Conselho Nacional de Desenvolvimento Científico e Tecnológico / The intensive use and/or inadequate management of pesticides have led to contamination of the aquatic ecosystem, reaching the biota living there, including the fish. Pesticides can affect toxicological parameters these animals, harming their survival. In this way, the aim this study has to evaluate biochemistry changes in carps (Cyprinus carpio) after exposure to a commercial formulation of the fungicide tebuconazole (Folicur®). At first, we performed a test of acute toxicity to determine the LC50 (96h) of tebuconazole for C. carpio. The animals were exposed to concentrations 1.0, 1.5, 2.0, 2.5 and 3.0 mg/L for 96 hours. After this period, were anesthetized and euthanized. Using the whole-body method, we investigated the levels of TBARS, protein carbonyls, activities of enzymes GST, CAT, SOD and AChE, levels of non-enzymatic antioxidants GSH and AsA and metabolic parameters, among which, lactate, glycogen, glucose, ammonia, amino acids and protein. Secondly, fish were exposed to the fungicide during seven days, both in field conditions (rice paddy field) as a laboratory. The concentrations used in the laboratory (33.5 and 36.2 mg/L) were close to the concentration used for rice farming (31.9 mg/L). After the experimental periods, we sampled brain, liver and muscle of fish. The biochemical parameters analyzed were as follows: TBARS levels in brain, liver and muscle, protein carbonyl in liver, enzyme activities CAT and GST in liver and AChE in brain and muscle. In the first experiment was obtained as LC50 (96h) of tebuconazole for C. carpio concentration 2.37 mg/L. The body levels of TBARS showed to be high at all concentrations tested. In contrast, the enzymes GST, CAT and SOD showed reduction in their activities. The levels of GSH and AsA were also decreased after the same period. Regarding the activity of AChE and the levels of protein carbonyls, there were no statistically significant changes. Among the metabolic parameters, there was an increase in the levels of glycogen and glucose concentration 1.5 mg/L, whereas protein levels were reduced at concentrations 2.0 and 2.5 mg/L. In the second experiment, we observed increased levels of TBARS in brain only in the field condition, while in liver and muscle this increase was observed both in field and laboratory. Likewise, the levels of protein carbonyls increased in both experimental conditions. The activity of AChE in brain was increased only in the field condition, whereas in the laboratory no significant change was observed. The activities of CAT, GST and AChE in muscle showed no significant change in both experimental conditions. The results of this research, considering the two experiments show that this fungicide causes disorders in antioxidants and metabolic parameters in fish exposed, indicating the occurrence of oxidative stress, which can compromise their survival in the wild. The evaluation of TBARS levels and protein carbonyl in the liver of carp can be used as a biomarker of exposure to tebuconazole. / O intenso uso e/ou manejo inadequado de pesticidas têm levado à contaminação do ecossistema aquático, atingindo a biota ali existente, incluindo os peixes. Pesticidas podem afetar parâmetros toxicológicos desses animais, prejudicando sua sobrevivência. Neste sentido, o objetivo deste trabalho foi avaliar alterações bioquímicas em carpas (Cyprinus carpio) após exposição a uma formulação comercial do fungicida tebuconazole (Folicur®). Num primeiro momento, foi realizado um teste de toxicidade aguda a fim de determinar a CL50 (96h) do tebuconazole para C. carpio. Os animais foram expostos às concentrações 1,0, 1,5, 2,0, 2,5 e 3,0 mg/L, durante 96 horas. Após esse período, foram anestesiados e eutanasiados. Utilizando o método whole-body, foram investigados os níveis de TBARS, proteína carbonil, atividades das enzimas GST, CAT, SOD e AChE, níveis dos antioxidantes não enzimáticos GSH e AsA e parâmetros metabólicos, dentre os quais, lactato, glicogênio, glicose, amônia, aminoácidos e proteína. Num segundo momento, os peixes foram expostos ao fungicida durante sete dias, tanto em condição de campo (lavoura de arroz irrigado) como de laboratório. As concentrações utilizadas em laboratório (33,5 e 36,2 μg/L) foram próximas à concentração utilizada na lavoura de arroz (31,9 μg/L). Decorridos os períodos experimentais, foram amostrados cérebro, fígado e músculo dos peixes. Os parâmetros bioquímicos analisados foram os seguintes: níveis de TBARS em cérebro, fígado e músculo; proteína carbonil em fígado; atividades das enzimas CAT e GST em fígado e da AChE em cérebro e músculo. No primeiro experimento obteve-se como CL50 (96h) do tebuconazole para C. carpio a concentração 2,37 mg/L. Os níveis corporais de TBARS mostraram-se elevados em todas as concentrações testadas. Ao contrário, as enzimas GST, CAT e SOD mostraram redução em suas atividades. Os níveis de GSH e AsA também se mostraram diminuídos após o mesmo período. Em relação à atividade da AChE e aos níveis de proteína carbonil, não foram observadas alterações estatisticamente significativas. Dentre os parâmetros metabólicos, houve aumento nos níveis de glicogênio e glicose na concentração 1,5 mg/L, enquanto que os níveis de proteína foram reduzidos nas concentrações 2,0 e 2,5 mg/L. No segundo experimento, observou-se aumento nos níveis de TBARS em cérebro somente na condição de campo, enquanto que em fígado e músculo esse aumento foi verificado tanto em campo quanto em laboratório. Da mesma forma, os níveis de proteína carbonil aumentaram em ambas as condições experimentais. A atividade da AChE em cérebro se mostrou aumentada somente em condição de campo, enquanto que em laboratório nenhuma alteração significativa foi observada. As atividades da CAT, GST e AChE em músculo não apresentaram mudanças significativas em ambas as condições experimentais. Os resultados da presente investigação, considerando os dois experimentos realizados, mostram que esse fungicida provoca desordens em parâmetros antioxidantes e metabólicos nos peixes expostos, indicando a ocorrência de estresse oxidativo, o que pode comprometer sua sobrevivência no meio natural. A avaliação dos níveis de TBARS e de proteína carbonil em fígado de carpas pode ser utilizada como biomarcador de exposição ao tebuconazole.

Orientador: Leonardo Fernandes Fraceto / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-26T15:38:39Z (GMT). No. of bitstreams: 1 Campos_EstefaniaVangelieRamos_M.pdf: 7315526 bytes, checksum: e92d25c76302901307a5c47fb9437573 (MD5) Previous issue date: 2015 / Resumo: O Brasil tornou-se um dos maiores consumidores de defensivos agrícolas em função do seu crescimento no cenário mundial na produção agrícola. Embora esses produtos tragam benefícios para a produção agrícola, têm sido encontrados em concentrações alarmantes nos recursos hídricos e no solo, levando a necessidade de se buscarem alternativas para a redução de seus impactos negativos no meio ambiente. O presente trabalho teve como objetivo o preparo e caracterização de nanopartículas poliméricas (NC) e lipídicas sólidas (NLS) para o carreamento conjunto dos fungicidas carbendazim (MBC) e tebuconazol (TBZ), a fim de aumentar a sua eficácia biológica, além de diminuir os processos físico-químicos de degradação que estes ativos sofrem no ambiente. Os dois sistemas carreadores preparados apresentaram alta eficiência de encapsulação para ambos os fungicidas, superior a 99 %. As NC contendo fungicidas apresentaram diâmetro médio em torno de 550 nm, índice de polidispersão inferior a 0,2 e potencial zeta de aproximadamente -17 mV. Enquanto as NLS apresentaram diâmetro médio em torno de 270 nm, índice de polidispersão superior a 0,2 desde o tempo inicial e potencial zeta de -20 mV. As nanopartículas foram capazes modificar o perfil de liberação dos fungicidas. A liberação do MBC quando associado as nanopartículas em 6 dias foi em torno de 30 % Já para o TBZ no mesmo período de tempo a liberação a partir das nanopartículas foi de 50 %. O modelo matemático que mais se ajustou a liberação do MBC de ambas as nanopartículas foi o de Higuchi. Considerando a liberação do TBZ a partir das nanopartículas o modelo de primeira ordem melhor se ajustou. Os ensaios de liberação em solo demonstrou que as nanopartículas reduzem a porcentagem de lixiviação dos fungicidas, onde para o MBC houve liberação de 17 % a partir da formulação comercial e quando associado as nanopartículas esse valor foi inferior a 5 % com um total de 10 lavagens do solo. Já para o TBZ a formulação comercial liberou 20 % e quando associado as nanopartículas foi em torno de 15 %. Ensaios de viabilidade celular demostraram que as partículas diminuem a toxicidade dos fungicidas à célula animal , sendo os valores de IC50 de 67, 89 e 81 µg/mL para a formulação comercial, NC e SLN respectivamente. Ensaios na espécie P. vulgaris evidenciaram que os fungicidas quando encapsulados causam menos impactos no desenvolvimento das plântulas. Os resultados obtidos mostraram que foi possível preparar sistemas carreadores para os fungicidas sendo, desta forma, obtidos bons sistemas carreadores com modificação no perfil de liberação e diminuição da citotoxicidade, desta forma sendo uma alternativa para o combate de pragas que acometem as culturas na agricultura / Abstract: Brazil has become a major consumer of pesticides according to their growth on the world stage in agricultural production. Although these products bring benefits to agriculture, they cause many impacts to the environment because they are found in high concentrations in soil and water resources. This work aimed to prepare and characterize nanoparticles (polymeric and solid lipid) in order to encapsulate two fungicides carbendazim and tebuconazole. This strategy aims to minimize their impacts to the environment and increase their biological effectiveness, besides of decreasing the physicochemical process of degradation of these compounds into the environment. Both carrier systems showed high encapsulation efficiency for both fungicides (higher than 99 %). The polymeric nanocapsules (NC) with fungicides showed mean diameter around 550 nm, polidispersity lower than 0.2 and zeta potential around - 17 mV. While solid lipid nanoparticles (NLS) showed mean diameter around 270 nm, polidispersity higher than 0.2 since initial time and potential zeta around - 20 mV. Nanoparticles were able to modify the release profile of fungicides. The release of the MBC when associated nanoparticles in 6 days was around 30%. For the TBZ in the same period of time the release from the nanoparticles was 50%. The mathematical model that most adjusted the release of MBC of both nanoparticles was to Higuchi. Whereas the release of the nanoparticles from the TBZ model best fit was observed by the first-order. Cell viability assays showed that particles decrease the toxicity of fungicides, which IC50 values of 67, 89 and 81 µg/mL for the commercial formulation, NC and SLN respectively. The plant assays with P. vulgaris showed that fungicides when encapsulated provoke less impact on seedling development in relation to commercial formulation. The results obtained with these formulations showed that it was possible to prepare nanoparticlescarrier systems for both fungicides in the same carrier and that the fungicide encapsulation decreased the cytotoxicity, increase fungicides effectiveness and thus being a possible alternative to control diseases that affect crops in agriculture / Mestrado / Bioquimica / Mestra em Biologia Funcional e Molecular

Η ανθρώπινη δραστηριότητα (γεωργική, βιομηχανική, αστική κ.α.) καθώς και η διαχείριση των λυμάτων στις μονάδες επεξεργασίας υγρών αποβλήτων (ΜΕΥΑ) επηρεάζουν τα επιφανειακά και υπόγεια υδατικά συστήματα όπως ποτάμια, λίμνες, λιμνοθάλασσες, θάλασσες κ.α. αποτελούν σημαντικές πηγές ρύπανσης και επιφέρουν επιπτώσεις στα οικοσυστήματα αυτά. Η μεταφορά, η κατανομή και η απομάκρυνση των οργανικών μικρορύπων όπως είναι τα παρασιτοκτόνα, οι φαρμακευτικές ενώσεις, οι ενώσεις που περιέχονται στα προϊόντα προσωπικής φροντίδας, κ.α., καθώς και οι μεταβολίτες τους, αποτελεί τις τελευταίες δεκαετίες αντικείμενο συστηματικής έρευνας προκαλώντας το ενδιαφέρον τόσο της επιστημονικής κοινότητας όσο και της κοινωνίας γενικότερα. Ο ποταμός Αχελώος (η κατώτερη λεκάνη απορροής του), που βρίσκεται στην Δυτική Ελλάδα καθώς και η μονάδα επεξεργασίας υγρών αποβλήτων (ΜΕΥΑ) της πόλης του Αγρινίου της οποίας τα επεξεργασμένα λύματα απορρίπτονται σε αυτόν, αποτελεί την περιοχή μελέτης της παρούσας διατριβής. Πρόκειται για ένα υδατικό σύστημα υψηλής περιβαλλοντικής σημασίας αφού συνδέεται με τις λίμνες Τριχωνίδα και Λυσιμαχία καθώς και με τις λιμνοθάλασσες Μεσολογγίου και Αιτωλικού. Επιπροσθέτως, το δέλτα του ποταμού προστατεύεται από διεθνείς περιβαλλοντικές συνθήκες (Ramsar και Natura 2000). Η κατώτερη λεκάνη απορροής του Αχελώου περιλαμβάνει μεγάλες εκτάσεις καλλιεργούμενης γης (πεδιάδες Αγρινίου και Νεοχωρίου-Κατοχής). Σκοπός της παρούσας διατριβής είναι: (α) η μελέτη των επιπέδων των συγκεντρώσεων της μεταβολής (χρονικής και χωρικής) και των επιπτώσεων παρασιτοκτόνων, φαρμακευτικών ενώσεων που ανήκουν σε διαφορετικές χημικές ομάδες και οργανικών ενώσεων προϊόντων προσωπικής φροντίδας στον ποταμό Αχελώο, (β) η απομάκρυνσή τους από την μονάδα επεξεργασίας υγρών αποβλήτων της πόλης του Αγρινίου, (γ) η μελέτη των επιπέδων ρύπανσης από τα επεξεργασμένα λύματα που καταλήγουν στον Αχελώο, (δ) η φωτολυτική και φωτοκαταλυτική αποικοδόμηση (κινητική, μηχανισμοί αποικοδόμησης, σχηματισμός παραπροϊόντων) επιλεγμένων οργανικών ενώσεων από τις παραπάνω ομάδες (του παρασιτοκτόνου tebuconazole και της βακτηριοκτόνου triclosan) στην υδατική φάση (απεσταγμένο νερό και επεξεργασμένα λύματα) ως εναλλακτική τεχνολογία επεξεργασίας για την μείωση των επιπέδων ρύπανσης από τις μονάδες βιολογικού καθαρισμού. Αναπτύχθηκαν και εφαρμόστηκαν πολυ-υπολειμματικές μεθοδολογίες προσδιορισμού των παρασιτοκτόνων και φαρμακευτικών ενώσεων σε δείγματα από τον ποταμό Αχελώο και από την ΜΕΥΑ του Αγρινίου. Η μεθοδολογία περιελάμβανε την απομόνωση των ενώσεων με την τεχνική της υγρής-στερεάς εκχύλισης και τον προσδιορισμό τους με τεχνικές αέριας χρωματογραφίας. Επιλέχτηκαν 32 ενώσεις παρασιτοκτόνων και μεταβολιτών τους (alachlor, atrazine, atrazine desethyl, EPTC, s-metolachlor, simazine, trifluralin, azinphos methyl, chlorpyrifos, chlorpyrifos methyl, chlorfenviphos, diazinon, dichlorvos, dimethoate, fenthion, fenthion sulfoxide, malaoxon, methidathion, methyl parathion, cyproconazole, pyrimethanil, triadimefon, pirimiphos methyl, isoproturon, diuron, carbofuran, quinalphos, triazophos, phosalone, pyrazophos, penconazole, tebuconazole) σύμφωνα με προηγούμενες και σύγχρονες χρήσεις τους στην λεκάνη απορροής του ποταμού Αχελώου. Για την ανίχνευση και την ποσοτικό προσδιορισμό τους χρησιμοποιήθηκε η αέρια χρωματογραφία με ανιχνευτή θερμοϊονισμού φλόγας (GC-FTD) και με φασματομετρία μάζας (GC-MS). Επίσης, έγινε επιλογή 12 φαρμακευτικών ενώσεων και μεταβολιτών τους (salicylic acid, paracetamol, clofibric acid, ibuprofen, phenazone, gemfibrozile, triclosan, naproxen, diclofenac, carbamazepine, caffeine, fenofibrate) σύμφωνα με τα δεδομένα ανίχνευσης και επιπέδων συγκέντρωσης που έχουν καταγραφεί στην Ευρώπη και σε ελάχιστες μελέτες για τον Ελλαδικό χώρο. Για την ανίχνευση και την ποσοτικό προσδιορισμό τους χρησιμοποιήθηκε επίσης αέρια χρωματογραφία με φασματομετρία μάζας (GC-MS). Η χωρική και χρονική διακύμανση των συγκεντρώσεων των παρασιτοκτόνων παρακολουθήθηκε από τον Μάρτιο του 2005 έως τον Φεβρουάριο του 2008. Η χρονική περίοδος δειγματοληψίας περιλαμβάνει ένα έτος πριν την κατάργηση της καλλιέργειας του καπνού, η οποία έγινε σταδιακά από 1/1/2006 μετά την αναθεώρηση της Κοινής Αγροτικής Πολιτικής (ΚΑΠ) σχετικά με τις καλλιέργειες καπνού, και δύο έτη μετά με συνέπεια οι αλλαγές στις γεωργικές χρήσεις γης να εμφανίσουν σημαντικές επιδράσεις στις εισροές παρασιτοκτόνων στον ποταμό Αχελώο. Τις μεγαλύτερες συχνότητες ανίχνευσης εμφάνισαν από τα εντομοκτόνα τα diazinon (78.6%) και fenthion (52.6%), από τα ζιζανιοκτόνα τα DEA (69.3%) και alachlor (50%) και από τα μυκητοκτόνα τα pyrimethanil (67,3%) και tebuconazole (44,7%). Η στατιστική ανάλυση των συγκεντρώσεων των ανιχνευθέντων παρασιτοκτόνων ανέδειξε σημαντικά μεγαλύτερες συγκεντρώσεις την περίοδο εφαρμογής τους, δηλαδή την άνοιξη και τις αρχές του καλοκαιριού, καθώς και μια αυξητική τάση κατά μήκος του ποταμού προς τα σημεία δειγματοληψίας που βρίσκονται στο δέλτα του ποταμού. Διαπιστώθηκε επίσης μείωση των μέσων τιμών των συγκεντρώσεων των παρασιτοκτόνων τα έτη 2006 και 2007 σε σχέση με το 2005 αποδεικνύοντας ότι η κατάργηση της καλλιέργειας του καπνού και η αλλαγή καλλιεργειών είχε σημαντική επίδραση στα επίπεδα ρύπανσης του ποταμού. Το συμπέρασμα αυτό επιβεβαιώθηκε και από την εκτίμηση του κινδύνου που υπολογίστηκε ξεχωριστά για κάθε έτος δειγματοληψίας με την προσδιοριστική μέθοδο του πηλίκου κινδύνου (Risk Quotient). Έτσι, το 2005 έξι παρασιτοκτόνα εμφάνισαν υψηλή επικινδυνότητα, ενώ το 2007 μόλις ένα. Η παρουσία και η απομάκρυνση των παρασιτοκτόνων από τη μονάδα επεξεργασίας υγρών αποβλήτων του Αγρινίου μελετήθηκε για χρονική περίοδο δεκατεσσάρων μηνών (Απρίλιος 2007-Μάιος 2008). Τα ζιζανιοκτόνα isoproturon, atrazine και ο μεταβολίτης DEA, alachlor, τα μυκητοκτόνα tebuconazole και cyproconazole και τα εντομοκτόνα diazinon, methidathion, chlorfenviphos και chlorpyriphos εντοπίστηκαν πιο συχνά στα δείγματα εισροής και εκροής. Οι εποχιακές διακυμάνσεις που παρατηρήθηκαν για τα περισσότερα παρασιτοκτόνα δείχνουν ότι στο παντοροϊκό σύστημα της μονάδας επεξεργασίας υγρών αποβλήτων καταλήγουν τα υπολείμματα των παρασιτοκτόνων από τις γεωργικές εφαρμογές στην περιαστική περιοχή καθώς και από την καταπολέμηση των παρασίτων στην πόλη, με υψηλότερες συγκεντρώσεις κατά τη διάρκεια της περιόδου κύριας εφαρμογής τους από τα μέσα Μαρτίου έως τα τέλη Ιουνίου. Εξαίρεση στην παραπάνω τάση παρατηρήθηκε στα μυκητοκτόνα της κατηγορίας των υποκατεστημένων αζολών, εκτός του triadimefon, όπου ανιχνεύσεις καταγράφηκαν για μεγαλύτερα χρονικά διαστήματα κατά τη διάρκεια του έτους, λόγω της χρήσεις τους ως συντηρητικά-βιοκτόνα σε επιφάνειες και επιχρίσματα. Μελετήθηκε επιπρόσθετα το ποσοστό της απομάκρυνσης των παρασιτοκτόνων κατά τα στάδια της δευτεροβάθμιας και τριτοβάθμιας επεξεργασίας των υγρών αποβλήτων. Η % απομάκρυνση συνολικά για όλα τα στάδια της επεξεργασίας, για τα ζιζανιοκτόνα κυμάνθηκε από 65% έως 77%, για τα alachlor και trifluralin αντίστοιχα, ενώ για το DEA έφτασε το 82%, για τα εντομοκτόνα κυμάνθηκε μεταξύ 62% και 98% για τα chlorfenviphos και dichlorvos, αντίστοιχα ενώ από όλα τα μυκητοκτόνα, το triadimefon εμφάνισε την μεγαλύτερη μέση απομάκρυνση (93%) και το pyrimethanil την μικρότερη (46%). Γενικά, η πρωτοβάθμια επεξεργασία στην ΜΕΥΑ δεν συμβάλει σημαντικά στην απομάκρυνση των παρασιτοκτόνων από τα υγρά λύματα, ενώ η τριτοβάθμια είναι αποτελεσματική για ένα μικρό αριθμό παρασιτοκτόνων. Αναδείχτηκε ότι μια σημαντική πηγή εισόδου παρασιτοκτόνων στον ποταμό Αχελώο αποτελούν και οι εκροές από την ΜΕΥΑ του Αγρινίου, αφού μόνο μερικές από αυτές τις ενώσεις απομακρύνονται σε υψηλά ποσοστά κατά τη διάρκεια της επεξεργασίας των αποβλήτων. Με στόχο την διερεύνηση της ρύπανσης του ποταμού Αχελώου από τις 12 επιλεχθέντες φαρμακευτικές ουσίες, έγιναν μηνιαίες δειγματοληψίες για χρονικό διάστημα ενός έτους ή 14 μηνών. Σύμφωνα με τα αποτελέσματα που προέκυψαν, το salicylic acid, η paracetamol, το carbamazepine και η caffeine ανιχνεύθηκαν στο 100 % των δειγμάτων, ενώ η υψηλότερη συγκέντρωση που ανιχνεύθηκε ήταν 350,13 ng/L για την paracetamol. Σύμφωνα με την στατιστική επεξεργασία των αποτελεσμάτων δεν παρατηρήθηκαν εποχιακές διαφορές στις μέσες τιμές των συγκεντρώσεων των φαρμακευτικών ενώσεων. Αντίθετα, για την χωρική κατανομή τους παρατηρήθηκε στατιστικά σημαντική διαφορά (Ρ<0,050) για το σημείο δειγματοληψίας, που βρίσκεται μετά την έξοδο της ΜΕΥΑ της πόλης του Αγρινίου για το σύνολο των ενώσεων που μελετήθηκαν. Τέλος, πραγματοποιήθηκε η εκτίμηση του κινδύνου για δύο σενάρια: της γενικής (RQm) και της ακραίας υπόθεσης (RQex) όπου η διάμεση και η μέγιστη συγκέντρωση που προσδιορίστηκαν, χρησιμοποιήθηκαν για τον υπολογισμό του πηλίκου κινδύνου. Τα αποτελέσματα ανέδειξαν και για τα δύο σενάρια ότι μόνο το triclosan εμφανίζει υψηλή επικινδυνότητα ενώ οι υπόλοιπες ενώσεις εμφανίζουν χαμηλή ή μέτρια επικινδυνότητα. Τα επίπεδα συγκέντρωσης της ομάδας των φαρμακευτικών ουσιών καθώς και η απομάκρυνσή τους από τη ΜΕΥΑ του Αγρινίου εξετάστηκαν κατά τη διάρκεια μιας δεκατετράμηνης δειγματοληψίας. Οι περισσότερες από αυτές (salicylic acid, clofibric acid, paracetamol, caffeine, gemfibrozil, triclosan, diclofenac και carbamazepine) ανιχνεύτηκαν σε ποσοστό 100% των αναλυθέντων δειγμάτων. Οι υψηλές συχνότητες ανίχνευσης οφείλονται είτε στην συχνή τους κατανάλωση (π.χ. salicylic acid, clofibric acid, paracetamol, caffeine και gemfibrozil), είτε στα μικρά ποσοστά απομάκρυνσής τους κατά τη διάρκεια της επεξεργασίας στη ΜΕΥΑ (π.χ. carbamazepine), είτε και στους δύο παραπάνω λόγους (π.χ. triclosan και diclofenac). Οι μέσες τιμές απομάκρυνσης των φαρμακευτικών ενώσεων από τη ΜΕΥΑ υπολογίστηκαν μεταξύ 46,3% και 96,8%, με τις υψηλότερες τιμές να καταγράφονται για το naproxen (96,8%) και την caffeine (96%) ενώ οι χαμηλότερες για το carbamazepine (46,3%) και το triclosan (63,2%). Κατά τη διάρκεια των χειμερινών μηνών παρατηρήθηκαν μικρότερες μέσες τιμές απομάκρυνσης λόγω των χαμηλότερων θερμοκρασιών αλλά και λόγω των βροχοπτώσεων που προκαλούν αραίωση από τη μια και μικρότερους υδραυλικούς χρόνους παραμονής των λυμάτων στη ΜΕΥΑ από την άλλη. Μελετήθηκε η φωτολυτική και φωτοκαταλυτική αποικοδόμηση (κινητική, μηχανισμοί αποικοδόμησης, σχηματισμός παραπροϊόντων) επιλεγμένων οργανικών ενώσεων από τις παραπάνω ομάδες (του παρασιτοκτόνου tebuconazole και της βακτηριοκτόνου triclosan) στην υδατική φάση (απεσταγμένο νερό και επεξεργασμένα λύματα) ως εναλλακτική τεχνολογία επεξεργασίας για την μείωση των επιπέδων ρύπανσης από τις μονάδες βιολογικού καθαρισμού. Το tebuconazole επιλέχθηκε λόγω της μεγάλης εφαρμογής του τα τελευταία χρόνια σε γεωργικές και αστικές χρήσεις, της μικρής του απομάκρυνσης κατά την επεξεργασία των υγρών αποβλήτων στη ΜΕΥΑ και της μεγάλης ανθεκτικότητάς του στο περιβάλλον. Το triclosan παρόλο που δεν ανιχνεύτηκε σε μεγάλες συγκεντρώσεις, επιλέχθηκε επειδή είναι η μόνη από τις φαρμακευτικές ενώσεις που μελετήθηκαν που εμφανίζει υψηλή επικινδυνότητα για το περιβάλλον ,καθώς επίσης και λόγω της χημικής του δομής, που είναι παρόμοια και μπορεί να είναι πρόδρομη με ιδιαίτερα τοξικές ενώσεις όπως τα πολυχλωριωμένα φουράνια και οι διοξίνες. Επιδεικνύει ανθεκτικότητα στην αποικοδόμησή του ενώ αποδεδειγμένα προκαλεί ενδοκρινική διαταραχή σε έμβια όντα. Ως πρώτος στόχος της μελέτης της φωτοκαταλυτικής αποικοδόμησης ήταν η διερεύνηση των βασικών πειραματικών παραμέτρων που επιδρούν στο ρυθμό και την απόδοση της φωτοκαταλυτικής οξείδωσης (συγκέντρωση του καταλύτη, αρχική συγκέντρωση του ρύπου, ένταση της ακτινοβολίας) και η εύρεση των βέλτιστων συνθηκών για τη μέγιστη φωτοκαταλυτική δραστικότητα με τη χρήση Κεντρικού Σύνθετου Πειραματικού Σχεδιασμού (Central Composite Design, CCD) και μεθοδολογία απόκρισης επιφανείας. Εν συνεχεία μελετήθηκε η κινητική της αποικοδόμησής τους σε διάφορες πειραματικές συνθήκες, η εκτίμηση του βαθμού ανοργανοποίησης των ρύπων και τέλος η μελέτη του μηχανισμού της αποικοδόμησής τους. Τα πειράματα της φωτόλυσης και της φωτοκατάλυσης πραγματοποιήθηκαν σε φωτο-αντιδραστήρα με χρήση οργανολογίας (SUNTEST XLS+, ATLAS) που προσομοιώνει την ηλιακή ακτινοβολία. Ο ποσοτικός προσδιορισμός των tebuconazole και triclosan για τη μελέτη της κινητικής της αντίδρασης διάσπασης σε υδατικά διαλύματα πραγματοποιήθηκε με υγρή χρωματογραφία υψηλής απόδοσης (HPLC). Στα δείγματα που λαμβάνονταν σε τακτά χρονικά διαστήματα εκτός από τη συγκέντρωση του ρύπου, προσδιορίζονταν και ο ολικός οργανικός άνθρακας (TOC) καθώς και οι συγκεντρώσεις των ανόργανων ιόντων που προκύπτουν από την διάσπαση της μητρικής ένωσης με τη χρήση ιοντικού χρωματογράφου. Επίσης, για την διερεύνηση του μηχανισμού των αντιδράσεων της φωτοκαταλυτικής διάσπασης των επιλεχθέντων ρύπων έγινε κινητική μελέτη με τη χρήση παρεμποδιστών ενώ παράλληλα προσδιορίστηκαν τα ενδιάμεσα προϊόντα της διάσπασής τους με ταυτοποίηση τους σε αέριο χρωματογράφο με φασματοσκοπία μάζας (GC-MS). Από τα αποτελέσματα των πειραμάτων του Κεντρικού Σύνθετου Πειραματικού Σχεδιασμού (CCD), οι βέλτιστες συνθήκες για την αποδόμηση του tebuconazole βρέθηκαν να είναι Cteb=1ppm, CTiO2=550ppm, I=650W/m2 ενώ για το triclosan Ctric=1ppm, CTiO2=550ppm, I=700W/m2. Στις βέλτιστες συνθήκες πραγματοποιήθηκαν τα πειράματα κινητικής μελέτης με ή χωρίς παρεμποδιστές, μελέτης των ενδιάμεσων προϊόντων της φωτοκαταλυτικής τους διάσπασης καθώς και της ανοργανοποίησής τους. Η διάσπαση των ρύπων ακολούθησε κινητική ψευδό-πρώτης τάξης. Τα αποτελέσματα από τα πειράματα κινητικής με χρήση παρεμποδιστών κατέδειξαν ότι η φωτοκαταλυτική αποικοδόμηση του tebuconazole λαμβάνει χώρα κυρίως μέσω των ελευθέρων ριζών υδροξυλίου και με μικρότερη συμμετοχή και των θετικών οπών. Αντίστοιχα, για το triclosan η φωτοκαταλυτική αποικοδόμηση λαμβάνει χώρα κυρίως μέσω ελευθέρων ριζών υδροξυλίου. Σχεδόν πλήρης ανοργανοποίηση επιτυγχάνεται μετά το τέλος της φωτοκαταλυτικής διεργασίας για το triclosan, ενώ για το tebuconazole, το άζωτο προσδιορίστηκε στο 80% της θεωρητικά αναμενόμενης ποσότητάς του που απελευθερώνεται στο διάλυμα με τη μορφή ΝΟ3- και ΝΟ2-, γεγονός που υποδηλώνει ότι είτε τα τελικά οργανικά προϊόντα διάσπασης του tebuconazole (αζολικοί δακτύλιοι) απαιτούν μεγάλους χρόνους ακτινοβόλησης για την αποικοδόμησή τους είτε ότι σχηματίζεται ελεύθερο Ν2 μετά τη διάνοιξη του αζολικού δακτυλίου. Τέλος, μετά την ανίχνευση και ποσοτικοποίηση των ενδιαμέσων προϊόντων των αντιδράσεων της φωτοκαταλυτικής διάσπασης των δύο ρύπων προτάθηκαν οι μηχανισμοί αποικοδόμησής τους σε υδατικά συστήματα. Σύμφωνα με τα αποτελέσματα της φωτοκαταλυτικής οξείδωσης των δύο ρύπων-μοντέλων που μελετήθηκαν στην παρούσα διατριβή η εφαρμογή της φωτοκατάλυσης σε ευρύτερη κλίμακα και σε πραγματικά υδατικά απόβλητα και συστήματα επεξεργασίας φυσικών νερών είναι μια γρήγορη και πολύ αποδοτική τεχνολογία επεξεργασίας οργανικών μίκρο-ρύπων. Απαιτείται όμως έλεγχος και βελτιστοποίηση των διαφόρων παραμέτρων που επιδρούν στην απόδοση της διεργασίας και η χρήση ηλιακής ακτινοβολίας, ώστε η εφαρμογής της να είναι πιο εφικτή τόσο από οικονομική όσο και περιβαλλοντική άποψη. Επιπλέον, η δυνατότητα συνδυασμού της μεθόδου αυτής με άλλες μεθόδους επεξεργασίας, βιολογικές ή/και φυσικοχημικές, θα μπορούσε να αποτελέσει μια πιο ολοκληρωμένη και οικονομική λύση σε προβλήματα περιβαλλοντικής ρύπανσης, σύμφωνα με τα σύγχρονα κριτήρια και τις απαιτήσεις. / Human activities (agricultural, industrial, urban, etc.) and wastewater disposal in sewage treatment plants affect surface and ground water bodies such as rivers, lakes, lagoons, sea waters etc., consisting major pollution sources leading to significant negative impacts on ecosystems. The transportation, distribution and elimination of organic micropollutants, such as pesticides, pharmaceutical and personal care products compounds, etc., and their metabolites has been the subject of systematic research in recent decades, arousing a great interest for both the scientific community and modern societies. The lower part of Acheloos River basin, located in Western Greece, and the wastewater treatment plant (WWTP) of Agrinio city, the effluents of which discharge into the river, are selected to be studied in the present thesis in order to monitor the occurrence, patterns and effects of organic micropollutants such as pesticides and pharmaceutical compounds in the sewage-impacted river as well as the removal of such pollutants using conventional (WWTP) and alternative advanced oxidation (photocatalysis) technologies. Acheloos River is one of the most important water resources in Greece with a great environmental significance since it is connected with the lakes Trichonida, Lysimachia and the Messolonghi and Etoliko lagoons. In addition, river's delta is protected by international environmental conventions (Ramsar and Natura 2000). The lower basin Acheloos includes large areas of cultivated land (Agrinio and Neochori-Katochi plains). The aims of the present study were the study of: (a) the occurrence and patterns (temporal and spatial distribution), of selected pesticides, pharmaceutical compounds and personal care products, belonging to different chemical groups in Acheloos River as well as the assessment of the environmental risk associated with their levels in river waters; (b) the occurrence and the removal rates along the secondary and tertiary treatments in the wastewater plant of Agrinio city; (c) the pollution levels from wastewater discharges into Acheloos River; (d) the photolytic and photocatalytic degradation (kinetic studies, degradation mechanisms, by-products formation, mineralization) of selected model compounds (pesticide tebuconazole and antimicrobial triclosan) within the detected micro-pollutants in different water substrates (distilled water and treated wastewater) as an alternative treatment technology for the reduction of pollution levels of the sewage treatment plants. For the achievement of the above aims and objectives two multi-residue methods have been developed and applied for the determination of pesticide and pharmaceutical compounds in water samples sampled from the river and the influent and effluents of WWTP. The analytical method included the isolation of compounds from the water matrices using solid phase extraction and the qualitative and quantitative determination by gas chromatography techniques using selective detectors (FTD) and mass spectrometry. Thirty-two pesticides including metabolites were selected (i.e. alachlor, atrazine, atrazine desethyl, EPTC, s-metolachlor, simazine, trifluralin, azinphos methyl, chlorpyrifos, chlorpyrifos methyl, chlorfenviphos, diazinon, dichlorvos, dimethoate, fenthion, fenthion sulfoxide, malaoxon, methidathion, methyl parathion, cyproconazole, pyrimethanil, triadimefon, pirimiphos methyl, isoproturon, diuron, carbofuran, quinalphos, triazophos, phosalone, pyrazophos, penconazole, tebuconazole) according to previous and current agricultural and urban uses in the basin river. In addition, twelve pharmaceutical compounds and metabolites (i.e. salicylic acid, paracetamol, clofibric acid, ibuprofen, phenazone, gemfibrozil, triclosan, naproxen, diclofenac, carbamazepine, caffeine, fenofibrate) were chosen according to the occurrence and data reported in the open literature for other water bodies in Europe and few studies in Greece. Gas chromatography mass spectrometry (GC-MS) was also used for the detection and the quantification of pharmaceutical compounds. Spatial and temporal variations in pesticide concentrations were monitored during the period March 2005 - February 2008. Sampling period began one year before the restrictions imposed in tobacco cultivation (from 1/1/2006), the main agricultural activity in the area, within the framework of Common Agricultural Policy, and lasted two years after the restriction in order to reveal probable changes in pesticide uses and inputs to the river flow. The most frequently detected pesticides within the monitoring period were diazinon (78.6%) and fenthion (52.6%), from the group of insecticides, DEA (69.3%) and alachlor (50%) from the group of herbicides and pyrimethanil (67.3%) and tebuconazole (44.7%) from the group of fungicides. Statistical analysis of the detected pesticide concentrations showed significantly higher concentrations during the spring and early summer coinciding frequently with the application periods, and an increasing trend along the river flow with the sampling sites located in the river's delta presenting the higher concentration levels. It was also found that mean concentrations of pesticides in years 2006 and 2007 were lower compared with the year 2005 proving that the restrictions of tobacco cultivation and changes in land uses had a significant effect on Acheloos River pesticide pollution levels. This is further confirmed by conducting the risk assessment for each year of the sampling period using the risk quotient method. Six pesticides showed high risk, in 2005 while just one in 2007. The occurrence and the removal of pesticides from the wastewater treatment plant of Agrinio were studied for a period of fourteen months (April 2007-May 2008). The herbicides isoproturon, atrazine and its metabolite DEA, alachlor, the insecticides diazinon, methidathion, chlorfenviphos and chlorpyriphos and the fungicides tebuconazole, cyproconazole were the most frequently detected in influent and effluent samples. Seasonal variations for the majority of pesticides showed that pesticide inputs in wastewater treatment plant from agricultural cultivations in the suburban area and pesticides control in the city, presented higher concentrations during the main period of applications from mid-March to late June. Fungicides belonging to the chemical group of substituted azoles were the exception to the above trend, except triadimefon, and were detected for longer periods during the year because of their uses as preservatives-biocides to surfaces and coatings of buildings. Additionally, the removal rates of pesticides during secondary and tertiary treatment of the WWTP were studied. Mean removal rates (%) after primary and secondary treatment ranged between 31% for pyrimethanil to 97% for dichlorvos. Mean cumulative removals (%) for the whole treatment ranged between 46% pyrimethanil to 93% for triadimefon. Generally, primary treatment in WWTP did not remove significantly pesticides from wastewater, while tertiary treatment was effective for a small number of pesticides. Based on the results of the pesticide occurrence and removal rates, the WWTP of Agrinio city should be considered as a significant point source of pesticides in Acheloos River. The occurrence and the removal of pharmaceuticals along wastewater treatment plant of Agrinio were also studied for the same sampling period (April 2007-May 2008). Most of the analyzed compounds (salicylic acid, clofibric acid, paracetamol, caffeine, gemfibrozil, triclosan, diclofenac and carbamazepine) were detected in 100% of the samples. The observed high detection frequencies were associated either to their frequent consumption (e.g. salicylic acid, clofibric acid, paracetamol, caffeine and gemfibrozil), or low elimination during treatment (e.g. carbamazepine), or both of the above reasons (e.g. triclosan and diclofenac). The highest mean removals after tertiary treatment were measured for naproxen (96.8%), caffeine (96%) and ibuprofen (92.3%), (except phenazone removals were not determined due to the low frequency of detection), while the lowest for carbamazepine (46.3%) and triclosan (63.2%). Between secondary and tertiary treatment mean removal efficiencies ranged from 18.3% for carbamazepine to 67.4% for naproxen. Lower mean removal rates were determined during winter probably due to low temperatures and high rainfall, causing dilution and lower hydraulic residence times in WWTP. Regarding the occurrence of pharmaceutical compounds along the river flow salicylic acid, paracetamol, carbamazepine and caffeine were detected in 100% of samples analyzed, while the highest detected concentration was 350,13 ng/L, recorded for paracetamol. Statistical analysis did not revealed seasonal differences in mean concentrations of pharmaceutical compounds. In contrary, spatial distribution showed statistically significant difference (P <0.050) for the sampling station located afterwards the discharge of WWTP for all studied compounds. Finally, risk assessment was estimated using the risk quotient method for different scenarios: the general (RQm) and extreme (RQex), where median and maximum concentrations were used respectively. The obtained results using both scenarios revealed that only triclosan exhibited high risk while the rest of the compounds presented low or moderate risk. Photolytic and photocatalytic degradation (kinetic studies, degradation mechanisms, by-products formation, mineralization) of the pesticide tebuconazole and the antibacterial compound triclosan were studied in different aqueous matrices (distilled water and treated wastewater) Tebuconazole was chosen due to its wide application in recent years in agricultural and urban uses, low removal efficiency during treatment in WWTP and high persistence in aquatic environment. Although triclosan was not extensively detected, it was selected due to the high risk determined, and because of its chemical structure, which is similar to highly toxic compounds such as polychlorinated furans and dioxins and may be a precursor for the formation of them. Triclosan exhibited also resistance to degradation and causes endocrine disruption in living organisms. Photolysis of tebuconazole was a very low process, thus the environmental significance for the degradation of tebuconazole is considered negligible. On the contrary, significant photolysis rates were determined for triclosan with half lives of about 7 days using mean solar irradiance intensity for Greece. Primary goal of the photocatalytic study was to explore experimental parameters of photocatalytic degradation that affect the rate and efficiency of oxidation reaction (catalyst concentration, initial pollutant concentration, radiation intensity) and to find the optimum conditions for maximum photocatalytic activity, using Central Composite Design (CCD) and response surface methodology. The kinetics of the photocatalytic degradation and the degree of mineralization were studied in different experimental conditions. Photolytic and photocatalytic experiments were performed using SUNTEST XLS+ instrument, from ATLAS, simulating solar radiation. Quantification of tebuconazole and triclosan for the kinetic studies in aquatic solutions was carried out by high performance liquid chromatography (HPLC). Samples taken periodically during photocatalytic process were filtered and analyzed for determining the concentration of the pollutant, the concentration of total organic carbon (TOC) using a TOC instrument, as well as the concentration of inorganic ions resulting from the decomposition of the parent compound using ion chromatography. The determined optimum experimental conditions using CCD and RSm methodologies were Cteb=1 ppm, CTiO2=550 ppm, I=650 W/m2 for tebuconazole and Ctric=1 ppm, CTiO2=550 ppm, I=700 W/m2 for triclosan. Degradation kinetics of both pollutants followed pseudo-first order model. In order to investigate the major reactive species taking part of the photocatalytic degradation mechanism of the selected pollutants, kinetic studies using scavengers were conducted, while by-products were identified by means of gas chromatography-mass spectrometry (GC-MS). Kinetic experiments using scavengers showed that the photocatalytic degradation of tebuconazole occurred primarily via hydroxyl radical and to a lesser extent by positive holes. Triclosan photocatalytic degradation took place mainly through hydroxyl radicals. Almost complete mineralization was achieved for triclosan, while for tebuconazole, more than 80% of the theoretically expected amount of nitrogen was determined as NO3- and NO2-, suggesting either that the final organic decomposition products of tebuconazole require longer irradiation times for degradation or that free N2 was formed during degradation of azole rings. Finally, the mechanisms of the photocatalytic degradation of both compounds were proposed based on the detection and semi-quantification of by-products performed by gas chromatography-mass spectrometry. According to the results obtained for the photocatalytic oxidation of the two model-pollutants, photocatalysis could be a very fast and efficient treatment technology for the removal of organic micro-pollutants from real wastewaters and natural waters. However, before implementation it is necessary to control and optimize various parameters that affect the efficiency of process such as the catalyst loading, the intensity of solar radiation etc., in order to be more feasible both economically and environmentally. Moreover, the possibility of combining this method with other conventional biological and/or physicochemical methods of treatment, could result in a more integrated and cost-effective solution for environmental pollution problems in accordance with modern standards and requirements.

Submitted by (lucia.rodrigues@ufrpe.br) on 2017-02-17T14:46:49Z No. of bitstreams: 1 Cicero Nicolini.pdf: 204101 bytes, checksum: 7157eb0dfbcf608b64b0ae9bbbaf5c05 (MD5) / Made available in DSpace on 2017-02-17T14:46:49Z (GMT). No. of bitstreams: 1 Cicero Nicolini.pdf: 204101 bytes, checksum: 7157eb0dfbcf608b64b0ae9bbbaf5c05 (MD5) Previous issue date: 2008-03-03 / The Alternaria black spot is one of the most common and destructive diseases of brassica species. This disease can be caused by several species of Alternaria, although A. brassicicola is predominant species in both conventional and organic crops in Brazil. Since commercial cultivars of brassica with acceptable levels of disease resistance are not available, the disease control in conventional production system is based on the fungicide applications, while in the organic production system the disease control on relays on cultural methods. The objective of this study is to assess the sensitivity of 112 isolates of A. brassicicola to fungicide groups: benzimidazoles (carbendazim), dicarboximides (iprodione), triazoles (tebuconazole) and strobilurines (azoxystrobin). The isolates were evaluated in vitro to obtain the concentration capable of inhibiting 50% of the mycelial growth (CL50) and separated in four classes depending on the sensibility to the tested fungicides. All the isolated of A. brassicicola were sensitive the iprodione, with CL50 values below to 0.1 mg i.a./L. Most of the isolates originating from conventional (92.9%) and organic (96.4%) were middling resistant the azoxystrobin, while an isolated (CFM-576) was highly resistant. In relation to tebuconazole, only isolated sensitive (42.9%) and lightly resistant (57.1%) were observed. There was nosignificant difference between the isolates of A. brassicicola originated either from conventional or organic systems and brassica types regarding the levels of sensitivity to the fungicides. / A alternariose é uma das doenças foliares mais comuns e destrutivas das brássicas, podendo ser causada por várias espécies de Alternaria, embora A. brassicicola seja a espécie predominante em plantios convencionais e orgânicos no Brasil. Como inexistem cultivares comerciais de brássicas com níveis aceitáveis de resistência à doença, no sistema de produção convencional o controle da doença se baseia na aplicação de fungicidas, enquanto no sistema orgânico em métodos culturais. O presente trabalho teve como objetivo avaliar a sensibilidade de 112 isolados de A. brassicicola oriundos de cultivos convencionais e orgânicos de brássicas a fungicidas dos grupos dicarboximidas (iprodione), triazóis (tebuconazole) e estrobilurinas (azoxystrobin). Os isolados foram avaliados in vitro visando obter a concentração capaz de inibir 50% do crescimento micelial (CL50) e separados em quatro classes dependendo da sua sensibilidade aos fungicidas testados. Todos os isolados de A. brassicicola foram sensíveis a iprodione, com valores de CL50 inferiores a 0,1 mg i.a./L. A maioria dos isolados oriundos de cultivos convencionais (92,9%) e orgânicos (96,4%) se comportou como medianamente resistente a azoxystrobin, enquanto um isolado (CFM-576) foi altamente resistente. Em relação a tebuconazole, foram constatados somente isolados sensíveis (42,9%) e ligeiramente resistentes (57,1%). Não foi encontrada diferença significativa quanto à sensibilidade aos fungicidas testados entre os isolados de A. brassicicola oriundos de cultivos convencionais e orgânicos, bem como, coletados de diferentes tipos de brássicas.

Sour rot of tomatoes, caused by Geotrichum candidum, occurs in the field and postharvest settings regularly, although postharvest losses are severe only in some years on the Eastern Shore of Virginia (ESV) and other tomato production regions. Fungicide products and cultural control methods are tested for efficacy utilizing a traditional wounding technique that does not properly reflect natural sour rot infections. A new inoculation technique was optimized for G. candidum using negative pressure to infiltrate the tomato stem scar with pathogenic spores. This new method creates consistently high rates of infection and more successfully creates infections in mature green and breaker fruit. The population of G. candidum on the Eastern Shore of VA (ESV) was characterized using multilocus sequencing technique. The resulting phylogenetic tree defines four distinct groups, including two with uncommon loci that distinguish them from the majority of the population. Thirty-seven G. candidum isolates were inoculated to media amended with ten fungicides and antimicrobial compounds commonly used in tomato production and postharvest treatments. Propiconazole and tebuconazole completely inhibited growth of all colonies. Cultivar trials were conducted to determine if resistance or tolerance to G. candidum occurs. Ten commonly grown round and Roma cultivars on the ESV were similarly susceptible to G. candidum, even at low inoculum levels. Field and postharvest surveys of sour rot on tomato fruit attempted to correlate disease incidence with weather conditions in order to better understand the cause of sporadic infection. Few patterns were seen consistently throughout harvest periods and years. Rainfall was positively correlated with disease 2-3 days before surveys and temperature was negatively correlated with disease 5-7 days before surveys. No in-field weather conditions were correlated with postharvest disease incidence. Greenhouse trials were conducted to assess the influence of water congested tomato fruit on susceptibility to sour rot. Tomato plants were exposed to water inundation to mimic rainfall and varying levels of irrigation, both in order to congest tomato fruit. Though water congestion was achieved, tomato fruit were equally susceptible to sour rot infections. / Ph. D.

Parmi les stratégies visant à réduire l’utilisation de pesticides, l’augmentation de l’efficacité des matières actives par du design de formulations ou par l’utilisation de cocktails de pesticides, épandus chacun à plus faible dosage, est une piste très suivie. Cependant, l’influence de mélanges de molécules sur le devenir, le comportement et la toxicité de chacune d’elle individuellement n’a été que rarement décrite. Nous avons donc étudié l’impact des formulations et du mélange d’un herbicide, la mésotrione, et d’un fongicide, le tébuconazole, sur leur biodégradation respective, en termes de cinétiques et de modulations de voies métaboliques de dégradation, mais aussi sur la croissance de différents microorganismes dégradant ou non. Un schéma général de biodégradation de la mésotrione commun à de nombreux microorganismes, Gram positif et négatif, a été proposé conduisant, dans nos conditions, à l’accumulation d’un métabolite, l’AMBA. L’étude comparée avec la formulation (Callisto®) a montré une forte influence des adjuvants sur la cinétique de biodégradation de la mésotrione, allant d’une forte inhibition pour les souches Gram positif testées jusqu’à une stimulation pour une souche d’E. coli. Pour le tébuconazole, seules 3 souches se sont montrées capables de dissiper ce fongicide et de nouveaux métabolites ont pu être mis en évidence. L’utilisation de la formulation (Balmora®) conduit à des effets variables sur sa biodégradation en fonction de la souche et de la concentration. Cependant, les adjuvants présentent toujours un effet inhibiteur, souvent même fort, sur la croissance des microorganismes testés, en particulier sur les souches bactériennes Gram positif, qui se sont toujours révélées plus sensibles. Les études sur le mélange de ces deux pesticides, purs ou formulés ont mis en évidence la complexité des phénomènes : effets inhibiteurs, neutres et souvent stimulation des cinétiques de biodégradation de chaque pesticide que le microorganisme testé dégrade ou non le composé en question ; effet synergique du mélange de pesticides, purs ou formulé, sur la toxicité. / Among the strategies aiming to reduce the use of pesticides, the increase in active ingredient efficiency by formulation design or pesticide mixture (each one being spread at a lower dose) is a well-established approach. Nevertheless, the effect of chemical mixture on the fate, behaviour and toxicity of each compound has not been widely studied. The impact of formulation and mixture of a herbicide, mesotrione, and a fungicide, tebuconazole, on their own biodegradation, in terms of kinetics and metabolic pathway, but also on microbial growth of several microbial strains degrading- or not was studied. A biodegradative pathway for mesotrione common to several Gram-positive and Gram-negative bacteria was proposed, leading under our conditions, to the accumulation of AMBA metabolite. The comparison with the formulation Callisto® showed a high effect of additives on the kinetics of mesotrione biodegradation, ranging from a strong inhibition for the Gram positive strains to stimulation for a strain of E. coli. For tebuconazole, only three bacteria were found capable of dissipating this fungicide and new metabolites were identified. The use of the formulation Balmora® lead to variable effects on tebuconazole biodegradation according to the strain and its concentration. Nevertheless, the additives always had an inhibitory effect on microbial growth, in particular on Gram positive bacteria. Studies on mixtures of both pesticides, pure or formulated, showed the complexity of the processes involved: variable effects but often increase of the pesticide biodegradation although the microorganism studied did or did not degrade them; a synergic toxic effect of pure or formulated pesticide mixture on microbial growth.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Due to the proximity of crop and fish culture areas, some agrichemicals that could be harmful for fish could enter into fishponds by different ways, such as by leaching through rain. The contamination of the aquatic environment by pesticides is a problem of global importance because of its toxic effects on non-target organisms. These products may affect toxicological endpoints in fish. Among the effects that pesticides can cause fish is the formation of reactive oxygen species (ROS) and changes in antioxidant enzymatic and nonenzyme, which may show a situation of oxidative stress. The formation of ROS may be associated with different pathological processes in fish exposed to pollutants such as pesticides. Several biochemical and physiological responses occur when a pesticide enters the body, resulting in the adaptation of the organism to the contaminant or if the body can not efficiently metabolize these products, they may induce toxicity. Rhamdia quelen (Teleostei) were exposed to sub-lethal concentrations of methyl parathion (MP), a glyphosate based herbicide (Gly), and tebuconazole (Teb). The liver of R. quelen exposed to MP and Teb showed enhanced levels of thiobarbituric acid reactive substances (TBARS), compared to the control fish (56% and 59%, respectively). In contrast, Gly did not alter the TBARS generation. The protein carbonyl content increased only in fish exposed to Teb. Fish exposed to the three agrichemicals showed a significant decrease of catalase activity (52%, 48%, and 67%, respectively) and increased glutathione-S-transferase (57%, 46%, and 160%, respectively) activity. Fish exposed to MP, Gly, and Teb showed higher reduced glutathione (151%, 472%, and 130%, respectively, when compared to the control levels) and ascorbic acid concentrations (121%,102%, and 184%, respectively),while the non-protein thiol content increased only in R. quelen exposed to tebuconazole. Fish exposed to MP and Teb showed several pathological changes in the liver, including hepatocyte degeneration and bile stagnation. The present work reports for the first time the toxicity of the pesticide MP and the fungicide Teb in R. quelen, and as in other works, suggests the relatively lower liver toxicity of Gly for fish. The data presented herein demonstrate that sublethal concentrations of MP and Teb cause changes in oxidative stress parameters as well as hepatic cell injuries in R. quelen, and that these parameters have the potential to be developed as bioindicators of exposure to these agrichemicals. / A contaminação do ambiente aquático por agrotóxicos é um problema de importância mundial, devido ao seu efeito tóxico em organismos não-alvo. Estes produtos podem afetar parâmetros toxicológicos em peixes. Entre os efeitos que os agrotóxicos podem causar em peixes está a formação de espécies reativas de oxigênio (EROs) e alterações em antioxidantes enzimáticos e não-enzimáticos, o que pode evidenciar uma situação de estresse oxidativo. Várias respostas bioquímicas e fisiológicas ocorrem quando um pesticida entra no organismo, resultando na adaptação do organismo ao contaminante ou se o organismo não consegue metabolizar com eficiência estes produtos, os mesmos podem induzir toxicidade. Alevinos de Rhamdia quelen foram expostos a concentrações subletais de parationato metílico (MP), um herbicida a base de glifosato (GLI) e tebuconazole (TEB). O fígado de R. quelen exposto a MP e TEB mostrou um maior nível de substancias reativas do ácido tiobarbitúrico (TBARS), superior nos peixes no grupo controle (56% e 59%, respectivamente). Em contraste, GLI não alterou a produção de TBARS. Ocorreu aumento da carbonilação pretéica apenas nos peixes expostos a TEB. Os peixes expostos aos três agrotóxicos mostraram uma diminuição significativa da atividade da catalase (52%, 48%,e 67%, respectivamente) e aumento da atividade da glutationa-S-transferase (57%, 46% e 160%, respectivamente). Os peixes expostos a MP, GLI e TEB apresentaram um aumento de glutationa reduzida (151%, 472% e 130%, respectivamente, quando comparado com os níveis do grupo controle) e concentrações de ácido ascórbico (121%, 102% e 184%, respectivamente), enquanto que o conteúdo de tióis-não-proteicos aumentou apenas em R. quelen expostos a tebuconazole. Os peixes expostos a MP e TEB mostraram diversas alterações histopatológicas no fígado, incluindo a degeneração dos hepatócitos e estagnação biliar. O presente trabalho relata pela primeira vez a toxicidade dos pesticidas MP e do fungicida TEB em R. quelen, e sugere que a toxicidade de GLI é relativamente menor para os peixes. Os dados apresentados demonstram que concentrações sub-letais de MP, TEB podem causar alterações nos parâmetros de estresse oxidativo, bem como lesões de células hepáticas em R. quelen e que estes parâmetros têm o potencial de serem desenvolvidos como biomarcadores de exposição a esses agrotóxicos.

Tebuconazole belongs to widely utilized plant fungicides, mostly in vineyards. Its traces are present in plant tissues, wine as well as in soil. The aim of the study was to determine stoichiometries and stabilities of metal (Cu, Zn, Cd, Ca and Ag) complexes with tebuconazole (1) by means of electrospray ionization mass spectrommetry (ESI-MS). It was found that the complexes of tebuconazole with copper ions are particularly stable ([Cu(1)]+ has appearance energy 224 kJ·mol-1 , corresponding with the dissociation of the water molecule from the tebuconazole molecule), while the stability of another tebuconazole complexes is lower, about 150 kJ·mol-1 . In real environmental samples, the detection of 1 itself is hampered by its spreading into various species. The method involved here is based upon a hyphenation of electrochemistry (amperometry) and electrospray-ionization mass spectrometry (ESI-MS). It uses the fact that Cu creates very stable complexes with 1. We demonstrate that at appropriate potentials (at higher than 500 mV) Cu cations can be generated in an electrochemical cell and subsequently form complexes with 1 present in the sample. The resulting complex [Cu(1)]+ is detected with ESI-MS. The intensity of detected complexes depends on the concentration of 1 and the oxidation level of the...

This study was undertaken to determine the sensitivity of grape downy mildew (DM, Plasmopara viticola) and powdery mildew (PM, Erysiphe necator) to commonly used single-site fungicides in Virginia and nearby states. DM and PM isolates were collected from 2005 to 2007. In grape leaf disc bioassays, 92% of the DM isolates were QoI (azoxystrobin)-resistant, but none were resistant to mefenoxam. Eighty-two percent of the PM isolates were QoI-resistant, but none were resistant to boscalid and quinoxyfen. The frequency of the G143A point mutation, which confers high levels of QoI resistance, was quantified in DM and PM isolates by real-time PCR. Most of the QoI-resistant DM and PM isolates contained >95% of the 143A allele. QoI-sensitive DM isolates contained less than 1% of 143A. One out of 145 and 14 out of 154 QoI-resistant DM and PM isolates (able to grow on azoxystrobin concentration ï ³ 1 µg/ml), respectively, contained less than 1% 143A. Most PM isolates exhibited reduced sensitivity to five DMI fungicides when compared to a sensitive subgroup (n=9) and compared to published reports for unexposed populations; the resistance factor (median EC50 of the entire isolate collection divided by median EC50 of sensitive subgroup) was highest for tebuconazole (360) and myclobutanil (350), followed by triflumizole (79), triadimefon (61), and fenarimol (53). Sensitivities to all five DMI fungicides, but also azoxystrobin, were moderately to strongly correlated (pairwise r-values ranging from 0.60 to 0.88). / Master of Science in Life Sciences

Leaky buildings are those that show elevated moisture contents of the framing timber, which can subsequently lead to the establishment of fungal and bacterial decay. Prior to this study, the causative agents of the decay in these leaky buildings were unknown, though it was suspected to be one or more species of decay fungi. Therefore, the overall goal of this multi-disciplinary PhD thesis research was to determine the causative agents of decay in leaky buildings of New Zealand in an effort to develop solutions for both their remediation and future prevention. Use of molecular biology methodology and classical mycological techniques based on morphology enabled identification of decay fungi from framing timber and air samples of leaky New Zealand buildings and provided insight into relative importance based on isolation frequency. In most cases, fungi colonising Pinus radiata D. Don were isolated to produce pure cultures. Mycelia from these cultures on agar media were collected to extract DNA. To identify the fungi to the species level, polymerase chain reaction (PCR) with fungal specific DNA primer pairs were performed followed by DNA sequencing of the internal transcribed spacer (ITS) region. Identification was by BLAST (Basic Local Alignment Search Tool) search on sequences in known GenBanks. In total, 421 samples from leaky buildings were processed, predominately untreated P. radiata decayed framing timber and also fibre cement boards and building paper. From these, sixty-eight fungal identifications were made. The only taxa that were isolated with significant frequency were identified as 4 basidiomycete species, as follows, along with the number of times they were isolated from the 421 samples: • Gloeophyllum sepiarium (Wulf.: Fr.) Karst. 13x • Oligoporus placenta (Fries 1865) Gilb. In Ryv.1985 11x • Antrodia sinuosa (Fr.) Karst. 8x • Gloeophyllum trabeum (Fr.) Murr. 4x Although these species were identified repeatedly, in total they represent less than 10% of the total samples and, therefore, it is concluded that the leaky building decay samples represent high fungal biodiversity. An aerial spore study of internal air, wall cavity air and exterior air of leaky buildings was carried out using a Merck MAS-100 instrument which collects spores directly onto selective media plates. Viable fungal aerial spores were detected at every sampling location tested at the leaky buildings, by the criteria of culturing, with a highest mean of 3714 colony-forming units (CFU) per cubic metre found in the cavities of water-damaged walls. This aerial spore study in conjunction with isolation from decayed wood samples from the same leaky buildings enabled identification of G. sepiarium and A. sinuosa at the same test site. The use of carboxymethylcellulose medium further demonstrated the presence of potential cellulose-degrading fungi within and around the location. Overall, the combination of direct sampling of timber and air sampling proved useful for detection of fungal species variability at a multi-unit building. Four decay fungi isolated from New Zealand leaky buildings and two standard control decay fungi (Coniophora puteana and Serpula lacrymans) were submitted to laboratory wood block testing to determine the effectiveness of currently used wood framing preservatives under laboratory conditions before and after a standard leaching regime. P. radiata blocks were treated with water based boron copper azole and solvent based IPBC propiconazole plus tebuconazole (1:1) preservatives and exposed to the basidiomycetes for 12 weeks. Mass loss for the fungal decay-infected samples was recorded of up to 55% for preservative-treated samples, up to 62% mass loss for leached samples and up to 58% mass loss for un-preservative treated samples. Additionally, well defined dosage responses and approximate toxic thresholds were obtained for all preservatives tested. Results suggested that the minimum IPBC retention specified by Hazard Class 1.2 of NZS3640:2003 (0.025% m/m) is on the low side, and demonstrated after the 2 week leaching regime complete loss of efficacy of boron at 0.4% m/m boric acid equivalent (BAE). This PhD research gave a first overview of fungi occurring in New Zealand leaky buildings, and it demonstrated the following key aspects of wood preservation: 1. The isolated test fungus Antrodia sinuosa was more difficult to control with propiconazole plus tebuconazole at retention 0.007% m/m than the known tolerant fungus Oligoporus placenta; 2. Boron at Hazard Class 1.2 retention of 0.4% m/m BAE was not toxic to Oligoporus placenta; 3. Serpula lacrymans exhibited tolerance to the highest retention of 0.06 %m/m tebuconazole plus propiconazole; and 4. Gloeophyllum species appeared susceptible to all wood preservatives. In order to correlate fungal colonisation and wood decay, colonised wood blocks were studied using light microscopy (LM) and field- emission scanning electron microscopy (FE-SEM). Microscopic observations of P. radiata wood blocks following a standard wood decay test of twelve weeks of fungal colonisation by Serpula lacrymans, Antrodia sinuosa, Oligoporus placenta and Gloeophyllum sepiarium revealed that the two microscopic techniques employed were complementary by allowing features such as pit membranes, chlamydospores or S3/S2 compound middle lamella interface to be photographed in greater detail, allowing for more precise analyses and interpretation of key findings, as follows: 1. Brown rot fungi directly target their apical growth towards degraded pit apetures; 2. Reliance on light microscopy and observed birefringence as a tool to record changes in cell wall crystallinity associated with brown rot decay alone could be misleading; 3. Presence of fine (≤ 1 m) to wide (≥ 3.5 m) bore-hole and hyphal size ranges, and nearly unchanged cell wall thickness of all wood/test fungal combinations, confirmed active decay at moderate to late stages; 4. Some ray parenchyma cells for Antrodia sinuosa, Oligoporus placenta and Gloeophyllum sepiarium colonised blocks were intact throughout late stages of decay, outlining that they were not preferentially degraded early in the brown rot decay process, and 5. Presence of bore-holes, clamp and medallion clamp formation and resting spores (chlamydospores and arthrospores) are fungal specific, can aid in their differentiation and identification, and should be recorded during wood decay studies, as especially resting spores are an important factor when planning remediation strategies. In summary, this PhD thesis research provided the first comprehensive investigation into the biodiversity of fungi from leaky New Zealand buildings, identified the dominant species and presented details about their micromorphology and their decay patterns. It also demonstrated substantial differences in efficacy of preservative formulations currently (December 2008) approved for framing treatments in New Zealand and possible deficiencies where framing may be subjected to severe leaching. This study also provided the first comparative analyses of viable fungal aerial spores between leaky wall cavities and the surrounding air environment. Subsequently, this research added to the knowledge of the decay fungal species diversity in and around New Zealand leaky buildings, outlined their capabilities to degrade treated and un-treated P. radiata framing timber and illustrated the efficacy of New Zealand approved wood preservatives for their potential as remedial treatment and future prevention.