Les matériaux au contact des aliments (MCDA) constituent un enjeu majeur pour les industriels européens de l’alimentation et de l’emballage d’autant que les MCDA doivent être produits selon les bonnes pratiques de fabrication et être en accord avec les règlementations, notamment le règlement cadre européen 1935/2004. Les MCDA constituent une source potentielle de contamination des aliments car ils peuvent transférer des constituants aux aliments par migration. Parmi les MCDA, les matériaux papiers-cartons sont les plus utilisés après les matériaux plastiques et leur taux de recyclage en Europe était de 72% en 2015 et il ne cesse d’augmenter. En plus des substances dites intentionnelles (SI), des substances non intentionnelles (SNI) peuvent migrer du MCDA vers l’aliment ; elles peuvent être des impuretés, des produits de dégradation, des contaminants provenant des matériaux recyclés etc. Actuellement, de nombreuses SNI ne sont pas identifiées. La présence potentielle de SNI et la nature complexe de ces matériaux rendent difficile l’étude des MCDA papiers-cartons recyclés. Ce projet de thèse a pour objectif d’évaluer le danger lié de ces matériaux. Pour cela, des papiers-cartons recyclés ont été prélevés le long d’une chaine de production (du papier-carton à recycler de début de chaine au papier-carton issu du processus de recyclage), extraits dans l’eau puis lyophilisés. Des analyses chimiques ont été réalisées par spectrophotométrie et par des méthodes chromatographiques (HPLC, GC-MS, LC-MS, ICP-MS). Ces analyses ont montré la présence d’un plus grand nombre de substances dans le papier-carton de début de chaine par rapport au papier-carton de fin de chaine de recyclage. Parmi les composants détectés, des substances naturelles ont été identifiées dans les deux extraits étudiés, provenant notamment du bois qui est la matière première utilisée pour la fabrication du papier (polymères de cellulose, d’hémicellulose et de lignine, acides gras, sucres, acides résiniques ou encore des phénols). D’autres substances identifiées comme additifs ou contaminants ont été caractérisées dans les extraits étudiés, dont bisphénols, des molécules appartenant à la famille des phthalates, des amines aromatiques ou encore des hydrocarbures saturés d’huiles minérales. Il est à souligner qu’une grande partie des substances identifiées dans le produit de début de chaine étaient absentes du produit de fin de chaine. En parallèle des analyses chimiques, des biotests ont été réalisés afin d’identifier des mécanismes d’action et des potentiels effets « cocktail ». Grâce à une batterie de biotests, les effets des extraits sur des cibles toxicologiques ont été étudiés sur différentes lignées cellulaires humaines (deux lignées hépatiques HEpG2 et HepaRG, la lignée hERα-HeLa-9903 et la lignée MDA-kb2): cytotoxicité, génotoxicité et potentiel perturbateur endocrinien. A la plus forte concentration testée, seul l’extrait de produit de fin de chaine a entrainé un effet cytotoxique sur la lignée MDA-kb2. Cependant, les mêmes conditions de test ont montré que les produits de départ et de fin de chaine pouvaient entrainer un stress oxydant dans les lignées hépatiques utilisées. L’évaluation de la génotoxicité a montré des effets génotoxiques des deux papiers-cartons étudiés. Cependant, contrairement aux lésions du matériel génétique induites par le produit de départ, les dommages de l’ADN dus au produit de de fin de chaine pourraient ne pas être réparables pas les systèmes de réparation cellulaires et entrainer des mutations chromosomiques. Enfin, l’étude du potentiel perturbateur endocrinien a mis en évidence des activités oestrogéniques et antiandrogéniques dose-dépendantes des extraits étudiés. Ainsi, la mise en corrélation des résultats d’analyses chimiques et toxicologiques permet d’offrir une stratégie pertinente pour l’évaluation du danger lié aux MCDA, afin d’aider les industriels dans l’évaluation du risque des potentiels NIAS présents dans ces matériaux. / Food contact materials (FCM) are a major issue of concern for European packaging and food companies as FCM must be produced in accordance with the good manufacturing practices and be in compliance with European Regulation 1935/2004/UE. FCM could be a potential source of food contamination because they could release chemicals by a phenomenon called migration. Among FCM, paperboards are the most common materials after plastics and the recycling rate of paperboards is high and was 72% in Europe in 2015. In addition to started substances also called Intentionally Added Substances (IAS), Non-Intentionally Added Substances (NIAS) such as impurities, degradation products, contaminants from recycled materials etc, are also able to migrate from the FCM to food and many of them are not always fully characterized. Assessing the risk of recycled paperboards FCM is difficult because of NIAS and as recycled paperboards can be complex mixtures of substances. This thesis project uses a methodology combining chemical analysis and in vitro bioassays to evaluate the potential hazard of recycled food contact paperboard extracts. For this study, recycled paperboards have been sampled at different steps a food packaging production chain (the starting paperboard of the beginning of the production chain and the end paperboard issued from the recycling process), extracted with water and the dry matter content was recovered by freeze-drying. Chemical analysis of the extracts was performed by spectrophotometry and by chromatographic methods (HPLC, GC-MS, LC-MS and ICP-MS). Chemical analysis showed the presence of a higher number of substances in the starting paperboard than the end paperboard issued from the recycling process. Among these substances, several natural substances were detected in both extracts such as wood extractives (polymers of cellulose, hemicellulose and lignin, fatty acids, sugars, resin acids and phenols). Other substances identified as additives and contaminants were characterized in both extracts such as bisphenols, phthalates, aromatic amins or mineral oil saturated hydrocarbons. It is important to precise that a large number of the substances of the starting paperboard were not detected in the end paperboard. In parallel to chemical characterization, bioassays were used as relevant tools to identify mechanisms of action and potential “cocktail effects”. A battery of bioassays has been performed using different human cell lines (two hepatic cell lines HepG2 and HepaRG, the hERα-HeLa-9903 cell line and the MDA-kb2 cell line) to check toxicological endpoints which are relevant to low exposure: cytotoxicity, genotoxicity and potential endocrine disruption. No cytototoxic effects of both extracts were observed on both human hepatic cell lines HepG2 and HepaRG neither on the HeLa 9903 cell line; whereas the end paperboard extract had cytotoxic effects on MDA-kb2 cells at the highest tested concentration. However, in the same conditions, the starting and the recycled end paperboard extracts were able to induce oxidative stress in the used cell lines. The results of the genotoxicity study showed genotoxic effects of the starting paperboard and the end paperboards. The DNA damage induced by the starting paperboard in the used cellular models might be repaired by cell repair systems. In contrast, data of this study suggest that the end paperboard was able to induce DNA damage that might turn into chromosomal mutations. Data of the endocrine disruption potential showed that the tested FCM paperboard extracts induced significant agonist and dose dependent estrogenic and antiandrogenic activities in the used cell lines. The correlation of the results of the in vitro toxicological study and chemical analysis offers a relevant strategy for hazard assessment of FCM in order to help manufacturers to manage the risk of NIAS.

L?utilisation de produits phytosanitaires est à l?origine d?un problème majeur à l?interface de l?agriculture et de la préservation des ressources en eau. Dans ce contexte, les effets de différents métaux (Cu, Zn), métalloïdes (As) et pesticides (Azimsulfuron, Oxadiazon, Prétilachlore, Fipronil et Alphacyperméthrine) ont été étudiés en terme de biodisponibilité voire de toxicité sur des algues d?eaux douces (Pseudokirchneriella subcapitata et Chlorella sp.) sensibles aux pollutions diffuses d?origine agricole, en prenant en compte l?influence des ligands naturels et l?impact d?une multipollution. L?étude de la relation potentielle entre spéciation, biodisponibilité et toxicité a été menée, sur trois sites d?étude associés à une pollution d'origine agricole. Des échantillons d'eaux, collectés dans des canaux de drainage et des rizières (pollution organique), de drainage prélevés sur des parcelles soumises ou non à des épandages de lisiers de porcs (Cu, Zn) et de ruissellement issus de parcelles viticoles (multipollution) ont été caractérisés et analysés. Les résultats de toxicité ont été confrontés avec ceux mesurés sur des solutions standards en conditions contrôlées et les résultats analytiques ; dosage de résidus de pesticides et des métaux et métalloïdes traces. L?analyse d?échantillons non toxiques menée conjointement, après additions de Cu, par biotests et DPASV a permis d?approfondir les relations entre la toxicité et les espèces électrochimiquement labiles détectées. Elle a souligné la présence de complexes labiles du cuivre (CuL), responsables de la toxicité. Les études de laboratoire et de terrain illustrent de la complexité de l'évaluation de l'exposition dans des contextes de multipollution, chaque molécule nécessitant une évaluation spécifique de son comportement et de ses effets dans un milieu donné. Par ailleurs, les divers produits phytosanitaires présents sont susceptibles d'interagir entre-eux (toxicité de mélanges complexes) et avec les ligands naturels (complexation).

Este trabalho foi desenvolvido nos reservatórios, em cascata, que fazem parte do Médio e Baixo Rio Tietê, incluindo os tributários, os Rios Tietê, Piracicaba e Bauru. Foram considerados os pontos de coleta, localizados à montante, jusante, bem como os pontos intermediários dos reservatórios estudados. Considerando-se os períodos referentes a outubro de 1999 fevereiro, maio e julho de 2000, as pesquisas nestes locais abordaram a qualidade ambiental dos reservatórios, com características limnológicas (água, sedimento), a fauna de macroinvertebrados bentônicos, além de testes ecotoxicológicos (Chironomus xanthus). As análises dos sedimentos permitiram evidenciar as frações de silte e argila, o elevado teor de matéria orgânica nos reservatórios situados no início da cascata, além do nitrogênio e fósforo e metais potencialmente biodisponíveis. Elevadas concentrações de nutrientes nitrogenados e fosfatados, material em suspensão (orgânico e inorgânico) e clorofila-a, foram observadas nas amostras de água, o que permitiu classificar os reservatórios como eutróficos, mesotróficos e oligotróficos, de acordo com sua posição no sistema bem como do período amostrado. As concentrações de alguns dos metais encontrados na água estiveram acima do permitido pela Resolução CONAMA (20/96), considerando-se os períodos em estudo. Quanto às relações bióticas, foram obtidas através do índice de Diversidade (H\'), da equitabilidade, a qual foi evidenciada pela distribuição agregadados organismos e a presença de indivíduos pertencentes à mesma espécie, evidenciou a baixa riqueza de taxa. Através do IET/Densidade de Oligochaeta constatou-se para os reservatórios em cascata e os principais tributários características de eutrofizados. A fauna bentônica dos rios Tietê, Piracicaba e Bauru e dos reservatórios dos locais de estudo caracterizou-se pela presença de Tubificidae (Oligochaeta) além de Chironomidae (Díptera), e do grupo Mollusca. Entre os Tubificidae, a espécie Limnodrilus hoffmeisteri, Branchiura sowerbyi e Limnodrilus udekemianus ocorreram em considerável dominância numérica no período de estudo. A Família Chironomidae, foi representada pelos gêneros Goeldichiromus e Coelotanypus e o grupo Mollusca pelo gênero, Haylacostoma e espécie Melanoides tuberculatus (Thiaridae), Corbicula fluminea (Corbiculidae), o gênero Biomphalaria (Planorbidae) e o gênero Lymnaea collumela, (Lymnaiidae). Quanto à Família Naididade foi representada pelas espécies (Prístina americana, Dero nívea, Dero digitata e Allonais chelata), pela família Alluroididae (Brinkhurstia americanus) e pela Família Opistocystidae (Opistocysta funiculus). Os testes de toxicidade com Chironomus xanthus confirmaram os resultados obtidos através das análises físicas e químicas, conferindo características de impactados aos reservatórios situados no início da cascata (Barra Bonita, Bariri e Ibitinga) e aos tributários (Tietê, Piracicaba e Bauru) moderadamente poluídos aos reservatórios intermediários (reservatório de Nova Avanhandava e Promissão) e, com ausência de poluição, o Reservatório de Três Irmãos, situado no final da cascata. Os resultados decorrem dos usos e ocupação do solo, atividades antropogênicas na Bacia Hidrográfica, do aporte de materiais para o sistema na qual se inserem os rios e os reservatórios estudados. / This work was developed in the reservoirs, at waterfalls, which are part of the Medium and Low Tiete River, including its tributaries, the Tiete, the Piracicaba river and the Bauru river. The collecting sites considered were upstream, downstream and those intermediary sites of the reservoirs. Taking into account the different periods of October 1999 and February, May and July of 2000, the researches in those sites were about the environmental quality of the reservoirs with limnologic characteristics (water, sediment), the bentonic macroinvertebrate fauna besides ecotoxilogic tests (Chironomus xanthus). The sediment analysis allowed to evidence the silt and clay fractions, the high organic matter content in the reservoirs located at the waterfall head in addition to nitrogen, phosphur and potentially bio-available metals. High nitrogened and phosphated nutrients, suspended matter (organic and inorganic) and chlorophyl-a were observed in the water samples, which allowed to classify the reservoirs as euthrophic, mesotrophic and oligotrophic according to their location in the system and the sampling period. The concentration of some metals found the water was above the level permitted by CONAMA Resolution (20/96) taking into account the periods of study. As to the biotic relations, they were obtained by means of the Diversity index (H\') of the equitability, which was evidenced by the aggregated distribution of the organisms and the presence of individuals belongingto the same species evidenced the low taxa richness. Through the IET/Oligochaeta abundance, the reservoirs and the tributaries were found to be eutrophitized. The Tiete, Piracicaba and Bauru rivers and the reservoirs studied sites bentonic fauna was characterized by the presence of Tubificidae (Oligochaeta) besides Chironomidae (Diptera) and the Mollusca group. Among the Tubificidae, the Limnodrilus hoffmeisteri, Branchiura sowerbyi e Limnodrilus udekemianus species occurred in considerable numerical dominance in the period of study. The Chironomidae family was represented by the genus Goeldichiromus and Coelotanypus and the Mollusca group by the genus Haylacostoma and Melamoides tuberculatus (Thiaridae) species, Corbicula fluminea (Corbiculidae), the genus Bionphalaria (Planorbidae) and the genus Lymnaea collumela (Lymnaiidae). The Naididae family was represented by the species (Pristina americana, Dero nivea, Dero digitata and Allonais chelata), by the family Alluroididae (Brinkhurstia americanus) and the family Opistocystidae (Opistocysta funiculus).The toxicity tests with Chironomus xanthus confirmed the results obtained through physical and chemical analyses granting characteristics of impacted to the reservoirs located at the waterfall head (Barra Bonita, Bariri and Ibitinga) and the tributaries (Tiete, Piracicaba and Bauru) moderately polluted to the intermediate reservoirs (Nova Avanhandava and Promissão reservoirs) and, absenceof pollution, the Tres Irmãos Reservoir located at the end of the waterfall. The results arise from the uses and occupation of the soli, anthropogenic activities in the hydrographic basis, disclosure of materials into the system in which are the rivers the reservoirs under study are located.

Este trabalho foi desenvolvido nos reservatórios, em cascata, que fazem parte do Médio e Baixo Rio Tietê, incluindo os tributários, os Rios Tietê, Piracicaba e Bauru. Foram considerados os pontos de coleta, localizados à montante, jusante, bem como os pontos intermediários dos reservatórios estudados. Considerando-se os períodos referentes a outubro de 1999 fevereiro, maio e julho de 2000, as pesquisas nestes locais abordaram a qualidade ambiental dos reservatórios, com características limnológicas (água, sedimento), a fauna de macroinvertebrados bentônicos, além de testes ecotoxicológicos (Chironomus xanthus). As análises dos sedimentos permitiram evidenciar as frações de silte e argila, o elevado teor de matéria orgânica nos reservatórios situados no início da cascata, além do nitrogênio e fósforo e metais potencialmente biodisponíveis. Elevadas concentrações de nutrientes nitrogenados e fosfatados, material em suspensão (orgânico e inorgânico) e clorofila-a, foram observadas nas amostras de água, o que permitiu classificar os reservatórios como eutróficos, mesotróficos e oligotróficos, de acordo com sua posição no sistema bem como do período amostrado. As concentrações de alguns dos metais encontrados na água estiveram acima do permitido pela Resolução CONAMA (20/96), considerando-se os períodos em estudo. Quanto às relações bióticas, foram obtidas através do índice de Diversidade (H\'), da equitabilidade, a qual foi evidenciada pela distribuição agregadados organismos e a presença de indivíduos pertencentes à mesma espécie, evidenciou a baixa riqueza de taxa. Através do IET/Densidade de Oligochaeta constatou-se para os reservatórios em cascata e os principais tributários características de eutrofizados. A fauna bentônica dos rios Tietê, Piracicaba e Bauru e dos reservatórios dos locais de estudo caracterizou-se pela presença de Tubificidae (Oligochaeta) além de Chironomidae (Díptera), e do grupo Mollusca. Entre os Tubificidae, a espécie Limnodrilus hoffmeisteri, Branchiura sowerbyi e Limnodrilus udekemianus ocorreram em considerável dominância numérica no período de estudo. A Família Chironomidae, foi representada pelos gêneros Goeldichiromus e Coelotanypus e o grupo Mollusca pelo gênero, Haylacostoma e espécie Melanoides tuberculatus (Thiaridae), Corbicula fluminea (Corbiculidae), o gênero Biomphalaria (Planorbidae) e o gênero Lymnaea collumela, (Lymnaiidae). Quanto à Família Naididade foi representada pelas espécies (Prístina americana, Dero nívea, Dero digitata e Allonais chelata), pela família Alluroididae (Brinkhurstia americanus) e pela Família Opistocystidae (Opistocysta funiculus). Os testes de toxicidade com Chironomus xanthus confirmaram os resultados obtidos através das análises físicas e químicas, conferindo características de impactados aos reservatórios situados no início da cascata (Barra Bonita, Bariri e Ibitinga) e aos tributários (Tietê, Piracicaba e Bauru) moderadamente poluídos aos reservatórios intermediários (reservatório de Nova Avanhandava e Promissão) e, com ausência de poluição, o Reservatório de Três Irmãos, situado no final da cascata. Os resultados decorrem dos usos e ocupação do solo, atividades antropogênicas na Bacia Hidrográfica, do aporte de materiais para o sistema na qual se inserem os rios e os reservatórios estudados. / This work was developed in the reservoirs, at waterfalls, which are part of the Medium and Low Tiete River, including its tributaries, the Tiete, the Piracicaba river and the Bauru river. The collecting sites considered were upstream, downstream and those intermediary sites of the reservoirs. Taking into account the different periods of October 1999 and February, May and July of 2000, the researches in those sites were about the environmental quality of the reservoirs with limnologic characteristics (water, sediment), the bentonic macroinvertebrate fauna besides ecotoxilogic tests (Chironomus xanthus). The sediment analysis allowed to evidence the silt and clay fractions, the high organic matter content in the reservoirs located at the waterfall head in addition to nitrogen, phosphur and potentially bio-available metals. High nitrogened and phosphated nutrients, suspended matter (organic and inorganic) and chlorophyl-a were observed in the water samples, which allowed to classify the reservoirs as euthrophic, mesotrophic and oligotrophic according to their location in the system and the sampling period. The concentration of some metals found the water was above the level permitted by CONAMA Resolution (20/96) taking into account the periods of study. As to the biotic relations, they were obtained by means of the Diversity index (H\') of the equitability, which was evidenced by the aggregated distribution of the organisms and the presence of individuals belongingto the same species evidenced the low taxa richness. Through the IET/Oligochaeta abundance, the reservoirs and the tributaries were found to be eutrophitized. The Tiete, Piracicaba and Bauru rivers and the reservoirs studied sites bentonic fauna was characterized by the presence of Tubificidae (Oligochaeta) besides Chironomidae (Diptera) and the Mollusca group. Among the Tubificidae, the Limnodrilus hoffmeisteri, Branchiura sowerbyi e Limnodrilus udekemianus species occurred in considerable numerical dominance in the period of study. The Chironomidae family was represented by the genus Goeldichiromus and Coelotanypus and the Mollusca group by the genus Haylacostoma and Melamoides tuberculatus (Thiaridae) species, Corbicula fluminea (Corbiculidae), the genus Bionphalaria (Planorbidae) and the genus Lymnaea collumela (Lymnaiidae). The Naididae family was represented by the species (Pristina americana, Dero nivea, Dero digitata and Allonais chelata), by the family Alluroididae (Brinkhurstia americanus) and the family Opistocystidae (Opistocysta funiculus).The toxicity tests with Chironomus xanthus confirmed the results obtained through physical and chemical analyses granting characteristics of impacted to the reservoirs located at the waterfall head (Barra Bonita, Bariri and Ibitinga) and the tributaries (Tiete, Piracicaba and Bauru) moderately polluted to the intermediate reservoirs (Nova Avanhandava and Promissão reservoirs) and, absenceof pollution, the Tres Irmãos Reservoir located at the end of the waterfall. The results arise from the uses and occupation of the soli, anthropogenic activities in the hydrographic basis, disclosure of materials into the system in which are the rivers the reservoirs under study are located.