 3 Melt intercalation and compression molding (Shahlari and Lee, 2012) PBAT/PLA Cloisite 30B 0, 3 Extrusion and EB hot-pressing E, TS, EB (Singh et al. 2010) PLLA Cloisite 30B 0, 1, 3, 5 Melt compounding E, TS, EB Melt compounding and flat film E, TS, EB (Hadj-Hamou et al., 2014) PBAT, PVC Cloisite 30B 0, 3 Extrusion and injection molding E, TS, EB Melt extrusion (Sinha Ray et al., PBSA Cloisite 30B 0, 3, 6, 9 and compression E, TS, EB 2005) molding extrusion (Boonprasith et al., 2013) PBS/Cassava starch MMT-Na + , Cloisite 30B 0, 5 Melt compounding and compression molding (Hassannia-Kolaee et al., 2016) Whey protein MMT-Na + 0, 1, 3, 5 Solution casting TS, EB (Slavutsky et al., Brea gum MMT-Na + 0, 1, 3, 5 Solution casting E, TS, EB 2014) E, TS, EB (Bordes et al., 2008) PHB Cloisite 30B 0, 1, 2.2, 5 Injection molding E, TS, EB PHBV 0, 0.6, 2.6, 4.4 (Botana et al., 2010) PHB MMT-Na + , Cloisite 30B 0, 5 Melt intercalation E, TS (Branciforti et al., 2013) PHBV Cloisite 30B 0, 3 Melt blending, extrusion and injection molding E, EB (Bruzaud et al., 2007) PHBV Cloisite 15A 0, 1, 2.5, 5 Solution casting E, TS, EB (Carli et al., 2011) PHBV Cloisite 30B, halloysite 0, 1, 3, 5 Melt compounding and injection molding E, TS, EB (Chang et al., 2002) PLA C16-MMT, DTA-MMT, Cloisite 25A 0, 2, 4, 6, 8 Solution casting E, TS, EB (Chen et al., 2002) PHBV OMMT 0, 1, 3 Solution intercalation E, TS (Chen et al., 2011) PBAT MMT-Na + , ODA-MMT 0, 1, 5, 10 Solution casting E, TS, EB (Chen and Yang, 2015) PBAT, MA-g-PBAT MMT-Na + , ODA-MMT, DHA-MMT 0, 3 Melt blending E, TS, EB (Chieng et al., 2010) 70%PBS/30%PBAT MMT-Na + , ODA-MMT, DDOA-MMT 0, 1, 3, 5, 7 (Issa et al., 2018) Sweet potato starch Nanomer MMT-Na + , 0, 3 Solution casting E, TS, EB (Kasirga et al., 2012) Chitosan MMTK-10 0, 0.5, 1, 2.5, 5 Solution casting TS, EB (Koh et al., 2008) PLA Cloisite 15A, 20A, 30B (Someya et al., 2003) PBS ODA-MMT, LEA-MMT, HEA-MMT, 0, 3 Melt E, TS, EB intercalation 0, 0.1, 0.2, 0.4, 0.8 ALA-MMT Solution casting E, TS, EB (Krishnamachari et al., 2009) PLA MMT-Na + , Cloisite 30B 0, 1 Melt E, TS (Someya et al., PBAT Melt mixing and MMT-Na + 0, 3, 5, 10 injection E, TS 2005) molding compounding (Kumar et al., 2010b) Soy protein isolate Cloisite 20A, 30B 0, 5, 10, 15 Extrusion and solution casting TS, EB MMT-Na + , (Sothornvit et al., Whey protein Cloisite 30B, 0, 5 Solution casting E, TS, EB 2009) 20A (Lavorgna et al., 2010) Chitosan MMT-Na + 0, 3, 10 Solution casting E, TS, EB (Lee et al., 2002) APES Cloisite 30B, 10A (Sothornvit et al., 2010) Whey protein Cloisite 30B 0, 5, 10, Solution casting TS, EB 20 0, 1, 3, 5, Melt blending E, TS, EB (Steeves et al., 2007) PBSA OMMT 0, 5 Extrusion E 10, 20, 30 (Lee and Kim, 2010) Soy protein isolate MMT-Na + 0, 3, 6, 9, Solution casting TS, EB (Strawhecker and Manias, 2000) PVA MMT-Na + 0, 2, 4, 6, 8 Solution casting E, TS 12, 15 (Luduena et al., 2013) PCL MMT-Na + , Cloisite 30B, 20A 0, 5 Melt compounding E, TS, EB (Tang et al., 2008) Corn starch, wheat starch, potato starch Melt blending MMT, Nanomer 0, 3, 6, 9 and solution TS, EB I30E casting and compression molding (Luecha et al., 2010) Zein MMT Nanomer I.34 TCN 0, 1, 3, 5, 10 Solution casting, Blown extrusion (Thellen et al., 2005) PLA Cloisite 25A 0, 5 Melting and TS, EB blowing E, TS, EB (Martino et al., 2010) PLA+15%plasticizer, PLA+20%plasticizer Cloisite 30B 0, 1, 3 Compression molding (Tunc et al., 2007) Wheat gluten Nanofil EXM 757 0, 2.5, 5, Solution casting E, TS, EB 7.5 E, EB (Memis et al., 2017) Fenugreek seed MMT-Na + , Nanomer I.44 P 0, 2.5, 5, 7.5 (Xie et al., 2016) PBAT OLDH 0, 0.5, 1, Solution casting TS, EB 2, 4, 6, 8 Solution casting TS, EB (Mohan et al., 2016) Corn starch Cloisite 30B 0, 1, 2, 3, 5, 7, 10 (Zembouai et al., PHBV/PLA Cloisite 30B 0, 3 Melt mixing E, TS, EB 2014) Solution casting E, TS, EB (Zhao et al., 2013) PLA/PHBV Cloisite 30B 0, 4 E, TS, EB Melt compounding and compression molding E, TS (Chivrac et al., 2010) PBAT MMT-Na + , Cloisite 20A 0, 3, 6, 9 Melt intercalation E, TS, EB (Choi et al., 2003) PHBV Cloisite 30B 0, 1, 2, 3 Melt blending and injection E, TS, EB (Müller et al., 2012) Cassava starch MMT-Na + , Cloisite 30B 0, 3, 5 Extrusion and hot-pressing E, TS, EB (Park et al., 2002) Potato starch The analysis of these publications dealing with mechanical properties of bio-MMT-Na + , Cloisite 30B, 0, 5 Injection molding TS, EB nanocomposite materials is presented in Figure I.10, which reports the augmentation or 10A, 6A (Phua et al., 2012) PBS Melt mixing and reduction in mechanical property according to the percentage of 2:1 phyllosilicate. The OMMT Nanomer I.30TC 0, 2, 10 compression E, TS, EB variation rate of each characteristic value is calculated by the following equation: molding molding (Chung et al., 2010) Corn starch MMT-Na + 0, 5 Hot-pressing E, TS, EB (Corrêa et al., 2012) PHBV Cloisite 30B 0, 3 Injection (Rhim et al., 2006) Chitosan MMT-Na + , Cloisite 30B 0, 5 Solution casting TS, EB (I.4) MMT-Na + , E, TS (Rhim et al., 2009) PLLA Cloisite 30B, 0, 5 Solution casting TS, EB molding (Daitx et al., 2015) PHBV MMT-Na + 0, 3 Melt compounding molding (Dean et al., 2009) PBSA FHT-MAE, Na-FHT-MTE, Na-FHT-MEE 0, 1, 2.5, 5 Injection molding E, TS, EB and film blowing 2014) PHBV/PBAT Halloysite 0, 5 compounding TS, EB (Scarfato et al., Melt Na-FHT, Na-(Rodríguez-Marín et al., 2013) flour MMT 0, 5 Slution casting TS, EB Banana flour, rice and injection E, TS, EB 20A (Rhim, 2011) Agar MMT-Na + Where is the mechanical property ( , tensile strength or elongation at 0, 2.5, 5, 10, 15, 20 Solution casting TS, EB break) of polymer nanocomposite and , the mechanical property of the neat polymer.

Table I

 I

		grafted PCL/TPS			compression
		(70/5/25 wt%)			molding
						BOD -Aqueous
					Melt	conditions -EN ISO
	(Iggui et al., 2015)	PHBV	Cloisite 30B	0, 1, 3, 5	compounding and cast-film	14851 method and weight loss Compost
					extrusion	medium -EN ISO
						20200 standards
	(Issa et al., 2018)	Sweet potato starch	MMT	0, 3 Solution casting	Weight loss -Soil or Enzymatic media
	(Lee et al.,	Biodegradable	Cloisite 30B,	0, 3, 5,	Melt	Weight loss -Activated
	2002)	APES	10A	10, 20, 30	intercalation	soil
	(Luduena et al., 2013)	PCL	Cloisite Na+, 30B, 20A	0, 5 Melt blending	Weight loss -Indoor soil burial
	(Maiti et al., 2003)	PHB	MMT, fluoromica	0, 3 Melt extrusion	Weight loss -Composted manure
	(Maiti et al., 2007)	PHB	Fluoromica	0, 2	Melt blending and compression molding	Weight loss -Composted manure
	(Mohanty and Nayak, 2010)	PBAT, MA-grafted PBAT	MMT-Na+, Cloisite 20A, 30B, Bentonite 109	0, 1, 3, 5 Melt blending	BOD -Compost medium -ASTM D5338
	(Shayan et al., 2015)	PLA/MTPS (70/30, 60/40, 50/50)	Cloisite 30B	0, 2, 4	Melt blending and compression molding	Weight loss -Activated sludge -ASTM D5271-02
	(Sinha Ray et al., 2003)	PLA	Fluorine mica	0, 4 Melt blending	Respirometric method -Compost medium
	(N. K. Singh et al., 2010)	PCL	Cloisite 30B 18A	0, 4 Solvent blending	Weight loss -Organic manure
	(Steeves et al., 2007)	PBSA	Modified MLS	0, 5 Melt extrusion	Respirometric method -Soil medium -ASTM standard D5988
	(Swain et al., 2014)	Starch rice	Cloisite 30B	0, 5, 8, 10 Solution casting	Weight loss -Sludge water
	(Thellen et al., 2005)	PLA	Cloisite 25A	0, 5	Compounding and blown-film processing	Respirometric method -Soil medium
	(Wang et al., 2005)	PHBV	PMMT	0,3, 5, 10	Solution blending	Weight loss -Soil suspension
	Reference (Wu et al., 2009)	Polymeric matrix PCL	Type of nanoclay MMT-Na+	Nanoclay (wt%) content 0, 1, 2, 4 Solution casting method Processing	evaluation method Biodegradability Weight loss -Compost medium and phosphate buffered solution
	(Balaguer et al., 2016)	PLA	DHA-MMT	0, 3.9 Melt blending	Respirometric method -Composting conditions -ISO 14855-1: 2012
	(Chen and Yang, 2015)	PBAT, MA-grafted PBAT	MMT, ODA-MMT, DHA-MMT	0, 3 Melt blending	Weight loss -Enzymatic degradation
	(Fukushima et al., 2009)	PLA	Cloisite 30B, Nanofil 804	0, 5	Melt blending and compression molding	Weight loss -Industrial compost
	(Fukushima et al., 2010)	PCL	Cloisite 30B, Nanofil 804	0, 5	Melt blending and compression molding	Weight loss -Industrial compost
	(Guaras et al., 2016)	PCL/TPS (75/25 wt%), PCL/MA-	Cloisite Na+, 30B, 20A	0, 1, 3	Melt intercalation and	Weight loss -Soil medium

.4: Analysis of the publications related to the study of biodegradability of biodegradable polymers reinforced with 2:1 phyllosilicates.

  , T., Xie, T.,Yang, G., 2009. Preparation and characterization of poly( -caprolactone)/Na+-MMT nanocomposites. Appl. Clay Sci. 45, 105 110. https://doi.org/10.1016/j.clay.2009.02.009 Xia, Y., Rubino, M., Auras, R., 2014. Release of nanoclay and surfactant from polymer-clay nanocomposites into a food simulant. Environ. Sci. Technol. 48, 13617 13624. https://doi.org/10.1021/es502622c Xie, J., Zhang, K., Wu, J., Ren, G., Chen, H., Xu, J., 2016. Bio-nanocomposite films reinforced with organomodified layered double hydroxides: Preparation, morphology and properties. Appl. Clay Sci. 126, 72 80. https://doi.org/10.1016/j.clay.2016.02.025 Xu, J.-T., Zhao, Y.-Q., Wang, Q., Fan, Z.-Q., 2005. Isothermal crystallization of intercalated and exfoliated polyethylene/montmorillonite nanocomposites prepared by in situ polymerization. Polymer (Guildf). 46, 11978 11985. https://doi.org/10.1016/j.polymer.2005.10.004 Yahiaoui, F., Benhacine, F., Ferfera-Harrar, H., Habi, A., Hadj-Hamou, A.S., Grohens, Y., 2014. Development of antimicrobial PCL/nanoclay nanocomposite films with enhanced mechanical and water vapor barrier properties for packaging applications. Polym. Bull. https://doi.org/10.1007/s00289-014-1269-0 Zembouai, I., Bruzaud, S., Kaci, M., Benhamida, A., Corre, Y.-M., Grohens, Y., Lopez-Cuesta, J.-M., 2014. Synergistic effect of compatibilizer and cloisite 30B on the functional properties of poly(3-hydroxybutyrateco-3-hydroxyvalerate)/polylactide blends. Polym. Eng. Sci. 54, 2239 2251. https://doi.org/10.1002/pen Zenkiewicz, M., Richert, J., 2008. Permeability of polylactide nanocomposite films for water vapour, oxygen and carbon dioxide. Polym. Test. 27, 835 840. https://doi.org/10.1016/j.polymertesting.2008.06.005 Zhao, H., Cui, Z., Wang, X., Turng, L.S., Peng, X., 2013. Processing and characterization of solid and

renforcement des propriétés barrières et mécaniques des matériaux biodégradables destinés à emballage alimentaire -argiles. Globalement, leur ajout renforce Pour les propriétés mécaniques, plus particulièrement la résistance étudiée dans ce travail grâce au module de Young, à la résistance à la traction et à paramètres en même temps et un compromis doit donc être trouvé. Cette étude a également montré que liés à l ajout de ces renforts. nanocomposites au contact alimentaire sont étudiés par la migration globale, la migration des nano-argiles ou de ces éléments constitutifs ou encore la migration spécifique de molécules. nano-) et les travaux réalisés sur la migration des nano-argiles ont ous leur forme intègre mais libérer leurs éléments constitutifs (silicium, aluminium et magnésium) après solubilisation dans le simulant alimentaire. Concernant la migration spécifique, elle est peu étudiée dans les matériaux biodégradables et les seules études recensées matériaux en PLA. Les risques liés à -argiles sur la biodégradation et écotoxicité de ces matériaux -argiles sur ce paramètre toxicité des nano-argiles mais il est également difficile de tirer des conclusions générales. Ainsi, les bé -argiles dans des matériaux biodégradables sont bien démontrés mais il y a un manque de connaissances sur, alimentaire de ces matériaux et autre part, leur fin de vie, notamment concernant de ces nanoparticules résiduelles. Wumicrocellular poly(lactic acid)/polyhydroxybutyrate-valerate (PLA/PHBV) blends and PLA/PHBV/Clay nanocomposites. Compos. Part B Eng. 51, 79 81. https://doi.org/10.1016/j.compositesb.2013.02.034 CHAPTER II. MATERIALS AND METHODS Chapter II. Materials and Methods Ce chapitre décrit les matériaux et les méthodes utilisés au cours de cette thèse. Tout la formulation des matériaux bio-nanocomposites sont décrits, les matrices polymères : PBSA et PHBV et les renforts : nano-argiles modifiées méthodes utilisées pour leur caractérisation (structurale, thermique et transfert aux gaz et à la . Une partie est ensuite

Table II .

 II 1: Main characteristics of PBSA and PHBV polymers.

		Method	Unity	PBSA	Value	PHBV
	General properties
	Density	ISO 1183		1.24		1.23
	MFI (190°C; 2.16 kg)	ISO 1183	g/10 min	4 -5		5 -10
	Optical properties			Translucent		Opaque
	Thermal properties
	Melting temperature	DSC	°C	88		170 -176
	Mechanical properties
	Stress at elastic limit	ISO 527	MPa	/		18.7
	Strain at elastic limit	ISO 527	%	/		1
	Stress at break	ISO 527	MPa	23		39,6
	Strain at break	ISO 527	%	350		3.2
		ISO 527	MPa	290		4200
	Flexural modulus	ISO 178	MPa	/		3800
	Charpy shock (unnotched)	ISO 179	kJ/m²	NB		5
	Hardness (15s)	ISO 868	Shore D	51		76

Table II .

 II 2: List of additives.

	Category	Code	Physical properties	CAS number	M (g.mol -1 )
	High molar	Uvitex OB	Non-volatile	7128-64-5	430.56
	mass	Irganox 1076	Non-volatile polar	2082-79-3	530.86
	contaminants	Irganox 1010	Non-volatile	6683-19-8	1177.63
		Toluene	Volatile non-polar	108-88-3	92.14
		Chlorobenzene	Volatile polar	108-90-7	112.56
	Low molar mass contaminants	Methyl salicylate Biphenyl Phenyl cyclohexane Benzophenone	Non-volatile polar Volatile non-polar Non-volatile non-polar Non-volatile polar	119-36-8 92-52-4 827-52-1 119-61-9	152.15 154.21 160.26 182.22
		Methyl stearate	Non-volatile non-polar	112-61-8	298.50
		DEHA	Non-volatile polar	103-23-1	370.57
	1.4 Food simulants
	1.4.

1 Liquid food simulant

  Two different microbial medium were used. The Potato Dextrose Agar (PDA) and De Man, Rogosa and Sharpe agar (MRS) plates (petri 90 mm) were delivered by VWR International. The PDA agar plates were inoculated with Penicillium Candidum supplied by Agro direct, France under the trade name PC NEIGE LYO 2 D. The MRS agar plates were inoculated with Lactobacillus Bulgaricus supplied by Pasteur Institute under the name Lactobacillus delbrueckii bulgaricus (CRBIP24.64). Anaerocult ® P sachets supplied by VWR International were used to maintain anaerobic conditions necessary for the growth of L. Bulgaricus. They contain components, which chemically bind oxygen quickly and completely, creating an

	1.4.2 Microbial food simulant
	Selected food simulant	Corresponding food categories
	10% ethanol (simulant A)	Hydrophilic foods
	3% acetic acid (simulant B)	Acidic foods
	20% ethanol (simulant C)	Alcoholic foods with an alcohol content of up to 20 %
	50% ethanol (simulant D1)	Alcoholic foods with an alcohol content of above 20 % or oil in water emulsion
	95% ethanol (simulant D2)	Fatty foods
	Isooctane (simulant D2)	Fatty foods
	Tenax (simulant E)	Dry foods

Six different food simulants (Table

II

.3) were chosen according to European Commission for food contact materials (EU 10/2011)

(European Commission, 2011)

in order to evaluate the suitability for food contact of bio-nanocomposite materials, each food simulant being representative of a food category. Acetic acid, ethanol and 2,2,4 trimethyl pentane (isooctane) were commercially provided by Sigma-Aldrich with a purity > 99% and were used to prepare these food simulants. 95% ethanol and isooctane were employed as substitutes of simulant D2 (initially vegetable oil) for compliance testing in contact with fatty food. Poly(2,6-diphenyl-pphenylene oxide) was supplied by Sigma-Aldrich under the trade name Tenax (60/80 mesh) and was used as solid food simulant (simulant E).

Table

II

.3: Selected food simulants and their corresponding food behavior to simulate.

  La migration globale vise à mesurer la quantité de substances non volatiles cédée par le matériau aux simulants de denrées alimentaires. La migration spécifique a pour but de déterminer la née, cédée par le matériau aux simulants alimentaires. Les propriétés de transfert peuvent être étudiées par ces tests de migration spécifique en réalisant des cinétiques de désorption de ces substances/additifs. Leur diffusivité dans la matrice peut être concentration de la substance dans le matériau et le simulant alimentaire.

	Chapter III. Impact of nanoclays on the properties of transfer and inertia
	plastiques conventionnels peuvent être applicables au cas particulier des polymères of materials biodégradables.
	-argiles sur les Enfin, la troisième partie (Article 3) a pour but de déterminer si les polymères
	biodégradables, plus particulièrement le PBSA, peut être envisagé pour une utilisation au
	globale et spécifique, dont les conditions sont définies dans le règlement européen N° 10/2011. tests de migration adaptés à cette étude sont mis en place en remplaçant les simulants
	alimentaires liquides habituellement utilisés par des simulants alimentaires microbiens. Ainsi,
	des géloses ensemencées avec deux microorganismes différents ont été sélectionnées. Des tests
	d	sont effectués afin de déterminer
		-argiles sur la migration de ces molécules. En
	parallèle, la dégradation du matériau est suivie au cours du temps de contact par perte de masse
	et observations microscopiques.
	CHAPTER III. IMPACT OF Migration Migration NANOCLAYS ON THE PROPERTIES -argiles sur les propriétés additifs nanoparticules
	OF TRANSFER AND INERTIA OF Exposition Danger Perméabilité 2 Perméabilité au CO 2 Biodégradation	Ecotoxicité
	règlement européen N° 10/2011 dédié aux matériaux non-dégradables sont appliquées. Un MATERIALS -à-des nano-argiles tend à augmenter la diffusivité des additifs. En augmentant le chemin de Propriétés barrières Gain matériau Fin de vie Usage
		illeurs été obtenue dans le cas des polymères
	Bénéfices	Risques	-feuillets
	gonflement du polymère dans les simulants alimentaires sont étudiés.
	PBSA et PHBV est étudiée. Le caractère biodégradable de ces matériaux, qui les rend instable
	dans le temps est un atout lors de leur fin de vie mais la question est de savoir quel en est
		-à-dire après leur production mais également après
		dié. Le but étant de déterminer si

Table III

 III

		.1: List of selected additives.
	Category	Code	Physical properties	CAS number	M (g.mol -1 )
	High molar	Uvitex OB	Non-volatile	7128-64-5	430.56
	mass	Irganox 1076	Non-volatile polar	2082-79-3	530.86
	contaminants	Irganox 1010	Non-volatile	6683-19-8	1177.63
		Toluene	Volatile non-polar	108-88-3	92.14
		Chlorobenzene	Volatile polar	108-90-7	112.56
		Methyl salicylate Non-volatile polar	119-36-8	152.15
	Low molar mass contaminants	Biphenyl Phenyl cyclohexane	Volatile non-polar Non-volatile non-polar	92-52-4 827-52-1	154.21 160.26
		Benzophenone	Non-volatile polar	119-61-9	182.22
		Methyl stearate	Non-volatile non-polar	112-61-8	298.50
		DEHA	Non-volatile polar	103-23-1	370.57

Table III .

 III 2: LDPE and PET specific parameters.

	Polymer
	LDPE	10	0
	PET	2.2	1577
	3 Results and discussion

Table III

 III

	.3: Solvent uptake of films after 10 days of contact with food simulant at
			40°C.
			Solvent uptake (%)
	Sample	Food simulant	95% ethanol Isooctane
	PBSA		3.7 ± 0.5	0
	PBSA NCP	4.7 ± 0.1	0
	PHBV		3.5 ± 0.1	1.3 ± 0.1
	PHBV NCP	3.8 ± 0.2	0.5 ± 0.1

Table III

 III

	.4: Degree of crystallinity of film samples without contact and after 10 days of
	contact with 95% ethanol or isooctane at 40°C.
		Degree of crystallinity (XC) (%)
	Sample	Without contact	95% ethanol	Isooctane
	PBSA	38.2 ± 0.0	36.7 ± 0.1	39.0 ± 0.1
	PBSA NCP	34.9 ± 0.4	34.1 ± 0.3	35.7 ± 0.1
	PHBV	62.8 ± 1.6	62.0 ± 0.1	62.9 ± 0.1
	PHBV NCP	55.0 ± 0.4	53.6 ± 0.3	55.7 ± 0.1

Table III .

 III 5: Crystallization temperature (Tc), melting temperature (Tm) and crystallinity degree (XC) of neat and nanocomposite PBSA and PHBV and the corresponding aged materials.

			Tc (°C)	Tm (°C)	XC (%)
	PBSA	Control UV treated	42.3 ± 0.1 39.8 ± 3.7	86.7 ± 0.4 88.4 ± 1.8	45.6 ± 0.6 53.7 ± 1.9
	PBSA NCP	Control UV treated	44.0 ± 0.3 33.4 ± 0.1	87.2 ± 0.6 86.6 ± 1.4	45.8 ± 0.5 53.4 ± 2.3
	PHBV	Control UV treated	122.1 ± 0.1 117.8 ± 0.1	169.1 ± 0.7 166.1 ± 0.4	54.1 ± 1.2 55.5 ± 0.8
	PHBV NCP	Control UV treated	122.3 ± 0.1 119.9 ± 0.4	171.5 ± 0.9 168.2 ± 1.8	52.7 ± 0.9 53.5 ± 0.7

  The selected clay was a commercial organo-modified montmorillonite, the Anaerocult ® P sachets supplied by VWR International were used to maintain anaerobic conditions necessary for the growth of L. Bulgaricus. They contain components, which chemically bind oxygen quickly and completely, creating an oxygen free environment and a CO2 atmosphere.

	An array of additives with a variety of chemical and physical properties as described in
	FDA and EFSA Regulations (Food and Drug Administration, 2006; European Food Safety
	Authority, 2011) was chosen. They include: toluene, chlorobenzene, methyl salicylate,
	biphenyl, phenyl cyclohexane, benzophenone, methyl stearate and DEHA, all provided from
	Sigma-Aldrich.
	Cloisite 30B (C-30B) supplied by BYK additives & instruments, Germany.
	Two different microbial medium were used. The Potato Dextrose Agar (PDA) and De Man,
	Rogosa and Sharpe agar (MRS) plates (petri 90 mm) were delivered by VWR International.

The PDA agar plates were inoculated with Penicillium Candidum (P. Candidum) supplied by Agro direct, France under the trade name PC NEIGE LYO 2 D. The MRS agar plates were inoculated with Lactobacillus Bulgaricus (L. Bulgaricus) supplied by Pasteur Institute under the name Lactobacillus delbrueckii bulgaricus (CRBIP24.64).

Table IV

 IV

	.2: Tolerated consumer exposure to additives by age range according to the
			Regulation (EC) No 10/2011.
			Tolerated consumer exposure (mgadditive.day -1 )
	Age range of consumers	Methyl salicylate	Benzophenone	DEHA	Uvitex OB Irganox 1076
	0 -10 years	50.65	1.01	30.39	1.01	10.13
	11 -17 years	64.49	1.29	38.69	1.29	12.90
	18 -79 years	88.24	1.77	52.94	1.77	17.65

Table IV .

 IV 3: Consumer exposure to additives by age range according to food diet if all foods and beverages are packed using biomaterials. PHBV or PHBV NCP material. The values are logically lower than those in scenario (1) since it simply reflects a lower level of consumption of foods packed in these materials. The changes generated by nanoclays remain the same in proportion and the consumer exposure to benzophenone decreased by 30% but remains too high in all cases.TableIV.4: Consumer exposure to additives by age range according to food diet if all foods are packed using biomaterials (without considering beverages).

	Consumer exposure (mgadditive.day -1 ) Benzophenone DEHA Uvitex OB 2.59 ± 0.07 1.40 ± 0.14 0.62 ± 0.05 0.37 ± 0.17 Irganox 1076 2.90 ± 0.09 1.59 ± 0.11 0.77 ± 0.00 0.56 ± 0.02 2.69 ± 0.12 1.35 ± 0.11 0.52 ± 0.01 0.38 ± 0.02 2.14 ± 0.09 0.90 ± 0.07 0.48 ± 0.03 0.30 ± 0.03 2.99 ± 0.08 1.48 ± 0.15 0.60 ± 0.06 0.32 ± 0.02 3.34 ± 0.10 1.67 ± 0.13 0.73 ± 0.00 0.46 ± 0.02 3.28 ± 0.15 1.46 ± 0.12 0.52 ± 0.01 0.33 ± 0.02 2.57 ± 0.09 0.94 ± 0.06 0.49 ± 0.03 0.26 ± 0.03 4.28 ± 0.12 1.78 ± 0.28 0.62 ± 0.07 0.32 ± 0.02 4.67 ± 0.13 1.87 ± 0.15 0.76 ± 0.00 0.47 ± 0.02 4.89 ± 0.21 1.66 ± 0.14 0.54 ± 0.01 0.34 ± 0.02 3.77 ± 0.14 1.05 ± 0.06 0.51 ± 0.03 0.26 ± 0.03 The consumer exposure to additives on the basis of the scenario (2) was calculated in Table Age range of consumers Additive Material Methyl salicylate 0 -10 years PBSA 3.23 ± 0.23 PBSA NCP 3.68 ± 0.17 PHBV 3.05 ± 0.30 PHBV NCP 2.53 ± 0.14 11 -17 years PBSA 3.85 ± 0.27 PBSA NCP 4.39 ± 0.20 PHBV 3.76 ± 0.37 PHBV NCP 3.11 ± 0.19 18 -79 years PBSA 5.51 ± 0.41 PBSA NCP 6.40 ± 0.30 PHBV 5.53 ± 0.56 PHBV NCP 4.46 ± 0.26 IV.4 considering that all the foods consumed except beverages are packed in PBSA, PBSA Age range of consumers Additive Material Methyl salicylate Benzophenone DEHA Uvitex OB Irganox 1076 0 -10 years PBSA 2.32 ± 0.15 1.87 ± 0.05 1.14 ± 0.08 0.55 ± 0.04 0.35 ± 0.02 PBSA NCP 2.60 ± 0.11 2.11 ± 0.07 1.33 ± 0.10 0.68 ± 0.00 0.53 ± 0.01 PHBV 2.16 ± 0.20 1.86 ± 0.10 1.10 ± 0.15 0.45 ± 0.01 0.36 ± 0.02 PHBV NCP 1.83 ± 0.11 1.51 ± 0.15 0.76 ± 0.20 0.40 ± 0.02 0.28 ± 0.02 11 -17 years PBSA 2.62 ± 0.16 2.02 ± 0.05 1.10 ± 0.09 0.48 ± 0.04 0.28 ± 0.02 PBSA NCP 2.93 ± 0.13 2.26 ± 0.07 1.29 ± 0.11 0.60 ± 0.00 0.42 ± 0.01 PHBV 2.57 ± 0.23 2.17 ± 0.11 1.10 ± 0.14 0.41 ± 0.01 0.29 ± 0.01 PHBV NCP 2.16 ± 0.14 1.71 ± 0.14 0.73 ± 0.17 0.38 ± 0.02 0.23 ± 0.02 18 -79 years PBSA 3.57 ± 0.25 2.74 ± 0.08 1.15 ± 0.18 0.42 ± 0.03 0.26 ± 0.01 PBSA NCP 4.10 ± 0.18 2.94 ± 0.07 1.20 ± 0.10 0.52 ± 0.00 0.39 ± 0.01 PHBV 3.67 ± 0.35 3.14 ± 0.13 1.03 ± 0.14 0.35 ± 0.01 0.27 ± 0.01 PHBV NCP 2.95 ± 0.18 2.43 ± 0.17 0.69 ± 0.18 0.31 ± 0.02 0.21 ± 0.02 Table IV.5 summarizes the consumer exposure according to the scenario (3) considered as the most realistic hypothesis. Only the foods already packed in plastic (tray, pot, bottle or bag) NCP, Consumer exposure (mgadditive.day -1 ) at this time were considered (Annex II, Table IV.8). Again, it is consistent to see all exposure

Table IV .

 IV 5: Consumer exposure to additives by age group according to food diet if foods are packed using biomaterials only when it is realistic.

				Consumer exposure (mgadditive.day -1 )
	Age range of consumers	Additive Material	Methyl salicylate	Benzophenone	DEHA	Uvitex OB	Irganox 1076
		PBSA	1.60 ± 0.10	1.26 ± 0.03	0.84 ± 0.04 0.33 ± 0.04 0.16 ± 0.01
	0 -10 years	PBSA NCP PHBV	1.79 ± 0.09 1.46 ± 0.14	1.50 ± 0.06 1.22 ± 0.08	1.07 ± 0.10 0.40 ± 0.00 0.23 ± 0.01 0.91 ± 0.13 0.29 ± 0.01 0.17 ± 0.01
		PHBV NCP 1.27 ± 0.09	0.98 ± 0.12	0.57 ± 0.16 0.28 ± 0.02 0.13 ± 0.02
		PBSA	1.86 ± 0.10	1.41 ± 0.03	0.89 ± 0.03 0.36 ± 0.04 0.18 ± 0.01
	11 -17 years	PBSA NCP PHBV	2.05 ± 0.09 1.79 ± 0.16	1.64 ± 0.07 1.47 ± 0.09	1.13 ± 0.10 0.45 ± 0.00 0.26 ± 0.01 0.97 ± 0.13 0.32 ± 0.00 0.19 ± 0.01
		PHBV NCP 1.56 ± 0.11	1.16 ± 0.11	0.61 ± 0.14 0.31 ± 0.02 0.15 ± 0.02
		PBSA	1.72 ± 0.09	1.28 ± 0.03	0.77 ± 0.03 0.30 ± 0.03 0.16 ± 0.01
	18 -79 years	PBSA NCP PHBV	1.89 ± 0.08 1.71 ± 0.14	1.47 ± 0.06 1.36 ± 0.08	0.98 ± 0.09 0.37 ± 0.00 0.23 ± 0.01 0.83 ± 0.12 0.26 ± 0.01 0.16 ± 0.01
		PHBV NCP 1.47 ± 0.10	1.07 ± 0.11	0.53 ± 0.15 0.25 ± 0.02 0.13 ± 0.02

of environmental benefits related to the addition of nanoclays in biopolyesters-based food packaging (Article 5)

  Anaïs Lajarrige a , Sébastien Gaucel a , Valérie Guillard a , Nathalie Gontard a and Stéphane Peyron a

	Fish dishes Vegetable-based dishes Dishes made from potatoes, cereals or legumes Fast foods (sandwichs, pizzas, toasts, etc.) Sandwiches, toasted bread pizza and the like containing any kind of foodstuff: 10% ethanol In an oily medium: 10% ethanol In an aqueous medium: 20% ethanol -3% acetic acid (*) Vegetables in the form of purée, preserves, pastes or in its own juice (including pickled and in brine): 20% ethanol 10% ethanol Spices and seasonings in the natural state such as cinnamon, cloves, powdered mustard, pepper, Assessment a JRU IATE 1208, CIRAD, INRA, Montpellier SupAgro, University of Montpellier, 2 place
	Condiments, spices, sauces Pierre Viala, F-34060 Montpellier 01, France vanilla, saffron, salt and other: poly(2,6-diphenyl-p-phenylene oxide) With fatty character e.g. mayonnaise, sauces derived from mayonnaise, salad creams and other
	Meat substitutes 1 Introduction	oil/water mixtures e.g. coconut based sauces: 10% ethanol -3% acetic acid (*) 10% ethanol
	Prepared dishes and infant desserts	20% ethanol
	Infant drinks	Milk powder including infant formula (based on whole milk powder): poly(2,6-diphenyl-p-phenylene oxide)
	Annex II
	Table IV.6: Scenario 1	Table IV.7: Scenario 2	Table IV.8: Scenario 3
	Food group	Food group	Food group
	Cereal and cereal bar for breakfast	Cereal and cereal bar for breakfast	Cereal and cereal bar for breakfast
	Pasta, rice, grain	Pasta, rice, grain	Pasta, rice, grain
	Pastry, cake, biscuit	Pastry, cake, biscuit	Pastry, cake, biscuit
	Milk	Milk	Milk
	Yoghurt and cottage cheese	Yoghurt and cottage cheese	Yoghurt and cottage cheese
	Cheese	Cheese	Cheese
	Milky desserts	Milky desserts	Milky desserts
	Ice cream, sorbet, frozen dessert	Ice cream, sorbet, frozen dessert	Ice cream, sorbet, frozen dessert
	Fats	Fats	Meat

Table IV .

 IV 10: Characteristics of the materials used in the study.The global warming impact related to the package of fresh poultry in PP, PHBV or PHBV NCP trays was evaluated using global warming impact values of PP, PHBV and C-30B recovered from the literature (TableIV.10). Yates et al., shown that several LCAs about bioplastics including PHBV are available in the literature and that the results can be very disparate

	Material	Density (g.cm -3 )	Weight (g) (18.6 cm 3 of tray)	Global warming impact (kg CO2eq/kg)
	PP	0.91	16.93	2.29 (David, 2019)
	PHBV	1.23	22.88	3.47 (David, 2019)
	C-30B	1.98	-	1.41 (Joshi, 2008)
	PHBV NCP	1.270	23.62	3.37 (calculated)

Table IV

 IV

		.11: Oxygen and carbon dioxide permeabilities
	Sample	PO2 (mol.m/(m².s.Pa)) PCO2 (mol.m/(m².s.Pa))
	PBSA	1.62×10 -16 ± 0.02×10 -16 2.62×10 -15 ± 0.09×10 -15

Table IV .

 IV 12: Summary of global warming impacts of the consumption of fresh poultry meat in accordance with the different case studies (written in brackets are the reduction percentages compared to the use of PP).

		Global warming impact (kg CO2eq/kg)
		Initially for 103 trays/person/year
	PP	PHBV		PHBV NCP
	126.59	130.77		130.79
		Impact of material savings
		PHBV	PHBV NCP
	125.04 (-1.2%)	124.49 (-1.7%)
		Impact of losses and waste
	Distribution	Consumption
	PHBV	PHBV NCP	PHBV	PHBV NCP
	125.47 (-0.9%) 125.49 (-0.9%) 124.90 (-1.3%) 123.02 (-2.8%)

Table V .

 V 1 gathers the characterizations made on the different samples: the film thickness,

the carbon content of cellulose, neat and nanocomposite PBSA and PHBV and the nanoclay content of the two nano-reinforced films.

Table V .

 V 1: Characterization of materials: thickness and composition.

	Sample	Thickness (µm)	C (%)	Nanoclay content* (%)
	Cellulose	-	40.6	-
	PBSA	180 ± 10	56.7
	PBSA NCP	220 ± 25	55.1	4.6 ± 0.1
	PHBV	145 ± 20	55.6	-
	PHBV NCP	140 ± 10	53.3	5.3 ± 1.2
	* Inorganic part of the nanoclays excluding the alkylammonium.

Remerciements

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Funding This work has been carried out in the framework of the Labex SERENADE (ANR-11-LABX-0064) funded by the « Investissements d'Avenir » French Government program managed by the French National Research Agency (ANR).

Compliance with ethical standards

The authors declare that there is no conflict of interest regarding the publication of this paper.

Chapter III. Impact of nanoclays on the properties of transfer and inertia of materials

Funding

The project leading to this publication has received funding from Excellence Initiative of Aix-

Funding

The project leading to this publication has received funding from Excellence Initiative of Aix-Marseille University -A*MIDEX, a French "Investissements d'Avenir" program, through its associated Labex SERENADE project.

Abbreviations

PBSA: poly(butylene succinate-co-butylene adipate) column guard was used. Compounds were separated with 55% acetonitrile at a flow rate of 0.3 mL.min -1 at 50°C. Absorbance was recorded at 230 nm to quantify the additive concentration. An external calibration was made in ethanol with concentrations of 1.25 to 40 mg.L -1 .

Consumer exposure

Five additives were selected: methyl salicylate, benzophenone, DEHA, Uvitex OB and Irganox 1076, all being approved for food contact and having a SML (Table IV.1).

The migration from food packaging into food or food simulant, in conjunction with the consumer food diet, determine the consumer exposure to these additives. In this way, each category of food consumed was associated to a food simulant according to the Regulation (EC)

No 10/2011. The consumer food diet was extracted from the data on food consumption and eating habits of individuals living in France issued from a food consumption surveys conducted by French Agency for Food, Environmental and Occupational Health & Safety in 2014[START_REF] Gianfreda | Enzymes of importance to rhizosphere processes[END_REF](ANSES, 2017). Three classes of population were studied according to their age rating: 0-10 years, 11-17 years and 18-79 years. The consumer exposure was calculated according to different case studies for the material used for the packaging. Thus, contamination level induced by the contact with PBSA, PHBV and their corresponding nanocomposites were distinctly taken into account. The consumer exposure was calculated using the following equations:

-First, the surface, S (dm².day -1 ) of material used to pack the foods eaten daily is calculated using the daily food consumption (kg.d -1 ) and the surface/mass ratio of 6 dm².kg -1 defined by the Regulation. They consider that 6 dm² of film is used to pack 1 kg of food.

-Then, the consumer exposure (mgadditive.d -1 ) is calculated using the specific migration (mgadditive.dm -2 material) and the surface of material calculated previously (Eq. IV.1). The specific migration is determined considering that materials are contaminated with 500 mg.kg -1 of each additive and using the results of the relative migration.

In the case where the food or the food category is associated with several simulants, the migration value considered is the highest value following a "worst case" scenario of consumer exposition. For example, for cheese it can be whole cheese with not edible rind, natural cheese without rind, with edible rind, processed cheese like soft cheese or cottage cheese, preserved As consequence and due to the lack of knowledge about the food contact ability of bionanocomposite materials, a case-by-case risk assessment, which is still proposed in the recent guidance on the risk assessment of nanotechnologies in the food and feed chain undoubtedly remains recommended.

Funding

The project leading to this publication has received funding from Excellence Initiative of Aix-Marseille University -A*MIDEX, a French "Investissements d'Avenir" program, through its associated Labex SERENADE project.

Compliance with ethical standards

The authors declare that there is no conflict of interest regarding the publication of this paper. which is a suitable method to assess the environmental performance of a product or process, can be applied to emerging biodegradable and nanocomposite materials to quantitatively verify environmental claims. Such approach investigates the inputs (i.e. resources and energy) and outputs (i.e. waste gases, wastewater and solid waste) across the entire life-cycle stages (cradleto-grave). Some studies applying LCA to the area of nanotechnology evaluated the potential environmental benefits related to their introduction in polymer matrices [START_REF] Roes | Environmental life cycle studies of poly(hydroxybutyrate)-and polypropylene-based composites[END_REF][START_REF] Joshi | Can nanotechnology improve the sustainability of biobased products?[END_REF][START_REF] Hischier | Life cycle assessment of engineered nanomaterials: State of the art and strategies to overcome existing gaps[END_REF]. They concluded that the reinforcement of a polymer with nanoparticles can lead to environmental benefits.

The objectives of this work are (i) to compare the performances and environmental impact of a conventional non-degradable polymer and biodegradable and bio-sourced emerging polymers and (ii) to determine the benefits of the addition of layered silicates in biodegradable matrices, for the packaging of oxygen-sensitive products. This study was conducted on fresh poultry meat as sensitive model food. In this way, the gain/loss in food shelf life obtained under MAP is studied using modelling tool [START_REF] Guillard | Validation of a predictive model coupling gas transfer and microbial growth in fresh food packed under modified atmosphere[END_REF] simulating gas exchanges in the packed food, which include permeation through the packaging material, solubilisation, desorption and diffusion into the food. The gain related to the addition of nanoclays on the permeability values of the nanocomposite is then evaluated and converted into an environmental benefit according to two distinct scenarios: (i) the possible reduction of food loss and waste and (ii) the reduction of the amount of polymer used to formulate the food packaging considering that the amount of material used to achieve target permeability value is lower in case of nanocomposite materials. The global warming impact, considered as the most appropriate criterion, was estimated using available data for each of scenario distinctly involving loss and waste reductions or material savings. Two distinct polymers: poly(butylene succinate-co-butylene adipate) (PBSA) and poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) were selected on the basis of their differing transfer properties, the PHBV being a highbarrier polymer (Crétois et al., 2014) contrary to PBSA (Phua et al., 2013). These biodegradable polymers were reinforced with 5 wt% organo-modified montmorillonite, Cloisite 30B. The polypropylene (PP), widely used for tray packaging was selected as reference material.

Chapter IV. Estimation of risks-benefits of adding nanoclays in biodegradable polymer matrices 2 Experimental

Materials

Two biodegradable and bio-sourced polymers, poly(butylene succinate-co-butylene adipate) (PBSA, PBE 001, density 1.24 g.cm -3 ) and poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV, PHI 002, density 1.23 g.cm -3 ) were commercially procured from Natureplast, France.

Cloisite 30B (C-30B), a montmorillonite clay modified by a methyl bis-2-hydroxyethyl ammonium quaternary salt was supplied by BYK additives & instruments, Germany.

Production of bio-nanocomposite films

PBSA and PHBV films containing 5 wt% C-30B (PBSA NCP and PHBV NCP) were synthetized by a melt extrusion process using an EuroLab 16XL co-rotating twin-screw TM , Germany) with a L/D ratio of 40 and a screw diameter of 16 mm as described previously (Lajarrige et al., 2019). The control samples of neat polymers were prepared following the same protocol used for nanocomposites in order to respect the same thermal processes. Films of 180 ± 10 µm for PBSA, 220 ± 25 µm for PBSA NCP, 145 ± 20 µm for PHBV and 140 ± 10 µm for PHBV NCP were produced.

Mass transfer characterization

Oxygen permeability

The O2 permeability of films was determined at 23°C and 50 % of relative humidity using an O2 permeation cell equipped with a PSt6-type sensor spot to detect the concentration of O2 manufactured by PreSens Precision Sensing GmbH (Regensburg, Germany). Films were placed in the middle of the cell and the lower and upper chambers were purge with nitrogen. Once the concentration of O2 in the cell is zero, a constant flow of 50 mL.min -1 of O2 was introduced in the lower chamber and the percentage of O2 crossing the film was directly measured in the upper chamber. O2 permeability (mol.m/(m 2 .s.Pa)) was calculated from Fick's first law, Eq.

IV.5.

(IV.5) J (mol.s -1 ) is the flux of gas that pass through the film, e is the film thickness (m), A, the permeation area (m²) , the gas (O2 in this case) pressure differential between the upper and lower chambers of the cell (Pa).

Influence of the material savings

Substitution of PP with PHBV-based packaging

The O2 permeability of PHBV is 70.1% lower than that of PP (Table IV .11). Considering that the O2 permeability of PP is sufficient for the service and that permeability is proportional to the thickness of the material, a reduction of 70.1% of the mass of PHBV can be envisaged.

On this basis, the tray mass decreases from 22.88 g to 6.84 g and the global warming impact of the tray initially equal to 0.0794 kg CO2eq is lowered to 0.0237 kg CO2eq. The global warming impact due to the consumption of 23.09 kg/person/year, which corresponds to 103 trays containing 225 g of fresh poultry, decreases by 1.2% (125.04 kg CO2eq/person/year).

Substitution of PHBV with PHBV NCP-based packaging

The O2 permeability of PHBV is decreased by 76.9% with the addition of 5 wt% C-30B (Table IV.11). Thus, the tray mass decreases from 23.62 g to 5.46 g and the global warming impact of the tray initially equal to 0.0795 kg CO2eq is reduced to 0.0189 kg CO2eq. The global warming impact due to the consumption of the 103 trays of fresh poultry consequently decreases by 1.7% (124.49 kg CO2eq/person/year).

However, this projection is free from considerations related to the respect of the mechanical strength of the materials, the thickness of the tray to be sufficient to ensure its functions. The thickness of the new tray made of PHBV is equal to 120 µm and that made of PHBV NCP to 92 µm, which is lower than the current benchmark and probably requires an additional control of its quality of use.

Influence of the extended shelf-life

In Europe, losses and waste of meat are estimated at 10% and 3.7% at the consumer and distributor level respectively [START_REF] Fao | Pertes et gaspillages alimentaires dans le monde -Ampleur, causes et prévention[END_REF]. The decrease of O2 permeability measured in the case of PHBV and PHBV NCP can be used to decrease these losses.

Substitution of PP with PHBV-based packaging

Considering that the extension of the product's shelf life leads to a proportional reduction in losses and waste, the substitution of PP with PHBV allows to extent the shelf life of fresh poultry by 2.9 days as evidenced in Section 3.2, which represents an increase of 41%. This shelf life gain is sufficient to consider that the food waste at the distributor level (3.7%) is suppressed, which avoid the waste of 4.2 trays/person/year and therefore reduce the global warming impact at 125.47 kg CO2eq/person/year. At the consumer level, this shelf life gain can allow to reduce

Compliance with ethical standards

The authors declare that there is no conflict of interest regarding the publication of this paper.

biodegradation were impacted. This can be induced by the mechanism of degradation inherent to each polymer.

The disintegration of films showed a good degradation of materials in a simulated composting environment. The composts resulting were used to amend a soil in which winter forage rapeseed were sown. The ecotoxic effect of the nanoclays was investigated on plant, soil and rhizosphere microbial enzymatic activities as proxy of soil quality. Using a global analysis, integrating all the data collected on the plant-soil system as proxy of the biocompatibility, a significant polymer-dependent impact was shown on biocompatibility of the nanoclay C30-B.

Funding

The project leading to this publication has received funding from Excellence Initiative of Aix-Marseille Universityassociated Labex SERENADE project.