F. graminearum est l’une des espèces prédominantes en tant que pathogène des céréales en Europe et dans les régions tempérées du monde. Cette moisissure appartient au genre Fusarium qui est l’un des genres qui produit le plus de métabolites secondaires, dont de nombreuses mycotoxines. Cependant, les connaissances sur son génome laissent présager que F. graminearum produit une très grande variété de métabolites qui sont encore inconnus. C’est pourquoi l’objectif de cette thèse a été de caractériser le métabolome de cette moisissure en identifiant de nouveaux métabolites ainsi qu’en étudiant leur toxicité. Pour cela, une méthode analytique combinant la spectrométrie de masse de haute résolution et le marquage des métabolites secondaires avec des isotopes stables a été développée. Celle‐ci à tout d’abord était validée avec le métabolome bien connu d’Aspergillus fumigatus avant d’être appliquée au métabolome de F. graminearum. Cette méthode a permis d’identifier 37 nouveaux métabolites, dont les fusaristatines A, C et D qui on été détectées pour la première fois chez F. graminearum. Le deoxynivalenol (DON) est une mycotoxine majeure produite par F. graminearum, c’est pourquoi sa toxicité a été comparée in vivo sur un modèle porcin à celle d’un mélange de fusariotoxines. Les résultats de cette étude ont montré une toxicité bien différente du mélange par rapport au DON seul, que ce soit par rapport à la performance des animaux ou à leur réponse immunitaire. Ceci suggère que certaines des fusariotoxines présentes dans le mélange auraient des répercutions biologiques et donc que leur toxicité reste encore à être élucidée. De plus, l’importance de la caractérisation de la toxicité du métabolome de F. graminearum a été soulignée par les résultats obtenus dans une seconde étude, qui ont révélés une possible implication du DON dans les maladies inflammatoires chroniques de l’intestin par l’intermédiaire des lymphocytes Th17. / F. graminearum is one of the most predominant cereal pathogen in Europe and temperate regions of the world. It belongs to the genera Fusarium, which is one of the most important in terms of the production of secondary metabolites, among which stand several mycotoxins. However, recent advances on the study of its genome suggest that only a small fraction of the secondary metabolites produced by F. graminearum have been identified so far. Therefore, the main objective of this thesis was to characterize the metabolome of this fungus by identifying new metabolites and by analyzing their toxicity. First, an analytical method combining high resolution mass spectrometry and isotopically labeled secondary metabolites was developed. The method was validated with the well known metabolome of Aspergillus fumigatus before it was applied to F. graminearum. As a result, 37 new metabolites were identified such as fusaristatin A, C and D, which were detected for the first time in the metabolome of F. graminearum. Deoxynivalenol (DON) is one of the major mycotoxins produced by this fungus. Therefore, its toxicity was compared to that of a blend of fusariotoxins in vivo in a pig model. The results of this study showed different effects of the blend compared to DON alone on animal performances and immune response. This suggests that some of the fusariotoxins present in the blend could be biologically active and their toxicity should be further investigated. Toxicity of the metabolome of F. graminearum has also been highlighted by the results of a second study which showed a possible implication of DON on the appearance of inflammatory bowel diseases by the action of Th17 lymphocytes.

La pourriture bleue causée par Penicillium expansum est l'une des maladies les plus dommageables des fruits pomaceae (pommes et poires). Outre des dégâts directs, cette maladie pose un problème de santé publique car l'agent pathogène produit des mycotoxines nocives pour l'homme et les animaux dont la plus sérieuse est la patuline. La croissance du champignon pathogène et la production de patuline requièrent des conditions physico-chimiques particulières. Les informations existantes à ce propos demeurent cependant modestes et insuffisantes pour envisager de développer des moyens de lutte contre l'apparition du champignon. Par ailleurs, la patuline reste avec l'ochratoxine A, les seules toxines dont la voie de biosynthèse n'a pas encore été complètement établie, tant sur le plan chimique que moléculaire. Cette étude apporte dans un premier temps des données complémentaires sur les facteurs physico-chimiques (température, pH….) qui conditionnent la croissance de P. expansum de même que sa capacité à produire la patuline. La connaissance de ces besoins et de ces conditions conduit en pratique à lutter et contrôler la contamination par la patuline tout le long de la chaine alimentaire. Dans un deuxième temps, cette thèse apporte des améliorations spectaculaires sur le plan fondamental, en termes d'élucidation de la voie de biosynthèse de la patuline. Le cluster des gènes impliqués dans la biosynthèse de cette mycotoxine chez l'espèce la plus préoccupante P. expansum a été entièrement identifié et caractérisé. Pour lever encore plus le voile sur la biosynthèse de cette mycotoxine, la caractérisation du facteur de régulation spécifique de cette voie (patL) a été également établie. Une perturbation de ce gène a provoqué une incapacité de production de patuline et une sévère diminution de l'expression des gènes Pat. De même, grâce à ce mutant déficient, il a été montré que la patuline pourrait agir comme facteur de virulence lors du développement de la moisissure dans les pommes. La caractérisation de la dernière étape de la voie de biosynthèse de la patuline a ensuite été entreprise par mutagenèse dirigée du gène patE du cluster de la patuline, chez la même espèce. Ce dernier code pour une Glucose méthanol Choline (GMC) oxydoréductase responsable de la conversion de l'ascladiol en patuline. L'ascladiol est également une molécule clé de la dégradation de la patuline par diverses espèces bactériennes ou de levures et plus particulièrement lors de la fermentation alcoolique. La non-toxicité de l'ascladiol accumulé chez le mutant ∆patE a été démontrée sur une lignée cellulaire intestinale humaine (Caco-2), suggérant que la patuline perd sa toxicité avec l'ouverture du deuxième cycle. Finalement, un système de détection et de quantification de P. expansum par PCR en temps réel a été développé en ciblant un gène hautement spécifique de la voie de biosynthèse de la patuline, patF. Cette approche préventive nous a ainsi permis d'avoir une estimation rapide de la contamination en patuline dans les pommes à partir de la quantification d'ADN de P. expansum. En conclusion, l'ensemble de ces travaux qui s'inscrivent dans le cadre de la gestion du risque « patuline » dans la filière fruit a permis d'amener des réponses tant sur le plan fondamental que sur le plan appliqué avec le séquençage du cluster, le développement d'un outil de diagnostic et la démonstration que l'ascladiol ne présentait aucune cytotoxicité. / Among diseases affecting apples, blue mold caused by Penicillium expansum is a major concern causing yield and quality losses due to the production of mycotoxins, of which patulin is the most alarming one. This mycotoxin was proven to be harmful for humans and animals. The pathogen growth and the patulin production occur under specific physico-chemical conditions (temperature, pH…). However, the description of these conditions in literature remains largely insufficient for the development of strategies to fight the development of the fungus. Furthermore, patulin remains, along with ochratoxin A, the only toxins for which the biosynthetic pathway is not fully established yet at both chemical and molecular levels. Firstly, this study provides supplementary data on the physico-chemical factors that modulate P. expansum growth and its ability to produce patulin. The acquaintance of these conditions leads, in practice, to the control of the patulin contamination along the food chain. Secondly, significant improvements were brought on the fundamental level, especially by elucidating the patulin biosynthetic pathway. The cluster of genes involved in the biosynthesis of this mycotoxin was fully identified and characterized in the species of greatest concern P. expansum. In order to reveal additional info on the biosynthesis of this mycotoxin, the specific factor of the pathway (patL) was characterized. The disruption of this gene has led to failure in patulin production and an important decrease in Pat genes expression. Furthermore, pathogenesis studies, using this same deficient strain showed that patulin potentially acts as a virulence factor during P. expansum development on apples. The last step of the patulin biosynthetic pathway was later characterized by site-directed mutagenesis of the patE gene in the same species. This gene encodes a Glucose Methanol Choline (GMC) oxidoreductase that is responsible for the conversion of ascladiol to patulin. Ascladiol is not only the last intermediate in the patulin pathway but also the main product of patulin degradation during the alcoholic fermentation of apple juice. The non-toxicity of ascladiol accumulated by the ΔpatE strain was proved against the human Caco-2 cell line. Finally a Real time PCR assay was developed to specifically detect and quantify P. expansum. This was done by targeting a highly specific gene from the patulin gene cluster in P. expansum, patF. This predictive approach allowed the quick estimation of the patulin content via the quantification of the P. expansum DNA in apples. To conclude, this thesis is part of the patulin's risk management study in the fruit sector; it provides significant improvements on both fundamental and practical levels. These advances are mainly characterized by the sequencing of the patulin gene cluster, the development of a molecular diagnostic tool and the demonstration of the non-cytotoxicity of ascladiol.

Quatro experimentos foram conduzidos com o objetivo de determinar os valores nutricionais de milhos de quatro qualidades, obtidos por meio de estratificação em mesa densimétrica. Os milhos foram designados como: MDA - milho de densidade alta; MDI - milho de densidade intermediária; MDB - milho de densidade baixa; e MDO - milho de densidade original, composto de 25% de MDA, 50% de MDI e 25% de MDB. O primeiro experimento objetivou determinar os valores de energia metabolizável aparente corrigida (EMAn), utilizando o método tradicional de coleta total de excretas com frangos de corte de diferentes idades. O primeiro ensaio metabólico foi conduzido com pintos Cobb de 14 a 17 dias, o segundo, com aves de 25 a 28 dias e o terceiro de 38 a 41 dias de idade. Os valores de energia metabolizável dos milhos de diferentes densidades obtidos por estratificação (MDA, MDI, MDB e MDO) foram respectivamente, 3562, 3382, 3198 e 3357kcal/kg na fase inicial, para frangos na fase de crescimento 3576, 3555, 3229 e 3416 kcal/kg e para a fase final 3610, 3554, 3354 e 3585 kcal/kg. A diferença da EMAn entre as idades das aves sugere melhor eficiência de utilização da energia com o avanço da idade. O segundo experimento teve com o objetivo determinar o desempenho de frangos de corte alimentados com rações formuladas com milhos de diferentes densidades na fase inicial (1-21 dias de idade), e avaliar rendimento de carcaça de frangos de corte aos 43 dias de idade. Nesse período de criação houve diferença significativa para a conversão alimentar, sendo que as aves que receberam ração formulada com a matriz do milho de baixa densidade tiveram uma conversão de 8% melhor que a obtida pelas aves que receberam ração formulada com o milho de alta densidade em substituição ao milho de densidade original. O rendimento de carcaça não diferiu entre os tratamentos, mas o rendimento de gordura foi mais elevado para o tratamento MDB apresentou maior deposição de gordura abdominal. O terceiro experimento teve como objetivo verificar o desempenho de frangos de corte alimentados com rações formuladas com milhos de diferentes densidades na fase final (35 42 dias de idade). Não foram observadas diferenças significativas para nenhuma das variáveis analisadas, portanto estes resultados demonstram que é possível utilizar milho de baixa densidade em substituição ao milho de densidade original na fase final, sem prejudicar o desempenho. O quarto experimento objetivou verificar a incidência de micotoxinas nos milhos estratificados;com relação as micotoxinas estudadas, foi detectada a presença da Fumonisina. A distribuição desta micotoxina variou com a densidade, sendo observada uma maior concentração no MDB, indicando eficiência da mesa gravimétrica na segregação de milho mais contaminado com esta micotoxina. / Four experiments were conducted with the objective of determining the nutritional value of corn segregated by densimetric table. The corn fractions have been designated as: MDA high density corn; MDI middle density corn; MDB low density corn; and MDO original density corn, consisting of 25% MDA, 50% MDI and 25% MDB. The first experiment determined the nitrogen-corrected apparent metabolizable energy (EMAn). The metabolism assays were conducted using the traditional method of total excreta collection with broilers different ages. Assay 1 was conducted with Cobb broiler chicks 14-17 days old, assay 2 was conducted with broilers 25-28 days old and in the final assay the birds were 38-41 days old. EMAn of corn obtained by stratification (MDA, MDI, MDB e MDO) were, respectively, 3562, 3382, 3198 e 3357 kcal/kg for the starter phase, 3576, 3555, 3229 e 3416 kcal/kg for the grower phase and 3610, 3554, 3354 e 3585 kcal/kg for the finisher phase. EMAn values increased with age of the broilers, indicating improved energy utilization. The second experiment was conducted to evaluate the broilers performance fed diets formulated with different densities corn in the initial phase (1-21 days old), and carcass yield of broilers at 43 days old. Feed conversion was significantly different, broilers fed diets formulated with the low density corn having better feed conversion than broilers fed high density corn. Carcass yield was not different between treatments; however fat yield was higher for MDB treatment. The third experiment was conducted to evaluate broiler performance fed diets formulated with different densities corn in the final phase (35-42 days old). Performance was not different among treatments, indicating that it is possible to use MDB replacing MDO in the final phase. The fourth experiment was conducted to evaluate the incidence of mycotoxins in the corn fractions after 18 month storage. Fumonisin was the only mycotoxin detected and higher concentration was found in MDB than in the other fractions, indicating that the gravimetric table was efficient in the corn segregation.

Philosophiae Doctor - PhD / Immunosensors are affinity ligand-based biosensor solid-state devices in which the immunochemical reaction is coupled to a transducer. The specificity of the molecular recognition of antigens by antibodies to form a stable complex is the basis of the immunosensor on the electrode. The development of such a sensor requires a better design and preparation of an optimum interface between the biomolecules and the detector material. The immunosensors were developed based on Polyaniline derivative composite. Novel water soluble PdTe quantum dots (QD) was synthesized and characterized by different physical techniques such as UV-Visible (UV-VIS), Fluorescence Spectroscopy (PL), Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM) and X-Ray Diffraction (XRD). The electroactivity of such synthesized quantum dots was studied by cyclic voltammetry in aqueous media. The synthesis of poly(2,5- dimethoxyaniline)-multi wall 'carbon nanotubes nanocomposite was carried out by electropolymerization in situ of 2,5-dimethoxyaniline - multi wall carbon nanotubes (PDMA-MWCNT) from aqueous dispersion containing acid-treated multi wall carbon nanotubes (MWCNT) and 2,5-dimethoxyaniline subsequently modifying a glassy carbon electrode in acid media. An undoped PDMA was also prepared for control. The composite for this work, consists of layer-by-layer method to form a multilayer film of QDs and PDMA-MWCNT. The method used was as follows; the drop coating of quantum dots followed by electrodeposition of poly(2,5- dimethoxyaniline )-carbon nanotubes onto surface of glassy carbon. The PDMA-CNT was characterized by UV-Visible (UV-Vis), Fourier Transform Infrared Spectroscopy (FTIR) and Scanning Electron Microscopy (SEM). The electrochemical characterisation of PDMA-CNT was carried out using cyclic voltammetry (CV) and Electrochemical Impedance Spectroscopy (EIS). The composite (QDs-PDMA-MWCNT) was also characterized using above mentioned techniques. The electrochemical immunosensor for fumonisin a mycotoxin was prepared by dropcoating of mycotoxins antibody onto the composite modified glassy carbon electrode. The response profiles of fumonisins sensors system were obtained from electrochemical impedance spectroscopy (EIS) measurements. The fumonisin immunosensor was used for the detection of fumonisins in certified com reference materials. For comparison reasons, analysis of such mycotoxins was carried out by using conventional analytical method enzyme-linked immunosorbent assay (ELISA). The EIS response of FBI immunosensor (GCEIPT-PDMA-MWCNT/anti-Fms-BSA) gave a linear range of 7 to 49 ng L-I and the corresponding sensitivity and detection limits were 0.0162 ka L ng-I and 0.46 pg L-I, respectively. Hence the limit of detection of GCEIPT-PDMA-MWCNT immunosensor for fumonisins in com certified material was calculated to 0.014 and 0.011 ppm for FBI, and FB2 and FB3, respectively. These results are lower than those obtained by ELISA, a provisional maximum tolerable daily intake (PMTDI) for fumonisins (the sum of FBI, FB2, and FB3) established by the Joint FAO / WHO expert committee on food additives and contaminants of 2 ug kg" and the maximum level recommended by the U.S. Food and Drug Administration (FDA) for protection of human consumption (2-4 mg L-I).

La fusariose est une des maladies les plus importantes altérant le blé en Bourgogne. L’espèce fongique Fusarium graminearum est l’un des agents principaux de la maladie. L’interaction hôte-pathogène peut entrainer la production de mycotoxines toxiques pour l’homme et l’animal.La seule alternative pour prévenir le développement de la maladie est de contrôler l’inoculum primaire dans son habitat naturel : les adventices, le sol et les résidus de culture. En raison de la tendance à la réduction du travail du sol, une attention particulière doit être portée au rôle des résidus de culture dans la survie et le développement de F. graminearum. Dans ce travail de thèse, deux approches ont été choisies pour mieux comprendre le développement saprotrophe du champignon et ses conséquences. La première, à travers un essai en champ dans le contexte pédoclimatique Bourguignon, avait pour but de définir la part relative des différentes sources d’inoculum dans le développement de la fusariose et l’accumulation des mycotoxines dans les grains. Cet essai devait en outre permettre de déterminer si des indicateurs précoces du développement de la maladie sur épi et de l’accumulation de mycotoxines pouvaient être identifiés. La seconde, à travers un essai en microcosmes, avait pour but de suivre le développement de F. graminearum dans le sol et les résidus.Ce travail a permis de souligner l’importance de la gestion des résidus de culture dans le développement de la fusariose du blé. Favoriser une décomposition biologique rapide des résidus de culture et/ou introduire des cultures intermédiaires assainissantes constituent des perspectives de recherche sur lesquelles doivent porter nos efforts / Fusarium Head Blight (FHB), mainly caused by the fungal species Fusarium graminearum, is one of the most important disease altering wheat crops in Burgundy. Moreover the plant-pathogen interaction leads to the production of mycotoxins potentially toxic for humans and animals.The only alternative to date to prevent the development of the disease is to control the saprotrophic development of F. graminearum in its natural habitat, i.e. weeds, soil and crops residues. Due to the trend of reduced tillage, special attention should be paid to the role of crop residues in the survival and development of F. graminearum.Two approaches were chosen to better understand the saprotrophic development of F. graminearum and its consequences towards FHB. i) The first through a field experiment in the Burgundian pedoclimatic context aimed at defining the relative importance of the different sources of inoculum in the development of FHB and the accumulation of mycotoxins in grains. The field experiment was also to determine whether early indicators of disease development on ears and accumulation of mycotoxins could be identified. ii) The second, through test microcosms, was to follow the development of F. graminearum in the soil and crop residues.This work highlighted the importance of crop residues management in the development of FHB and gave new understanding about the survival of the fungus on these residues. Improve the biological decomposition of crop residues at the soil surface or/and using suppressive intermediate crops could be the next prospective to investigate to limit the soil inoculum potential of saprotrophic F. graminearum

La fusariose de l’épi est une des maladies les plus importantes du blé. Elle est causée par un complexe d’espèces dont les plus courantes sont F. graminearum, F. culmorum, F. poae, M. majus et M. nivale. Ces champignons infectent les épis de blé au moment de la floraison mais l’impact de contaminations tardives sur les grains est peu compris. De plus, deux ou trois espèces sont susceptibles de co-infecter un même épi, ce qui pourrait modifier leur développement et leur production de toxines dans l’épi. Pour étudier ces problématiques, plusieurs isolats appartenant aux espèces citées ont été caractérisés selon leurs traits de vie et leur agressivité sur épi. Nous avons ensuite étudié le développement de souches de Fusarium inoculées 3 jours avant ou 0, 8, 18 ou 28 jours après la sortie des premières anthères. Les niveaux de maladie et toxines se sont révélés maximum autour de la floraison. La date d’inoculation semble conditionner la sévérité de la maladie et influencer le développement des souches. Des contaminations précoces et tardives seraient malgré tout possibles avec des isolats agressifs, ce qui ouvre la possibilité de telles infections au champ. Dans une deuxième expérimentation, nous avons étudié la compétition entre F. graminearum et les autres espèces (F. culmorum, F. poae, M. majus et M. nivale). Les réponses se sont avérées variables ; néanmoins, les souches agressives ne sont pas influencées ou légèrement favorisées par la présence d’un compétiteur alors que les souches peu agressives sont défavorisées par la présence de souches agressives. La production de toxines est, dans la plupart des cas, restée stable ou a diminué en co-inoculations par rapport aux inoculations simples. Dans une dernière expérimentation nous avons étudié la compétition entre souches associée à leurs mouvements dans l’épi. Inoculés seuls, les champignons progressent dans la totalité de l’épi alors que la présence d’un second champignon empêche leur développement, ce qui suggère une interaction compétitive entre eux. Cette étude apporte de nouveaux éléments nécessaires à la compréhension de l’épidémiologie de ces agents pathogènes et des niveaux de contamination à l’échelle du champ. / FHB is a potentially very damaging wheat disease, present in most wheat-growing areas and caused by a species complex from which F. graminearum, F. culmorum, F. poae, M. majus and M. nivale are the most common. These fungi may infect wheat spikes at flowering but infections at late stages of kernel development are poorly understood. Moreover, disease surveys have shown that co-infection of the same spike by two or three species is frequent and could modify their development in spikes. To investigate these questions, several isolates of the species cited above were characterised for their life traits and aggressiveness on spikes. We then investigated the development of Fusarium isolates inoculated on wheat spikes 3 days before or 0, 8, 18 and 28 days post anthers extrusion. The highest disease and toxin levels were found around anthesis. The inoculation date appeared to condition the disease severity and to influence the isolates development. Early or late contaminations were however possible with aggressive isolates, which suggest the possibility of such infections in field. In a second experiment, we studied the competition between F. graminearum and the other species (F. culmorum, F. poae, M. majus and M. nivale). The responses were variable; nevertheless, aggressive isolates were most of the time unchanged in their development or even promoted whereas less aggressive isolates were reduced against more aggressive ones. The toxin levels were generally unchanged or they decreased in co-inoculations compared to single inoculations. In a last experiment, we investigated the isolate competition and the movement of fungi in the spikes. Alone, isolates progressed in the whole spike whereas the presence of another fungus impaired their development, suggesting a competitive interaction. This study contributes to our understanding of these pathogens epidemiology and provides new elements for a better understanding of the contamination of wheat kernels at the field level.

Pyrenophora tritici-repentis (Ptr) is the necrotrophic fungus responsible for tan spot of wheat (Triticum aestivum). Ptr causes disease on susceptible wheat cultivars through the production and secretion of host-selective toxins (HSTs). HSTs are compounds that are only known to be produced by fungi and considered to be primary determinants of pathogenicity. Infiltration of these toxins into sensitive wheat elicits the same symptoms as the pathogen, which simplifies investigations of host- pathogen interactions due to exclusion of the pathogen. These characteristics make HSTs ideal molecules to dissect molecular plant-microbe interactions. Known HSTs of Ptr include Ptr ToxA (ToxA), Ptr ToxB (ToxB) and Ptr ToxC (ToxC). ToxA is the most characterized toxin of Ptr, as well as the first proteinaceous HST identified. The proposed mode-of-action for ToxA includes internalization into sensitive wheat mesophyll cells, localization to the chloroplast, photosystem perturbations and elicitation of high amounts of reactive oxygen species (ROS), all of which lead to necrosis. However, it is still unknown how ToxA is transported to the chloroplast. To identify additional interacting components involved in ToxA symptom development, genes were silenced in tobacco plants (Nicotiana benthamiana) using the tobacco rattle virus (TRV) virus-induced gene-silencing (VIGS) system. Four genes were identified that potentially could play a role in ToxA-induced cell death: a 40S ribosomal subunit, peroxisomal glycolate oxidase (GOX), a thiamine biosynthetic enzyme (Thi1), and the R-gene mediator, Sgt1. Ptr exhibits a complex race structure determined by the HST(s) produced and the symptom(s) elicited on sensitive wheat cultivars. Currently, there are eight characterized races and other HSTs and races have been proposed. Isolate SO3 was discovered in southern Oregon and elicits ToxA-like symptoms on a wheat differential set, yet lacks the ToxA gene. The transcriptome of SO3 was sequenced, assembled, and aligned to a ToxA-producing isolate, Pt-1C-BFP, which will aid in the identification of the protein(s) that may be responsible for these ToxA-like symptoms. SO3 contains a set of 497 sequences that were not found in the ToxA-producing isolate Pt-1C-BFP (BFP). These sequences should be further investigated to identify those that encode small secreted proteins (SSPs) and could potentially serve as HSTs and pathogenicity factors of SO3. / Graduation date: 2013

Aquest estudi s’ha realitzat en el marc del projecte titulat: “Ecotoxicidad, micotoxinas y degradación de nonilfenoles en lodos de depuradora y suelos tratados” finançat pel Ministeri d’educació i ciència amb número de referència: CTM2006-14163-C02-02, en col•laboració amb el Centre de Recerca i Aplicacions Forestals. La present Tesi Doctoral aborda els aspectes de determinació analítica de la presència de micotoxines en compost i l’evolució del nonilfenol en sòls. En concret, s’ha estudiat la presència de les aflatoxines G2, G1, B2, B1 i l’ocratoxina A, en tres mostres de compost produït en dues EDAR de Catalunya. Per això s’ha desenvolupat un mètode per a l’anàlisi simultània de les aflatoxines G2, G1, B2, B1 i l’ocratoxina A en compost. En primer lloc s’ha adaptat per aquestes mostres un procediment per HPLC-MS desenvolupat anteriorment en el nostre equip per a la determinació de les aflatoxines G2, G1, B2 i B1 aplicat en matrius de tipus alimentari, que s’ha estès a la determinació conjunta de les quatre aflatoxines i l’ocratoxina A. Aquest procediment ha mostrat problemes pel que fa a la recuperació i precisió dels resultats. Vistes les dificultats, s’ha desenvolupat un nou procediment per UHPLC-Fluorescència que permet una preparació de mostra més ràpida i simple, així com una millor sensibilitat. Aquest nou mètode ha estat validat i permet millorar el temps d’anàlisi i aconseguint eliminar l’etapa de purificació de mostra amb cartutxos de reblert polimèric, utilitzats en el mètode emprat com a punt de partida. Els dos procediment han permès demostrar que, en les mostres de compost estudiades no hi ha quantitats significatives de cap de les cinc micotoxines estudiades. Pel que fa al nonilfenol, s’ha estudiat la seva evolució temporal, durant 32 setmanes, en mostres de sòl, mescles de sòl amb torba i mescles de sòl amb compost, a les que s’ha addicionat nonilfenol a dos nivells de concentració. De la mateixa manera, s’ha estudiat la concentració de nonilfenol en els lixiviats de les mostres de sòl i de les mescles de sòl amb torba. Per això, s’ha posat al punt un procediment analític per HRGC-MS tant per a les mostres de sòls com per a les mostres aquoses dels lixiviats. S’ha posat de manifest la ràpida eliminació del nonilfenol en les tres mostres estudiades i que el nivell de pèrdua per lixiviació és molt baix. En els mateixos lixiviats s’ha determinat la matèria orgànica total i la seva toxicitat a través de l’assaig de Microtox. Les dues determinacions mostren que aquests lixiviats no presenten problemes ja que, complirien les exigències per poder ser abocats a les xarxes públiques de clavegueram. Finalment, es pot afirmar que l’ús del compost en agricultura és una pràctica segura, tant pel que fa a l’absència de les micotoxines, com per la ràpida degradació del nonilfenol en el sòl. / Este estudio se llevó a cabo en el marco del proyecto titulado, "Ecotoxicidad, micotoxinas y degradación de nonilfenoles en lodos de depuradora y suelos tratados" financiado por el Ministerio de educación y ciencia con número de referencia: CTM2006-14163-C02-01, en colaboración con el “Centre de Recerca i Aplicacions Forestals”. La presente Tesis Doctoral aborda los aspectos de determinación analítica de la presencia de micotoxinas en compost y la evolución del nonilfenol en suelos. En concreto, se ha estudiado la presencia de las aflatoxinas B1, B2, G1, G2 y la ocratoxina A en tres muestras de compost producido en dos EDAR de Catalunya. Por lo tanto, se ha desarrollado un método de análisis simultáneos de aflatoxinas B1, B2, G1, G2 y la ocratoxina A en compost. En primer lugar se ha adaptado a estas muestras un procedimiento por HPLC-MS desarrollado previamente en nuestro equipo para la determinación de aflatoxinas B1, B2, G1 y G2 aplicado a matrices alimentarias y se ha extendido a la determinación conjunta de las cuatro aflatoxinas y la ocratoxina A. Este procedimiento ha revelado problemas relativos a la recuperación y la precisión de los resultados. Vistas las dificultades, se ha desarrollado un nuevo procedimiento por UHPLC-fluorescencia que permite una rápida y simple preparación de muestra, así como una mejor sensibilidad. Este nuevo método ha sido validado y puede mejorar el tiempo de análisis y eliminar la etapa con cartuchos de purificación de muestra con cartuchos poliméricos, utilizados en el método de partida. Ambos procedimientos han permitido mostrar que, en las muestras del compost estudiado no hay cantidades significativas de ninguna de las cinco micotoxinas estudiadas. En cuanto a nonilfenol, se ha estudiado su evolución temporal, durante 32 semanas, en muestras de suelo, mezclas de suelo con turba y mezclas de suelo con compost, donde se ha adicionado nonilfenol en dos niveles de concentración. Asimismo, se ha estudiado la concentración del nonilfenol en las muestras de lixiviados de los suelos y mezclas de suelo con turba. Por lo tanto, se ha puesto a punto un procedimiento analítico por HRGC-MS tanto para muestras de suelo como para muestras de lixiviados acuosa. Se ha demostrado la rápida eliminación del nonilfenol en las tres muestras estudiadas y que el nivel de pérdidas por lixiviación es muy baja. Finalmente, en los mismos lixiviados se ha determinado la materia orgánica total y su toxicidad mediante el ensayo de Microtox. Ambas determinaciones muestran que estos lixiviados no presentan problemas ya que cumplen las exigencias para poder ser enviados a las redes de alcantarillado público. Se puede afirmar que el uso de compost en la agricultura es una práctica segura, tanto en cuanto a la ausencia de micotoxinas, como la degradación rápida de nonilfenol en el suelo. / This work was carried out in the framework of the project: “Ecotoxicidad, micotoxinas y degradación de nonilfenoles en lodos de depuradora y suelos tratados". This project was performed in collaboration with "Centre de Recerca i Aplicacions Forestals" and it was supported by “Ministerio de Educación y Ciencia” (contract number CTM2006-14163-C02-01). The present work is referred to the analytical determination of mycotoxins and the degradation of the nonylphenol in soils. Aflatoxins B1, B2, G1, G2 and ochratoxin A in three different samples of compost, produced in two sewage wastewater in Catalonia, has been studied. Also, a method for simultaneous determination of aflatoxins B1, B2, G1, G2 and ochratoxin A in compost has been developed. An analytical method using HPLC-MS has been developed to determinate aflatoxins B1, B2, G1, G2 and ochratoxin A in compost matrices. This method is based on a previous work performed in the Chromatographic Methods Lab at IQS to determinate aflatoxins B1, B2, G1, and G2 in food matrices. It is important to mention that the HPLC-MS method showed low recovery factors and precision problems in the compost samples analyzed. In a second set of experiments a new method using UHPLC-fluorescence has been designed to analyze aflatoxins B1, B2, G1, G2 and ochratoxin A in compost samples. UHPLC-fluorescence allowed quick and simple sample preparation, high sensitivity, short analysis time and the elimination of the purification step using polymeric cartridges. Finally, this method using UHPLC-fluorescence was validated for aflatoxins B1, B2, G1, G2 and ochratoxin A in compost matrices. It is important to mention that HPLC-MS and UHPLC-fluorescence methods verify the absence of mycotoxins in all compost samples analyzed. In a third set of experiments the temporal evolution of nonylphenol has been studied in soil samples, mixtures of soil with peat and soil with compost. The experiments were performed during 32 weeks in which nonylphenol was added at two levels of concentration in the samples at the beginning of the assay. Also, the concentration of nonylphenol in samples of leachates from soil and mixtures of soils with peat has been studied. An analytical method to analyze nonylphenol using HRGC-MS for soil samples and leachates has been designed. Rapid nonylphenol degradation in the three samples analyzed (soil, soil + compost and soil + peat) has been observed and very low nonylphenol levels in samples of leachates from soil and mixtures of soils have been obtained. Finally, Total Organic Carbon (TOC) of the leachates has been measured and also toxicity assays by Microtox test have been performed. Both techniques showed that the leachates analyzed can be dumped in the public sewerage networks because it’s low TOC and toxicity levels. To summarize, it can be assured that the use of compost in agriculture is a safe practice because the absence of mycotoxins and the rapid degradation of nonylphenol in soil.

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