Rozprawy doktorskie na temat „Degradation metabolites”

Plasticizers are widely used as additives for the production of PVC and other types of plastics. They have been observed to leach out of the solid matrix over the course of the lifetime of the finished product. These compounds have now been observed across the globe and in different environments. The main focus of this work is the study of the interactions of industrial plasticizers with soil microorganisms including bacteria, yeast and fungi.<br>This research is concerned with the microbial degradation of plasticizers such as di-2-ethylhexyl phthalate (DEHP) and di-2-ethylhexyl adipate (DEHA). In particular, the study has focused on the stable metabolites produced during biodegradation, including 2-ethylhexanol and 2-ethylhexanoic acid. The first step was to show that these toxic metabolites were found in significant concentrations in the environment. In addition, a series of experiments with a variety of organisms showed how wide spread the ability to produce these metabolites was. Most organisms tested were capable of interacting with the plasticizers and many of these produced the metabolites.<br>It was apparent that these metabolites could have appreciable stability and an in-depth study with one species of bacteria, R.rhodochrous , showed that the entire initial 2-ethylhexanol component incorporated in the original plasticizers could be accounted for. Some of this was volatile and found in the exit gas of the reactor. This included all of the 2-ethylhexanol and some of the 2-ethylhexanol. These compounds may contribute to the impairment of the quality of indoor air. An overall mass balance showed that while the bacterium could eventually oxidize the 2-ethylhexanol released by hydrolysis to 2-ethylhexanoic acid, it could not degrade this acid. Thus, a summation of the quantities of each of the various metabolites generated equaled the original amount of 2-ethylhexanol in the plasticizer.<br>A mathematical model was then constructed to include all of the above features of the interaction of R.rhodochrous with the plasticizers DEHA and DEHP. This model included terms for the biological interactions and enzyme kinetics as well as the toxicity and inhibition of bacterial growth by the plasticizers and their metabolites. The increased understanding of the interaction of microbes with plasticizers will lead to a better understanding of the environmental impact of these compounds and their metabolites. The results of this study also demonstrate that when assessing the environmental impact of a compound, it is essential that not only should the impact of the parent compound be considered, but it is essential that the assessment process must also account for impacts associated with degradation products.

Earlier work with pure cultures has shown that the interaction of microbes with plasticizers leads to the formation ofmetabolites including 2-ethylhexanol and 2ethylhexanoic acid that resist further degradation. The same studies have shown that these compounds exhibit acute toxicity. This work has shown that the ability of soil micro-organisms to produce these metabolites from the degradation ofplasticizers is a general phenomenon. It was also found that the ability of soil organisms to degrade 2ethylhexanoic acid does not seem to be as common. Taken together, it would be expected that partial de gradation products of plasticizers should be observed in the environment. This was confirmed in a variety of environmental samples including sediments, surface waters, tap water, and fresh precipitation. Thus, even in a complex ecosystem, when plasticizers were degraded, the breakdown is not complete and significant amounts of2-ethylhexanoic acid and 2-ethylhexanol were observed. Since it is already weIl established that plasticizers are ubiquitous in the environment, it is expected that their recalcitrant metabolites will also be ubiquitous. This is a concem because, while the plasticizers do not exhibit acute toxicity, their metabolites do.<br>Il a été démontré, lors d'études précédentes faites avec des cultures pures, que l'intéraction de microbes avec des plastifiants mène à la formation de certains métabolites résistant à une dégradation ultérieure, incluant le 2-éthylhexanol ainsi que l'acide 2éthylhexanoïque. Ces mêmes études ont aussi démontré que ces composés ont une toxicité aigue. Le présent ouvrage a démontré que l'habileté à produire ces métabolites à partir de la dégradation de plastifiants est un phénomène généralisé chez les microorganismes provenants des sols. Il a aussi été démontré que l 'habileté de ces microorganismes à dégrader l'acide 2-éthylhexanoïque ne semble pas être aussi répandue. À partir de ces observations, il semble que les produits de la dégradation partielle des plastifiants devraient être observables dans l'environnement. Ceci a été confirmé dans un éventail d'échantillons environnementaux incluant des sédiments, des eaux de surface, des eaux potables municipales et des précipitations. Donc même dans un écosystème complexe, lorsque les plastifiants sont dégradés, la décomposition n'est pas complète et des quantités notables d'acide 2-éthylhexanoïque et de 2-éthylhexanol sont observées. Puisqu'il a déjà été établi que les plastifiants sont omniprésents dans l'environnement, il est prévu que leurs métabolites récalcitrants y seront aussi omniprésents. Ceci présente un intérêt majeur puisque ces métabolites, contrairement aux plastifiants, possèdent une toxicité aigue fr

Human, animal, and in vitro data indicate significant vasoprotective activity of anthocyanins. However, few studies have investigated the activity of anthocyanin degradation products and metabolites which are likely to mediate bioactivity in vivo. The present thesis therefore examined the vascular bioactivity in vitro of anthocyanins, their phenolic degradants, and the potential for interactions between dietary bioactive compounds. Seven treatment compounds (cyanidin-, peonidin-, petunidin- & malvidin-3-glucoside, and protocatechuic, vanillic, and syringic acid) and two treatment combinations (cyanidin-3-glucoside or protocatechuic acid with epicatechin, quercetin, and ascorbic acid) were screened in a human endothelial cell model for effects on endothelial nitric oxide synthase (eNOS) activity (via ELISA & colourimetric assay), and NADPH oxidase (NOX)-mediated superoxide production (by cytochrome c reduction assay, optimised in-house). A bioactive treatment was then chosen to explore possible mechanisms of NOX inhibition, namely gene expression of NOX2, NOX4, p47phox, p67phox, p22phox, & haem oxygenase-1 (HO-1), and activation/expression of p47phox and HO-1 protein; using RT-qPCR and immunoblotting (optimised for cell stimulation conditions and qPCR reference genes). Differential bioactivity of parent anthocyanins and their phenolic degradants was observed at physiologically relevant concentrations, as only anthocyanins upregulated eNOS expression (by 4- to 7-fold; p < 0.01), whereas both anthocyanins and degradants appeared to reduce endothelial superoxide levels (by 1- to 8-fold; p < 0.05). The phenolic degradant vanillic acid significantly reduced (p < 0.05) superoxide by 2-fold at 1μM, and has been reported at low micromolar levels in human serum; therefore vanillic acid was selected to elucidate pathways potentially underlying observed bioactivity. Vanillic acid did not significantly modulate expression of NOX isoforms/subunits, but an apparent induction of the cytoprotective enzyme HO-1 by vanillic acid (2-fold increase) was observed in human umbilical vein and coronary artery endothelial cells, although changes were non-significant (p ≥ 0.3). In conclusion, anthocyanin phenolic degradants could enhance vascular function in vivo by decreasing superoxide production, and thus scavenging of the key mediator nitric oxide (NO). Vanillic acid might inhibit endothelial superoxide production through modulation of HO-1, thereby preserving NO bioavailability and vascular homoeostasis, and this pathway should be the focus of future research.

Civil Engineering<br>Ph.D.<br>Polychlorinated biphenyls (PCBs) are toxic and persistent chemicals that have been largely dispersed into the environment. The biological and abiotic transformations of PCBs often generate hydroxylated derivatives, which have been detected in a variety of environmental samples, including animal tissues and feces, water, and sediments. Because of their toxicity and widespread dispersion in the environment, hydroxylated PCBs (OH-PCBs) are today increasingly considered as a new class of environmental contaminants. Although PCBs are known to be susceptible to microbial degradation under both aerobic and anaerobic conditions, bacterial degradation of OH-PCBs has received little attention. The overall objective of this study is therefore to evaluate the transformation of mono-hydroxylated PCBs by the well characterized aerobic PCB-degrading bacterium, Burkholderia xenovorans LB400. In order to achieve our overall objective, a series of model mono-hydroxylated PCBs have been selected and they are used to determine the toxicity of hydroxylated congeners toward the bacterium B. xenovorans LB400. The biodegradation kinetics and metabolic pathways of the selected OH-PCBs by B. xenovorans LB400 are then characterized using GC/MS. To understand further the molecular basis of the metabolism of OH-PCBs by B. xenovorans LB400, gene expression analyses are conducted using reverse-transcription real-time (quantitative) polymerase chain reaction (RT-qPCR) and microarray technology. More formally, the specific aims of the proposed research are stated as follows: (1) To evaluate the toxicity of selected mono-hydroxylated derivatives of lesser-chlorinated PCBs toward the bacterium B. xenovorans LB400. (2) To assess the degradation of the selected OH-PCBs by B. xenovorans LB400. (3) To gain further understanding of the molecular bases of the metabolism of the selected OH-PCBs by B. xenovorans LB400. Three hydroxylated derivatives of 4-chlorobiphenyl and 2,5-dichlorobiphenyl, including 2'-hydroxy-, 3'-hydroxy-, and 4'-hydroxy- congeners, were significantly transformed by Burkholderia xenovorans LB400 when the bacterium was growing on biphenyl (biphenyl pathway-inducing conditions). On the contrary, only 2'-OH-4-chlorobiphenyl and 2'-OH-2,5-dichlorobiphenyl were transformed by the bacterium growing on succinate (conditions non-inductive of the biphenyl pathway). Gene expression analyses showed that only exposure to 2'-OH-4-chlorobiphenyl and 2'-OH-2,5-dichlorobiphneyl resulted in induction of key genes of the biphenyl pathway, when cells grown on succinate. These observations suggest that 2'OH-PCBs were capable of inducing the genes of biphenyl pathway. These results provide the first evidence that bacteria are able to cometabolize PCB derivatives hydroxylated on the non-chlorinated ring. Genome-wide transcriptional analyses using microarrays showed that 134 genes were differentially expressed in cells exposed to biphenyl, 2,5-dichlorobiphenyl, and 2'-OH-2,5-dichlorobiphneyl as compared to non-exposed cells. A significant proportion of differentially expressed genes were simultaneously expressed or down regulated by exposure to the three target compounds i.e., biphenyl, 2,5-DCB, and 2'-OH-2,5-DCB, which suggests that these structurally similar compounds induce similar transcriptional response of B.xenovorans LB400. Results of this study may have important implications for the natural attenuation of PCBs and fate of OH-PCBs in the environment. The recalcitrance to biodegradation and the high toxicity of some OH-PCBs may provide a partial explanation for the persistence of PCBs in the environment.<br>Temple University--Theses

Systemic insecticides are widely used for pests control and their success is due to their ability of protect the whole plant from roots to the upper leaf. In particular, seeds coating technique is very popular and it is applied to many crops (e.g. corn). However, the use of high amount of Active Ingridient (AI) for seeds coating is causing concern about negative effects to non-target animals. Pollinators insects are exposed to contaminated pollen and nectar, but also herbivore insects are exposed through contaminated leaf. In addition, these insecticides can leach from fields and contaminate wild plants or waterbodies. Therefore, also aquatic species are exposed to insecticides pollution and vertebrates like birds and small mammals could be exposed through coated seeds, seedling and insects. The aim of this study was to develop an UHPLC-HRMS method for the identification of insecticides and their degradation production in corn guttation drops. Particular attention was posed to metabolites, because few information are available in the literature about their presence in relevant matrix for eco-toxicological studies. In addition, some metabolites may have greater toxicity if compared with their parent compounds. In particular, neonicotinoids imine metabolites are characterised by an inversion of selectivity between insects and mammals. Therefore, they can be more toxic for mammals if compared to the neonicotinoids AI. Several metabolites were identify in corn guttation and an extraction procedure based on QuEChERS strategy coupled with a target UHPLC-MS2 method was developed and validated for the quantification of these compounds in corn leaf. High concentration of neonicotinoids thiamethoxam and thiacloprid were observed in corn seedling. In addition, high concentration of the thiamethoxam metabolite clothianidn was observed. Concerning the carbamate methiocarb, the AI was observed only at low concentration, but its metabolites were present at ug/g level. Particularly interesting was the presence of methiocarb sulfoxide, because this metabolite is more toxic of the parent compounds for some species. In conclusion, guttation analysis with UHPLC-HRMS is a powerful technique in order to assess the presence of insecticides metabolites in plants treated with systemic AI. However, UHPLC-MS2 still provide better performance for quantitative analysis, in particular for complex matrices as corn leaf. Therefore, HRMS and MS2 are complementary technique useful to provide levels of contamination and exposure.<br>Systemic insecticides are widely used for pests control and their success is due to their ability of protect the whole plant from roots to the upper leaf. In particular, seeds coating technique is very popular and it is applied to many crops (e.g. corn). However, the use of high amount of Active Ingridient (AI) for seeds coating is causing concern about negative effects to non-target animals. Pollinators insects are exposed to contaminated pollen and nectar, but also herbivore insects are exposed through contaminated leaf. In addition, these insecticides can leach from fields and contaminate wild plants or waterbodies. Therefore, also aquatic species are exposed to insecticides pollution and vertebrates like birds and small mammals could be exposed through coated seeds, seedling and insects. The aim of this study was to develop an UHPLC-HRMS method for the identification of insecticides and their degradation production in corn guttation drops. Particular attention was posed to metabolites, because few information are available in the literature about their presence in relevant matrix for eco-toxicological studies. In addition, some metabolites may have greater toxicity if compared with their parent compounds. In particular, neonicotinoids imine metabolites are characterised by an inversion of selectivity between insects and mammals. Therefore, they can be more toxic for mammals if compared to the neonicotinoids AI. Several metabolites were identify in corn guttation and an extraction procedure based on QuEChERS strategy coupled with a target UHPLC-MS2 method was developed and validated for the quantification of these compounds in corn leaf. High concentration of neonicotinoids thiamethoxam and thiacloprid were observed in corn seedling. In addition, high concentration of the thiamethoxam metabolite clothianidn was observed. Concerning the carbamate methiocarb, the AI was observed only at low concentration, but its metabolites were present at ug/g level. Particularly interesting was the presence of methiocarb sulfoxide, because this metabolite is more toxic of the parent compounds for some species. In conclusion, guttation analysis with UHPLC-HRMS is a powerful technique in order to assess the presence of insecticides metabolites in plants treated with systemic AI. However, UHPLC-MS2 still provide better performance for quantitative analysis, in particular for complex matrices as corn leaf. Therefore, HRMS and MS2 are complementary technique useful to provide levels of contamination and exposure.

Conselho Nacional de Desenvolvimento Científico e Tecnológico<br>Macrolide antibiotics are an important group of prescription drugs; as a consequence of the large and continuous use, they are commonly found in the environment. In the present study, it was developed and optimized a chromatographic method to assess the occurrence of macrolide antibiotics Azithromycin, Clarithromycin, Erythromycin and Roxithromycin in the effluent of the University Hospital of Santa Maria, in two sampling points, by applying high performance liquid chromatography coupled to mass detection with quadrupole ion trap (HPLC-MS/MS_QTrap) and clean-up/pre-concentration by solid phase extraction with the aid of Surface Methodology Response. The concentrations measured during a week in the hospital effluent were 1.32±0.13 and 0.22±0.06 μg L-1 for Azithromycin and Clarithromycin; in the receptor water sream was 1.12±0.20, 0.20±0.05 and 0.01±0.004 μg L-1 for Azithromycin, Clarithromycin and Erythromycin. Roxithromycin was not detected in all effluent samples. After this, it was done the evaluation of the risk quotient of the macrolide antibiotics. The value of the risk quotient for the hospital effluent for Azithromycin and Clarithromycin was 11 (high risk), and for the receptor water stream the risk quotient was 9.3 and 10.0 for Azithromycin and Clarithromycin; for Erythromycin, a quocient risk value of 0.5 (medium risk). For degradation of the antibiotics in aqueous solution, it was used UV-photolysis, by which the influence of pH (3-11) was evaluated. Azithromycin showed low degradation by acid pH; for other pH, as well, for all the other antibiotics, the degradation was above 70% after 60 min of treatment. It was conducted a kinetic study of the degradation process of macrolide antibiotics in different pHs, by which Azithromycin revealed a recalcitrant profile, and Roxithromycin, as the more easily degradable one. For identification of the products formed during the photolysis experiments it was used independent information acquisition and as precursor ions of fragments m/z 116 and 158, characteristic of the macrolide compounds, at three collision energies (30, 45, and 60 V). It was proposed fragmentation routes of the degradation products: 8 products for Azithromycin, 7 for Clarithromycin, 6 of Erythromycin and 8 Roxithromycin. Through the same experiments with independent information acquisition, it was investigated the presence of eventual metabolites in hospital effluent, and three metabolites were found. By applying photolysis to the hospital effluent fortified, at pH 7, it was observed that the degradation occurs above 80% for all compounds after 60 min of irradiation. It was observed the formation of degradation products previously determined by experiments in aqueous solution. It was also found three degradation products for Azithromycin, 2 for Clarithromycin, 1 for Erythromycin and 3 for Roxithromycin.<br>Os antibióticos macrolídeos são uma importante classe de fármacos preescritos no tratamento das mais variadas infecções, e como consequência se seu grande e continuo uso são comumente encontradas no ambiente. No presente estudo foi desenvolvido e otimizado método de cromatografia líquida de alta eficiência acoplada à detector de massas quadrupolo íon trap (HPLC-MS/MS_QTrap) e de clean-up/pré-concentração por extração em fase sólida com auxílio de Metodologia de Superfície de Resposta para avaliar a ocorrência dos antibióticos macrolídeos Azitromicina, Claritromicina, Eritromicina e Roxitromicina no efluente hospitalar do Hospital Universitário de Santa Maria em dois ponto de amostragem. As concentrações médias durante o ciclo de uma semana de amostragem no efluente do pronto atendimento foram de 1,32±0,13 e 0,22±0,06 g L-1 para Azitromicina e Claritromicina; no corpo recpetor foram de 1,12±0,20; 0,20±0,05 e 0,01±0,004 g L-1 para Azitromicina, Claritromicina e Eritromicina, respectivamente. Roxitromicina não foi detectada. Após foi feita a avaliação do quociente de risco dos antibióticos macrolídeos. O quociente de risco no efluente do pronto atendimento para Azitromicina e Claritromicina foi de 11, risco alto, o qual também foi evidenciado no corpo receptor com quociente de risco de 9,3 e 10 para Azitromicina e Claritromicna, e risco médio para Eritromicina de 0,5. Para degradação dos antibióticos foi utilizado fotólise artificial em solução aquosa, sendo avaliado a influência do pH de 3-11 na degradação destes compostos. Azitromicina apresentou baixa degradação em pH ácido, para os outros pH e demais compostos a degradação foi acima de 70% após 60 min de tratamento. Foi feito um estudo cinético do processo de degradação dos antibióticos macrolídeos em diferentes pH, observando-se que a Azitromicina apresentou um perfil recalcitrante para o processo, e Roxitromicina foi degradada com maior facilidade. Para a identificação dos produtos formados durante os experimentos de fotodegradação foram montados experimentos de informação independente de aquisição utilizando como íons precursores os íons de m/z 116 e 158 característicos dos compostos macrolídeos em três energias de colisão (30, 45 e 60 V). Foram identificadas e propostas rotas de fragmentação para 8 produtos de degradação de Azitromicina, 7 para Claritromicina, 6 para Eritromicina e 8 produtos de degradação de Roxiromicina. Através dos mesmos experimentos de informação independente de aquisição, foi investigada a presença de possíveis metabólitos no efluente hospitalar sendo encontrados 3 metabólitos. Com a aplicação de fotólise ao efluente hospitalar fortificado, em pH 7, observou-se que ocorre degradação acima de 80% para todos os compostos após 60 min de tratamneto. Foi observada a formação de produtos de degradação, que tinham sido previamente determinados em solução aquosa. Foram encontrados 3 produtos de degradação de Azitromicina, 2 para Claritromicina, 1 para Eritromicina e 3 produtos para Roxitromicina.

Soil samples which had been previously exposed to chlorinated aromatics were screened for ability to degrade dichlorobenzophenone (DBP). Of the samples tested soil from the Dow Elanco Agricultural Farm and soil from the AgResearch Winchmore Research Station showed apparent degradative capabilities. Degradation was not sustainable in these soil samples and a time scale study showed DBP was stable in soil over a 24 week period. Samples from Winchmore were further used to establish enrichment cultures capable of degrading DBP and its non-chlorinated analogue benzophenone (BP) through selection pressure. BP proved to be readily degraded but DBP degradation was only achieved after fungal suppressants were used. Degradation of DBP was enhanced with the addition of yeast extract and sodium salicylate to the enrichment cultures. Degradation of DBP was confirmed by capillary gas chromatography and the detection of the metabolite p-chlorophenyl acetic acid by gas chromatography-mass spectroscopy and thin layer chromatography. The enrichment cultures established on DBP also extensively degraded BP, p-chlorobenzophenone and p-chlorobenzoic acid. When inoculated back into soil, the enrichment cultures degradative capabilities were significantly reduced due to competition from other organisms, availability of alternative carbon sources and the bioavailability of DBP due to binding to soil particles. Two organisms capable of degrading BP as sole carbon source were isolated and identified as a Rhodococcus spp. and a Streptomyces spp. BP degradative capabilities were not maintained by these organisms and lost when subculturing on nutrient media Three presumptive DBP degraders were isolated and presumptively identified as two separate Streptomyces spp. and a Pseudomonas vesicularis. Of the three none were able to degrade DBP as sole carbon source in liquid culture although the P. vesicularis was able to co-metabolise DBP with the addition of yeast extract and sodium salicylate. Preliminary genetical studies of the P. vesicularis isolated were carried out.

L'objectif principal de la thèse est de développer une technologie efficace pour la dégradation des pesticides. L'herbicide atrazine a été étudié comme molécule modèle. L'atrazine a été dégradée dans l'eau par décharge électrique de haute tension (DEHT), et ses performances de dégradation ont été comparées aux technologies traditionnelles d'oxydation de Fenton et d'ultrasons (US). La détection et la quantification de l'atrazine et de ses métabolites ont été réalisées par chromatographie liquide haute performance et spectrométrie de masse à haute résolution (HPLC-HRMS). Une méthode d'analyse en ligne par HPLC-HRMS combinée à un échantillonnage automatique a été développée pour un suivi en temps réel du processus de dégradation. La technologie DEHT a dégradé efficacement l'atrazine et a réduit les métabolites toxiques générés au cours des processus d’oxydation de Fenton et d’US. Le procédé de DEHT est moins consommateur d'énergie que le procédé d’US tout en atteignant la même efficacité de dégradation de l'atrazine de 89%. Les mécanismes de dégradation de l'atrazine pour les différentes technologies ont été proposés. L'effet d'une matrice réelle (eau du robinet) par rapport à une matrice modèle (eau déminéralisée) sur la dégradation de l'atrazine a été étudié. Les résultats ont montré que dans le cas d’un traitement par DEHT, l'efficacité de dégradation de l'atrazine dans l'eau du robinet était inférieure à celle de l'eau déminéralisée, ce qui peut être lié à la conductivité de l'eau et au mécanisme de génération des arcs électriques dans un milieu conducteur. La toxicité aiguë (CL50) chez la daphnie Daphnia magna a été utilisée pour évaluer la toxicité des différentes solutions de traitement contenant initialement de l'atrazine. La toxicité de la solution d'atrazine traitée par oxydation de Fenton est supérieure à celle traitée par DEHT et US<br>The main goal of this thesis is to develop an efficient technology for the degradation of pesticides. For this purpose, the widely used herbicide atrazine was studied as a model molecule. Atrazine was degraded in water by high voltage electrical discharge (HVED), and its degradation performance was compared with traditional water treatment technologies Fenton oxidation and ultrasound (US). The detection and quantification of atrazine and its metabolites were achieved by high performance liquid chromatography-high resolution mass spectrometry (HPLC-HRMS). An online analysis method by HPLC-HRMS combined with automatic sampling was developed for real-time monitoring of the degradation process. The HVED technology efficiently degraded atrazine and reduced toxic metabolites generated during Fenton oxidation and US processes. HVED process has less energy consumption than US process while achieving the same 89% atrazine degradation efficiency. The mechanism pathways of atrazine degradation for different technologies were proposed. The effect of real matrix (tap water) versus model matrix (deionized water) on atrazine degradation was studied. Results showed that in HVED treatment, the degradation efficiency of atrazine in tap water was lower than that in deionized water, which may be related to the conductivity of the water and to the mechanism of electric arcs generation in a conductive medium. The acute toxicity (LC50) in Daphnia magna was used to evaluate the toxicity of different treatment solutions initially containing atrazine. The toxicity of atrazine solution treated by Fenton oxidation is higher than that treated by HVED and US

Mycocentrospora acerina est le principal agent pathogene de la carotte (daucus carota l. ) conservee au froid humide; ce travail decrit le processus infectieux d'isolats plus ou moins agressifs sur des cultivars de sensibilite differentes. L'objectif de cette etude, focalisee sur les composantes de l'agressivite du parasite et des defenses constitutives et induites de la plante, est de mieux comprendre la nature des echanges moleculaires s'etablissant lors de l'infection, afin de proposer des axes de recherches pour lutter contre ce nouveau probleme parasitaire. L'etude sur l'agressivite du champignon met en evidence l'importance quantitative des enzymes de degradation des parois cellulaires (edpc) dans l'intensite des necroses sur racines. Les modifications structurales observees par microscopie electronique des tissus racinaires inocules par m. Acerina s'accompagnent d'une apparition sequentielle d'activites edpc soulignant leur role dans la pathogenese. L'implication dans les defenses constitutives de l'hote, de la composition et de la structure des parois cellulaires, et plus particulierement des pectines de la lamelle moyenne est discutee. L'activite elicitrice des oligosaccharides d'origine parietale liberes sous l'action des edpc de m. Acerina montre que seuls les oligomeres presentant des degres de polymerisation >6 declenchent l'accumulation de phytoalexine. Enfin, dans la perspective d'une phytoprotection, l'effet d'une application d'oligosaccharides actifs sur l'acquisition d'une resistance induite a m. Acerina est testee

Pour examiner l'influence possible des conditions de croissance sur la traduction des arnm de e. Coli et l'existence eventuelle d'un couplage entre traduction et degradation et entre traduction et synthese des arnm, nous avons construit une collection de genes chimeriques lamb-lacz et gale-lacz ne differant entre eux que par des mutations ponctuelles affectant l'efficacite de traduction. Nos resultats indiquent que selon l'efficacite de traduction des arnm hybrides lacz, il se produit soit un couplage entre traduction et synthese, soit un couplage entre traduction et degradation dans lequel l'endonuclease rnase e est impliquee. Nous proposons un modele mecanistique pour le role de la rnase e et nous nous interrogeons sur la generalite et le role physiologique d'un couplage traduction-degradation-synthese des arnm. Nous montrons egalement que les efficacites relatives de traduction des arnm hybrides lacz peuvent etre largement modulees par les conditions de croissance. Selon leur reponse a un changement des conditions de croissance, nous classons les arnm en deux categories. Le changement des conditions de croissance modifie de nombreux parametres cellulaires. Nous testons donc l'effet des variations de differents parametres sur les efficacites de traduction des arnm hybrides lacz et discutons de l'eventuelle generalite de ce phenomene

The fate and behaviour of estrogens in the environment are of concern due to the compounds’ endocrine disruption potential. Estrogens, namely 17β-estradiol (E2), estrone (E1), and estrogen sulphates, i.e. 17β-estradiol-3-sulphate (E2-3S) and estrone-3-sulphate (E1-3S) excreted by livestock constitute a potential source for estrogen contamination in the environment. A method was developed to separate and quantify the hormones by high-performance-liquid-chromatography (HPLC) and ultraviolet detection (UV). A combination of dichloromethane (DCM) and dicyclohexylamine hydrochloride (DCH·HCl) gave recoveries from 97.3 to 107% for E1-3S extraction from aqueous solutions. The recoveries from soil samples ranged from 80.9 to 95.2% (E2-3S), and from 86.3 to 91.7% (E1-3S), respectively. Results of batch sorption studies showed that Freundlich isotherms were nonlinear (N ≠ 1) with Kf values ranging from 34.2 to 57.2, and from 3.42 to 4.18 mg¹-N LN kg⁻¹ for E1, and E1-3S, respectively, indicating the sorption affinity of E1-3S was about an order of magnitude lower than that of E1. The hydrophilic sulphate group of E1-3S possibly shielded the compound from hydrophobic interactions with the soil organic matter and allophanic clay minerals that were proposed as sorbents for E1. Contraction of clay minerals, “salting out” and competitive sorption of artificial urine constituents were likely to have been responsible for observed changes in Freundlich parameters when artificial urine was used as mediator matrix. Plotting the effective distribution coefficient as a function of hypothetical exposure concentrations facilitated the comparison of the sorption behaviour of both compounds as influenced by the mediator solution. The results emphasized that using the CaCl₂ matrix might result in false inferences for the sorption behaviour of these compounds in a dairying environment. The four hormones rapidly degraded in the agricultural soils under aerobic conditions, and the majority of the compounds degraded > 50% within the first 24 hrs. Soil arylsulphatase activities were directly correlated with degradation rate constants of the estrogen sulphates. Estrone was identified as a metabolite of E2 and E1-3S, and these three compounds were observed as metabolites of E2-3S. Single-first order (SFO) and double first-order in parallel (DFOP) kinetics were used to model the degradation and metabolite formation data. The results showed that the DFOP model was in most cases better able to predict the parent compound degradation than the SFO model, and also enabled to estimate accurate degradation endpoints. ER-CALUX® analysis revealed the formation of estrogenicity during E2-3S degradation, which could partly be explained by the formation of the metabolites E2 and E1. Transport studies with E1-3S and E1 showed that the transport and retention of both compounds were significantly influenced by the mediator matrix. While no breakthrough curves (BTCs) were recorded during hormone application in CaCl₂ (10 mM) both hormones were detected in the leachate when applied in artificial urine. Rate-limited sorption processes were proposed for the delayed arrival of the hormone BTCs compared with a conservative bromide tracer. Intense colouration of the leachate during the artificial urine experiments suggested the hormones were likely to be moved by colloid-facilitated transport. Furthermore, the detection of residue hormone and metabolite concentrations implied that degradation of E1-3S and E1 was hampered by urine constituents such as glycine and urea.

Les agents tensio-actifs de synthese constituent, a cause de leur large diffusion, une forme de pollution preoccupante atteignant tout le milieu naturel. Ce memoire vise a completer les donnees concernant le devenir, dans differents compartiments du milieu aquatique (eau, sediment), d'une variete de produits tensio-actifs tres utilises actuellement: les alkylbenzenesulfonates lineaires (las). Dans la premiere partie de cette etude sont presentees les connaissances existantes a propos des agents-tensio actifs et plus particulierement les las. Par la suite sont detaillees les diverses etapes de l'elaboration d'une methode analytique basee sur la chromatographie liquide haute performance (hplc), permettant de detecter simultanement les las et les metabolites qui sont susceptibles de se former au cours de leur biodegradation. Des techniques d'extraction ont ete mises, au point dans le but de concentrer et de purifier les echantillons environnementaux en vue de l'analyse chromatographique. En utilisant la methode de l'experimentation in-vitro, nous avons pu mettre en evidence la decomposition, en presence d'oxygene, d'un las et l'apparition de residus de degradation dans des echantillons d'eau et de sediment provenant d'un etang du milieu mediterraneen (l'etang de bolmon). Les metabolites de degradation observes ont ete identifies et ne sont pas persistants. Apres une investigation concernant le degre de pollution du bolmon par les detergents nous avons place un dispositif dans l'etang contenant un sediment artificiellement contamine par du las pour savoir si la biodegradation peut se produire dans le milieu naturel au sein meme du sediment. Les resultats obtenus montrent l'importance preponderante des phenomenes de ramaniement (hydrodynamisme, bioturbation) sur la regeneration du sediment

Le 5-fluorouracile (fu) est un des medicaments anticancereux les plus utilises dans le monde. Comme tous les cytostatiques, il presente des effets secondaires toxiques, en particulier hematologiques et digestifs, mais aussi neurologiques et cardiologiques. La cardiotoxicite observee chez certains patients traites au fu est en partie due a la presence d'impuretes fluorees presentes dans les solutions commerciales de fu. Ces impuretes sont en fait des produits de degradation du fu qui se forment avec le temps dans le milieu basique indispensable a la solubilisation de ce principe actif. Grace au modele de cur isole perfuse de lapin (cipl) et a la resonance magnetique nucleaire du fluor-19 (rmn #1#9f), nous avons etudie la cardiotoxicite d'une nouvelle forme galenique de fu qui est un lyophilisat a reconstituer immediatement avant l'injection. Une fois reconstitues, les lyophilisats de fu ne contenaient pas d'impuretes cardiotoxiques et n'ont provoque aucune manifestation cardiotoxique, contrairement aux solutions commerciales de fu qui ont induit une toxicite cardiaque severe. Toutefois, la frequence des accidents cardiaques chez les patients, trop importante d'apres nous pour etre expliquee uniquement par les faibles taux d'impuretes cardiotoxiques presentes dans les solutions commerciales de fu, nous a amene a montrer experimentalement grace au modele de foie isole perfuse de rat que le fu lui-meme etait metabolise en fluoroacetate et en acide 2-fluoro-3-hydroxypropionique, tous deux fortement cardiotoxiques sur le modele de cipl. La conclusion de notre travail est double. Pour limiter la cardiotoxicite du fu, il serait preferable d'utiliser en clinique une nouvelle forme galenique de fu depourvue de produits de degradation (lyophilisat par exemple). Cependant, pour supprimer cette toxicite, il faudrait bloquer la voie de degradation du fu en amont de son principal catabolite, l'-fluoro--alanine

碩士<br>弘光科技大學<br>環境工程研究所<br>95<br>Abstract Ozone, due its high oxidation and disinfection potential, has recently received much attention in water treatment technology. Despite several advantages of using ozone, it has a few disadvantages, which limit its application in water treatment technology. The main are relatively low solubility and stability in water. Because of both the high cost of ozone production and only partial oxidation of organic compounds present in water, the application of ozonation might not be feasible from an economic point of view. The objective of this study was to evaluate the heterogeneous catalytic ozonation process in mitigating the taste and odor causing compounds - 2-MIB. The surface water samples were collected from eutrophic reservoirs. Furthermore, the synthetic waters that matrix take from ground water and adding appropriate concentration of 2-MIB was also test in this study. The reaction types including batch, semi-batch and continuous flow reaction, respectively. The titanium dioxide (TiO2) was applied for catalyst in this work. Experimentally, it was found that combined use of O3 and TiO2 catalyst leads to a conspicuous 99% of 2-MIB degradation which compares favorably to the 20~30% obtained more than in the absence of the TiO2 catalyst. The electron paramagnetic resonance (EPR) was used to detect ozone-produced paramagnetic radicals. The experimental EPR spectra verified that more hydroxyl radicals (OH．) generated in the TiO2 catalytic ozonation process. In this system, elevated concentrations of OH． radical produced by the reaction of ozone with catalyst at the solid-liquid interface, is the main cause responsible for the improvement of ozonation induced by the presence of catalyst. At the constant dosage of TiO2 in the semi-batch tests, 2-MIB conversion rate can be approach to 85%, especially in the high pH conditions. In the continuous flow reactor which packing Raschig ring matrix (coating TiO2 films on the surface using Arc ion plating method), 2-MIB degradation rates was obvious high than ozone only reaction system. It was also found that the chlorophyll-a content in the O3/TiO2 effluent was lower than ozone only system, indicated that the high reactivity of OH． radicals were generated by O3/TiO2 during the oxidation process that effectively degraded algal cells and 2-MIB.

Algal infestation in water bodies causes the release of soluble organic compounds that impact negatively on the taste and odour of the water. With increasing pollution in water bodies and increasing nutrient loading from agricultural activities, most water reservoirs in South Africa and around the world have become affected by this problem. In this study, an advanced oxidation process (AOP), namely, photocatalysis was evaluated for its potential to degrade aromatic compounds; and taste and odour causing bi-cyclic compounds originating from algae. Semiconductor photocatalysis is an environmentally friendly technology requiring no chemical inputs which is capable of completely mineralising organic pollutants to CO2 and H2O thereby eliminating production of unwanted by-products. Although processes involved in the photo-degradation have been reported for a wide range of pollutants, the degradative pathway in this process has not been fully established. In this study, compounds including phenol, 2-chlorophenol, 4-chlorophenol and nitrophenol were successfully eliminated from simulated wastewater. Degradation of geosmin at an environmentally significant initial concentration of 220 ng/L to levels below the lowest detectable concentration was achieved with an optimum catalyst concentration of 60 mg/L at a rate of 14.78 ng/L/min. Higher catalysts loading above 60 mg/L resulted in a decrease in degradation rates. An increase in initial geosmin concentration resulted in a decrease in rates. Ionic species commonly found in surface waters (HCO3 -, and SO4 2-) significantly reduced the efficiency of geosmin degradation. Degradation of geosmin produced acyclic intermediates from ring fission tentatively identified as 3,5-dimethylhex-1-ene, 2,4-dimethylpentan-3-one, 2-methylethylpropanoate and 2-heptanal. The results obtained indicate that the degradation of organic pollutants in aqueous solution is as a result of synergic action from hydroxyl radicals, positive holes and direct photolysis by UV radiation, though the predominant pathway of degradation is via hydroxyl radicals in solution. Major aromatic intermediates of phenol degradation include catechol, resorcinol and hydroquinone produced in the order catechol > resorcinol > hydroquinone. All three are produced within 2 minutes of photocatalytic reaction of phenol and remain in solution until all phenol is degraded in aerated systems. Production of resorcinol in non-aerated systems is transient, further supporting the hydroxyl radical dominant reaction pathway.<br>Thesis (PhD)--University of Pretoria, 2014.<br>gm2014<br>Chemical Engineering<br>unrestricted

碩士<br>國立成功大學<br>環境工程學系碩博士班<br>100<br>The occurrence of cyanobacteria or blue-green algae in drinking water systems is potentially harmful for human health. Among the common problems associated with it include the production of toxins and undesirable taste and odor (T＆O). One of the most common toxin-producer among cyanobacteria is Microcystisaeruginosa. This species is capable of producing microcystins, a group of cyanotoxins which exhibit chronic effects on humans and wildlife by damaging the liver. Microcystis blooms can also produce β-cyclocitral, a malodor that gives a hay-tobacco odor.Oxidants, such as ozone, are mostly used for controlling cyanobacteria in drinking water treatment processes. The oxidation of the contaminants can occur via ozone’s decomposition into ozone (molecular ozone) or OH radicals (•OH). In this ozonationstudy, the degradation kinetics of the cell integrity and metabolites were evaluated. The Rctconcept,involving the use of a probe compound (para-chlorobenzoic acid), was applied to indirectly measure •OH. The cyanobacteria-laden water exhibited two phases of Rcts for the batch reactions, as expected from previous studies of natural waters. Rctis highly dependent on the initial ozone and cell concentrations. Semi-batch reactions were done to obtain rate constants for•OH and cell integrity and metabolites. A value of 6.35×109M-1s-1and 4.99×109M-1s-1were determined for k_(cell integrity/•OH )and k_(β-cyclocitral/•OH ),respectively. A model describing cell rupture and microcystin release and degradation was established for the ozonation of M. aeruginosa. The extracted parametersfor radical concentration estimation and cell rupture kinetics, and the literature value of microcystin degradation rate constant were used for the model predictions. The model was able to predict the kinetics of cell rupture and change of microcystin concentrations in the system, suggesting the model is reasonable.

A multi-faceted approach was used to investigate and overcome the PCB-degrading limitations of the Bph enzymes, responsible for the catabolism of biphenyl. First, the reactivities of four evolutionarily divergent extradiol dioxygenases towards mono-, di- and trichlorinated 2,3-dihydroxybiphenyls (DHBs) were investigated: 2,3-dihydroxybiphenyl dioxygenase (EC 1.13.11.39) from Burkholderia xenovorans sp. LB400 (DHBD[sub LB400]), DHBD[sub P6]-I and DHBD[sub P6]-III from Rhodococcus globerulus P6 and 2,2',3-trihydroxybiphenyl dioxygenase from Sphingomonas sp. RW1 (THBD[sub RW1]). The specificity of each enzyme for particular DHBs differed by up to 3 orders of magnitude. Moreover, each enzyme cleaved at least one of the tested chlorinated DHBs better than DHB. However, no enzyme was able to cleave 2',6'-diCl DHB or 3,4-DHB, two recalcitrant PCB metabolites. In the second facet of the study, biological selections were designed to facilitate the engineering of PCB-degrading enzymes via directed evolution. Although these selections failed to link the desired activities to host cell viability, they showed promise for other biocatalysts. In addition, a broadly useful high-throughput colorimetric screen was developed. The latter was applied to increase the specificity of DoxG, an extradiol dioxygenase from Pseudomonas sp. CI8, for 3,4-DHB. A single round of directed evolution yielded DOXG[sub SMA2], a variant that cleaved 3,4-DHB ∼1000-fold more specifically than the wild-type enzyme. DOXG[sub SMA2] contained three substituted residues: L190M, S191W and L242S. The crystal structure of the DoxG:3,4-DHB binary complex indicates that residues at position 190 and 242 occur on opposite sides of the DHB-binding pocket and may interact directly with the distal ring of the substrate. Kinetic analyses revealed that the substitutions are anti-cooperative. Finally, characterization of BphK, the glutathione S-transferase from the bph pathway of B. xenovorans sp. LB400, revealed that the enzyme catalyzes the glutathione-dependant dehalogenation of certain inhibitory CI HOPDAs. BphK catalyzed the dechlorination of 3-Cl HOPDA with a specificity constant of ~10⁴ M⁻¹s⁻¹ in a reaction that utilized a ternary complex mechanism and 2 equivalents of GSH. The identified product of the reaction, HOPDA, is the substrate of the hydrolase from the bph pathway. The relatively low specificity constant of BphK for 3-Cl HOPDAs corroborate genetic evidence that the enzyme was recently recruited to the bph pathway to facilitate PCB degradation.<br>Science, Faculty of<br>Microbiology and Immunology, Department of<br>Graduate

碩士<br>國立臺灣海洋大學<br>水產養殖學系<br>103<br>The objective of this study was to investigate the effect of different concentrations of chlorine dioxide (ClO2) on degradation of four nitrofuran metabolites (AOZ, AMOZ, SC and AH) in freshwater and seawater environments of water and sediments; also to understand the situation of nitrofuran metabolites residues in water and sediments. Experiment consisted of four parts, first was to establish the method for detecting the four nitrofuran metabolites in water and sediments, and the results showed that specificity, recovery, repeatability and limits of quantification were in line with the validation of Method of Test for chemistry in foods of Food and Drug Administration. Limits of quantification of four nitrofuran metabolites in freshwater, seawater and sediments were reached 0.5 ng/mL, and the recovery rate were between 90.10~109.97 %. Second was the different concentrations (1, 5 and 10 mg/L) of chlorine dioxide on 100 ng/mL nitrofuran metabolites degradation in freshwater and seawater. Results showed that 1 mg/L chlorine dioxide was effective immediately in 1 day after treatment for nitrofuran metabolites in freshwater and seawater, and treatment with 10 mg/L chlorine dioxide showed the most significant result both in freshwater and seawater which the concentrations of AOZ, AMOZ, SC and AH were below the limits of quantification after 10 mg/L chlorine dioxide treatment 2 day. Third was the different concentrations (10, 20 and 40 mg/L) of chlorine dioxide on 100 ng/mL nitrofuran metabolites degradation in sediments with freshwater and seawater. Results showed that 40 mg/L chlorine dioxide treatment effect was the most significant and effective immediately in 1 day after treatment for nitrofuran metabolites in sediments with freshwater and seawater. Fourth was to investigate the degradation of 100 ng/mL nitrofuran metabolites in water and sediments during 90 days. In freshwater and seawater, results indicated that AOZ was 42.07 ± 0.80 and 35.10 ± 0.35 ng/mL, AMOZ was 31.23 ± 0.08 and 21.08 ± 0.25 ng/mL, SC was 11.82 ± 0.53 ng/mL and below the quantification limit, and AH was 51.17 ± 0.29 and 7.03 ± 0.53 ng/ mL on ninetieth day; but SC was still 1.09 ± 0.19 ng/mL in seawater on thirtieth day. In sediments, outcome was that four nitrofuran metabolites were degraded immediately in 1 day. The degradation rate of nitrofuran metabolites became slow with time. In sediments with freshwater and seawater on ninetieth day, AOZ was 4.98 ± 0.12 and 5.49 ± 0.73 ng/mL, AMOZ was 31.23 ± 0.08 and 21.08 ± 0.25 ng/mL, SC was 1.52 ± 0.01 and 1.58 ± 0.09 ng/mL, and AH was 1.11 ± 0.03 and 1.31 ± 0.04 ng/mL. This experiment pointed that chlorine dioxide (ClO2) can degrade four nitrofuran metabolites (AOZ, AMOZ, SC and AH) in freshwater and seawater environments, and higher concentration was needed in sediments than in water. Four nitrofuran metabolites would remain in water and sediments for a long period, and the remaining time in seawater is longer than in freshwater.

碩士<br>國立高雄師範大學<br>生物科技系<br>106<br>2, 4, 6-Trinitrotoluene, the yellow-colored solid, is a useful explosive dynamite best known as TNT and massive utilized for military and industry. After widely used, it caused environmental pollution both for soil and groundwater contaminated. The influence of repopulation is difficult and expensive to remedy. In addition, the literature indicates that human exposure to TNT waste will stand a high probability of getting anemia and liver dysfunction. TNT and related metabolites also listed as carcinogens, for a reason that TNT contamination of remediation is an important environment issue currently. There are physical, chemical and biological technical treatments applied to TNT pollution remediation which biological treatment of environmental pollution remediation more moderate nowadays. In our previously study, we used the microbial remediation technique and selected TNT-degradable bacteria strains exhibited a significantly higher TNT degradation capability by treated stimulate amino-peptide. However, the acute toxicity of effluents and receiving waters test showed the toxicity of TNT metabolites is higher than TNT. Due to the department of phenolic compounds after degradation, the structure of metabolites with hydroxyl (-OH) on its benzene ring from TNT aerobic metabolism pathway. Laccase, are copper-containing oxidase enzymes that are found in many plants, fungi, and microorganisms. The enzyme orthologous genes in Escherichia coli called CueO and it has the potential to be oxidized hydroxyl groups on the benzene ring. Its strong oxidation widely used in dye contamination treatment. Because of the fact that we use biological model Escherichia coli K12 strains MG1655 as wild type and aim the CueO gene to treat as knockout and overexpression strains in this study. Accordingly we cultured these experiment strains in same nutrition with TNT and analysis results both degradation and metabolites toxicity. Preliminary results show that when E. coli reaching a level of TNT degradation, its metabolites toxicity higher than initial dose TNT. But comparison of wild type and knockout strains, the later has higher toxicity. Base on the results, we concluded that E.coli has TNT degradation capability and CueO indeed play a role in removing toxic metabolites of TNT. Our research continue to discuss and confirm the function of laccase (CueO) in TNT metabolism and how is it participate in degradation to decrease toxicity of TNT metabolites. keywords: 2,4,6-Trinitrotoluene, TNT、TNT degradation、bioremediation、laccase、E.coli CueO

碩士<br>國立臺灣大學<br>農業化學研究所<br>98<br>Dicarboximide fungicide vinclozolin was found to have the anti-androgen effects, and its metabolites, M1 (2-[[(3,5-dichlorophenyl)-carbamoyl]oxy]-2-methyl-3-butenoic acid) and M2 (3’,5’-dichloro-2-hydroxy-2-methylbut-3-enanilide), were found to have more affinity to androgen receptors than vinclozoline. In this study, the effect of vinclozolin metabolites in soil on its persistence and soil microbial populations diversity were investigated. The toxicity of vinclozolin on Daphnia pulex, were compared with 2 kinds of its metabolites. First, we extract vinclozolin from commmerical products and then perform chemical hydrolysis to collect the metabolites. The soil sample were collected from Taoyuan District Agricultural Research (Pu) and Extension Center and Hualien District Agricultural Research (Wl). The experimental concentration were 20.0 mg kg-1 for M1 and 5.0 mg kg-1 for M2. We used solvent phase extraction to extract residues from soil, and utilized HPLC-DAD to detect them. In sterilized soil, M1 and M2 have longer half-lives (10.9-126.6 and 87.1-296.5 day) than vinclozolin (8.7-35.0 day). The impact of vinclozolin metabolites on soil microbial community were analysed by PCR-DGGE. In the DGGE patterns, we could discover some original superior populations with the resisence to toxicity of metabolites, such as Acidobacterium sp. and Pseudomonas sp. in Pu soil, and Ammoniphilus oxalaticus in Wl soil had the resistence to M1 or M2. But some populations were disappeared or enlarged by metabolites treatment, such as Pseudomonas sp. and Bacillus sp. did enlarge their population in treated soil. We conducted 48 hr acute toxicity assay on daphnia. The result of toxicity are in the sequence of M2 >vinclozolin>M1. The metabolites from degradation of vinclozolin, still have the hazard on environment. Due to vinclozolin metabolites have characteristics of persistence and toxicity. When we assess the impact of vinclozolin on environment, the effect of metabolites should be taken into account.

碩士<br>國立成功大學<br>環境工程學系碩博士班<br>101<br>The proliferation of cyanobacteria in drinking water sources is problematic for water authorities as they can interfere with water treatment processes. Chlorination as a commonly used oxidation process in water treatment has shown the potential to lyse cyanobacterial cells, resulting in release of toxic metabolites and odorants. Unfortunately, the netabolites are sometimes difficult to be removed in conventional water treatment processes. Microcystis, a toxic genus of cyanobacteria often present in colonial forms under natural conditions is studied for the effect of chlorination on the cell integrity and metabolites release and degradation. In the oxidation experiments, the colonial Microcystis were sieved into three size groups and were oxidized in algae growth medium (ASM) and in the filtrated water from Cheng Kung Lake, NCKU. A fluorescence technique, combining SYTOX Green nucleic acid stain with flow cytometer, was successfully developed for the determination of cell integrity for colonial Microcystis. A solid-phade microextraction (SPME) concentration followed by a gas chromatograph (GC) and mass spectrometric detector (MSD) was employed to measure an odorous metabolite, β-cyclocitral, while an enzyme-linked immunosorbent assay (ELISA) was used to detect a toxic metabolite, microcystins. A series of chlorination of Microcystis-laden water was conducted at different chlorine dosages for different colony sizes. During the experiments, residual chlorine concentration, cell integrity, and metabolites concentration were monitored at different time. The results show that the bigger the colony is the slower the cell rupture kinetics was observed, meaning that the colonial Microcystis was more resistant to chlorine than single cells. A Delayed Chick Watson Model describe the experimental data very well for the kinetic of cell rupture. The lag time and rate constant of cell rupture increased and decreased with increasing colony size, respectively. It suggests that diffusion of chlorine into the intra-colonial cells and interaction between chlorine and extracellular polymeric substances (mucilage) may be the reason to retard the reaction. In the addition, experimental results obtained for those conducted in the two waters also confirmed that lag times and rate constants were also influenced by water matrix. For the toxic metabolite, chlorination may rupture the cells and cause the release of microcystins. The degradation of microcystins only ocuured when enough chlorine was dosed. The rate may be described by a second-order model, with a rate constant of 269-483 M-1s-1. For odorant, chlorine may inactivate β-carotene oxygenase and inhibit the production of β-cyclocitral. At short reaction time, low CT value of chlorine may stimulate the prodcution of β-cyclocitral by the cells. In addition, chlorine may also react with β-carotene directly to form β-cyclocitral, causing an increase of β-cyclocitral concentration later in the experimental time.

Benzodiazepines (Benzos) and ketamine (K) are compounds that have been encountered in Drug-Facilitated Sexual Assault (DFSA) cases. Due to the intimate nature of these crimes, evidence collection is often postponed due to delays and/or reluctance in reporting these crimes. Further delays in analysis may be encountered in laboratories with large caseloads and/or backlogs. Drug identification in biological samples is important to determine whether victims knowingly or unknowingly took an impairing substance, however, the results could be negative due to chemical degradation over a long storage period. The purpose of this project was to study if degradation could be prevented with a new preservation method at the time of collection. Urine samples were prepared by the addition of K and metabolites and selected benzos and metabolites that were subjected to different sample pre-treatment techniques, and were analyzed after storage at room temperature (25°C), refrigerator (4°C) and freezer (-20°). The samples were either pre-treated with preservative (0.5% toluene) or filtration sterilization (sterile filter kit) within two hours after sample collection, and a control group with no pre-treatment was incorporated into the study for comparison. The changes in concentrations over 50 days (Benzos group) and 210 days (K group) were evaluated between different pre-treated methods and different temperature conditions. Sample that were treated with 0.5% toluene showed the most degradation: 44% of oxazepam and 96% of diazepam degraded over 10 days, and 80% of dehydronorketamine degraded after storage of 150 days regardless the temperature conditions. Clonazepam and flunitrazepam concentrations were reduced by 80% of the original concentration when stored at room temperature for 10 days. The major benzodiazepines evaluated in this study were stable when stored in the freezer. In K group, ketamine and norketamine that were stored at room temperature and refrigerated over 210 days were stable, however, degradation was observed after 150 days when the samples were stored in the freezer. There was no statistically different change observed among the samples pre-treated with or without filtration sterilization. Each sample pH was measured and it was determined that those stored at room temperature had an average pH of 8.5, while samples stored in the refrigerator and freezer had an average pH of 6.7 and 6.5, respectively. This finding revealed that pH could be the major factor affecting compound degradation rather than the bacterial contamination with high pH contributing to degradation, and low pH potentially preventing sample lost.

碩士<br>國立中央大學<br>生命科學研究所<br>92<br>Alkylphenol polyethoxylates (APEOn), one kind of non-ionic surfactants, was extensively used in the industrial, agricultural and household activities. These compounds were frequently discharged into natural environment and transformed to alkylphenol (AP) and APEOn (n=1-2), which was more recalcitrant and has been demonstrated as the environmental hormone with estrogenic-like activity to aquatic organisms, wildlife and humans. Only a few of microorganism that can degrade alkylphenol polyethoxylates or alkylphenol were isolated. The biodegradation pathway of these compounds still remains unproven in previous study. The objectives of this study were aimed to isolate and characterize novel bacterial isolates able to grow on both APEOn and AP in order to elucidate the mechanism of disruption of estrogenic-like metabolites. In this study, 28 bacterial strains are found to be able to degrade alkylphenol and/or from different topsoil samples. All of these isolates were Gram-negative bacteria. They were identified by three methods, Biolog breathprinting, fatty acid fingerprinting and 16S rDNA sequence analysis. 64% of them was belonged to Pseudomonas genus. Other species included Alcaligenes defragrans, Bordetella brochiseptica, Zoogloea ramigera and Inquilinus limosus. Among them, a strain Pseudomonas putida TX2 was shown to have a novel activity to grow on alkylphenol polyethoxylates (0.05~20%) or alkyphenol (<0.02%) as the sole carbon source, which is the first pure culture showing the feature. The strain TX2 reveals an oxygen uptake activity of 192.6, 4.2 and 4.2 nmole/min for a 5 ml cell suspension at OD600 = 0.3 using octylphenol polyethoxylates, octylphenol and octylcatechol as substrates, respectively. Moreover, the oxygen uptake rate of strain TX2 decreased with the increase of carbon numbers in alkyl-chain in bulk alkylphenol and alkylcatechol. In addition, Western blotting further demonstrated an catechol 2, 3-dioxygenase-like aromatic ring-cleavage enzyme that was inducible when strain TX2 was grown on octylphenol polyethoxylates, octylphenol, or octylcatechol as the sole carbon source. Furthermore, LC/MS analysis of the TX2 transformed metabolites from OPEOn and AEO8 showed that octylphenol polyethoxylates could be degraded as a sequential cleavage of the polyethoxylate chain and then produced octylphenol, which was further transformed to form octylcatechol followed by generating a metabolite with a molecular weight of 206. It was suggested as a aromatic ring-cleavage product. These results suggested that strain TX2 was able to shorten the alky chain followed by the cleavage of aromatic ring in the degradation of octylcatechol.

Atrazine photochemical degradation in homogeneous phase using Fe(III)/UV/air system was studied. Two toxicity assessments, a Lemna minor growth inhibition test and a Daphnia magna acute immobilisation test, were employed to test potential toxicity of atrazine and its degradation products. The occurrence of atrazine in rivers from the Vltava River basin was evaluated from the analyses performed by Povodí Vltavy, State Enterprise.

<p>Parkinson’s disease (PD) is a detrimental neurodegenerative disorder characterized by the presence of large protein aggregates in the brain called Lewy bodies, which are primarily composed of the protein α-synuclein (αSyn). Due to the dysregulation of αSyn levels in PD, controlling its levels through the manipulation of protein degradation pathways has been suggested as a therapeutic avenue for the treatment of PD and related diseases. Although αSyn is known to be degraded through the autophagy and proteasome pathways, it is one of only a few known substrates of the ubiquitin-independent proteasome pathway, which utilizes the 20S core particle of the proteasome (20S CP) to degrade proteins. We therefore hypothesize that small molecule stimulation of the 20S CP will enhance αSyn degradation and reduce αSyn pathology, providing a therapeutic benefit in PD models.</p><p>We began our studies by developing a fluorescence resonance energy transfer (FRET) reporter assay to monitor 20S CP activity and screen for small molecule stimulators. This assay provides a greater dynamic range to detect 20S CP stimulation compared to the most commonly used assay to monitor proteasome activity. Using the FRET assay, we were able to identify a number of novel 20S CP stimulators that differ in structure as well as potency and degree of stimulation. We next evaluated the ability of four small molecule stimulators to enhance protein degradation by the 20S CP in a biochemical assay using 15 different purified proteins. These 15 proteins include known substrates of the 20S CP and vary in size and degree of disorder. From this assay, we demonstrate that a 20S CP stimulator is likely to enhance the degradation of highly disordered proteins, such as αSyn, but the effect on other protein levels appears to be distinct for each stimulator. Two of our more potent stimulators, AM-404 and miconazole, were used with the proteasome inhibitor bortezomib for subsequent studies in HEK-293T cells in which we corroborated the results of our biochemical assay. While both AM-404 and miconazole were shown to impact highly disordered proteins, there was not much overlap between the proteins shown to be affected by each stimulator. Due to the distinct effect of each stimulator on protein regulation by the 20S CP, this study indicates the potential of tailoring a small molecule 20S CP stimulator to enhance the degradation of particular substrates.</p><p>Since AM-404 and miconazole were shown to impact 20S CP activity in different ways, we next evaluated whether either stimulator would be able to prevent the αSyn-induced inhibition of the 20S CP. High levels of αSyn have been shown to lead to proteasome impairment in biochemical and cell studies. We confirm 20S CP impairment in the presence of micromolar amounts of αSyn, and we demonstrate that miconazole, but not AM-404, is effective at maintaining 20S CP activity in the presence of increasing concentrations of αSyn. We also show that αSyn-overexpressing PC12 cells (PC12 C4 cells) display reduced proteasome activity compared to the parent cell line. Miconazole and AM-404 increased proteasome activity in PC12 C4 cells, which were more sensitive to 20S CP stimulation than non-transfected PC12 cells, but miconazole was shown to be more effective at modulating αSyn phosphorylated at Ser129 in PC12 C4 cells. </p><p>Our results reveal the dynamic nature of the 20S CP and the ways in which its activity can be modulated to affect protein levels. While AM-404 is effective at stimulating the 20S CP to enhance the degradation of some proteins, miconazole was shown to be more efficient at modulating αSyn levels and impacting αSyn pathology, as it relates to 20S CP impairment. While the results described here mark the beginning of an exciting area of study, we do demonstrate the therapeutic potential of 20S CP stimulation to combat PD. </p><div><br></div>

Fluorotelomer alcohols (FTOHs, F(CF2)nCH2CH2OH) are emerging contaminants in the environment. Biodegradation of 6:2 and 8:2 FTOHs has been intensively studied using soils and activated sludge. However, little is known about the bacteria responsible for biotransformation of FTOHs. This study deciphered factors affecting biodefluorination of FTOHs and their metabolites, and developed three effective FTOH-degrading consortia. Two alkane-degrading Pseudomonas strains (P. oleovorans and P. butanovora) can defluorinate 4:2, 6:2 and 8:2 FTOHs, with a higher degree of defluorination for 4:2 FTOH. According to the identified metabolites, P. oleovorans transformed FTOHs via two pathways I and II. Pathway I led to formation of x:2 ketone (x = n-1), x:2 sFTOH and perfluorinated carboxylic acids (PFCAs). Pathway II resulted in the formation of x:3 polyfluorinated acid and relatively minor shorter-chain PFCAs. Conversely, P. butanovora transformed FTOHs by pathway I only. Mycobacterium vaccae JOB5 (a C1-C22alkane-degrading bacterium) and P. fluorescens DSM 8341 (a fluoroacetate-degrading bacterium) can transform 6:2 FTOH via both pathways I and II with the formation of odd-numbered short-chain PFCAs. In the presence of dicyclopropylketone or formate, P. oleovorans transformed 6:2 FTOH six times faster and produced odd-numbered PFCAs. P. butanovora, utilized both pathways I and II in the presence of lactate, and it also produced odd-numbered PFCAs. Unlike P. oleovorans, P. fluorescens DSM 8341 could slightly convert 5:3 polyfluorinated acid (a key metabolite during 6:2 FTOH degradation, [F(CF2)5CH2CH2COOH]) to 4:3 acid and PFPeA via one-carbon removal pathways. Three FTOH-degrading consortia transformed FTOHs, with enhanced removal of FTOHs in the presence of n-octane. A higher copy number of alkB gene was found to correspond to better removal of FTOHs, suggesting that alkane-degrading bacteria might be the key degraders in the enrichments. The three enrichment cultures showed a similar microbial community structure. This is the first study reporting that pure strains of alkane- and fluoroacetate-degrading bacteria can bio-transform FTOHs via different or preferred transformation pathways to remove multiple –CF2– groups from FTOHs to form shorter-chain PFCAs, and to other perfluorinated acids. The results of this study also suggest that enhanced FTOH biodegradation is possible through co-substrate addition and/or using enrichment cultures.

The present thesis was focused on degradation studies of five extensively and globally used pharmaceutical compounds: chloramphenicol, paracetamol, fluoxetine and 17α-ethynilestradiol which are considered emerging pollutants that enter in the environment due to its inefficient removal by the conventional wastewater treatments plants. Photodegradation and biodegradation assays were realized. The photodegradation of chloramphenicol and paracetamol was successfully performed by heterogeneous photocatalysis using PbS/TiO2 nanocomposites, as catalysts, which were synthesized using biological sulphide produced by sulphate reducing bacteria in batch cultures. Although photodegradation demonstrated to be a faster process, biodegradation is preferable since it occurs without the use of external compounds, such as catalysts, using only the microorganisms already present in the environment. Communities and bacterial isolates with ability to remove and use these drugs as unique carbon sources were obtained using sludge collected from an oxidation ditch and a lagoon system of WWTPs. An initial aerobic bacterial community suffered a shift to favor the genera Pseudomonas sp; Flavobacterium, Dokdonella and Methylophilus which displayed high efficiency in removing 50 mg/L paracetamol as sole carbon source and are described for the first time as paracetamol degraders. Bacteria capable of effectively biodegrade paracetamol were isolated in aerobic conditions, suggesting for the first time that members of “Bacillus cereus group”, [Brevibacterium] frigoritolerans, Corynebacterium nuruki and Enterococcus faecium can use paracetamol as carbon source, whereas the isolates obtained for fluoxetine and 17α-ethynilestradiol only partially removed these drugs. An anaerobic consortium which was able to degrade 20 mg/L of FLX, as unique carbon source, to values below the limit of detection was obtained. The final consortium was mainly composed by genera vadinBC27 wastewater-sludge group, Macellibacteroidetes, Dethiosulfovibrio, Bacteroides, Tolumonas, Sulfuricurvum, f_Enterobacteriaceae_OTU_18, which are for the first time assumed as FLX biodegraders. In the biodegradation of 20 mg/L FLX, a possible metabolite, 2,3-dimethyl-5-(trifluoromethyl) benzene-1,4-diol, was detected after 28 of assay. Several attempts to optimize an aerobic granular system were performed to degrade ibuprofen. This process is still ongoing.<br>A presente tese foca-se em estudos de degradação de 5 fármacos considerados poluentes emergentes, são estes: cloranfenicol, paracetamol, fluoxetina, 17α-etinilestradiol e ibuprofeno. Estes são considerados potenciais ameaças aos ecossistemas ambientais, à saúde e à segurança humana, especialmente devido ao seu uso extensivo a nível mundial. Os tratamentos de águas residuais convencionais têm-se mostrado ineficientes na remoção total destes fármacos resultando na sua libertação para o ambiente. Atualmente, uma das maiores preocupações é a contaminação incessante dos ambientes aquáticos com estes poluentes e os problemas ecológicos inerentes. Com o objetivo de conhecer, desenvolver ou melhorar os processos já existentes, foram realizados estudos de fotodegradação e biodegradação visando encontrar novas alternativas que sejam baratas e simples e que permitam uma completa remoção destes fármacos nas estações de tratamento de águas residuais (ETARs). Nos estudos de fotodegradação foram sintetizados com sucesso nanocompósitos de PbS/TiO2 (fotocatalisador heterogéneo) utilizando sulfureto produzido biologicamente pelas bactérias sulfato redutoras (BSR) em batch. A importância deste processo está no aproveitamento de resíduos tóxicos, como o sulfureto e iões chumbo em solução aquosa, para formulação de um catalisador eficiente na remoção destes poluentes (fármacos). Estes nanocompósitos, foram empregues com êxito como catalisadores na remoção de cloranfenicol e paracetamol, revelando uma elevada eficiência de remoção (>93%), após 240 minutos de irradiação. Nos ensaios de biodegradação obtiveram-se comunidades e isolados bacterianos com capacidade de utilizar os fármacos em estudo, como única fonte de carbono e que são descritos pela primeira vez como tendo um papel muito importante na remoção destes poluentes. Identificou-se uma comunidade bacteriana aeróbia que apresentou elevada eficácia na remoção de paracetamol como única fonte de carbono (97 ± 3%). Ao longo do ensaio verificou-se um “shift” na população inicial levando a um favorecimento dos géneros Pseudomonas sp; Flavobacterium, Dokdonella e Methylophilus, revelando pela primeira vez o seu possível envolvimento na degradação do paracetamol. Os metabolitos identificados nesse processo foram 4-aminofenol, hidroquinona e um composto desconhecido. Isolaram-se bactérias com capacidade de biodegradar paracetamol utilizando como inóculo as lamas recolhidas numa vala de oxidação, sugerindo pela primeira vez que membros de “Bacillus cereus group” (associados Bacillus pacificus ou Bacillus paranthracis), [Brevibacterium] frigoritolerans, Corynebacterium nuruki e Enterococcus faecium têm a capacidade de usar este fármaco como única fonte de carbono. Uma espécie pertencente ao “Bacillus cereus group” degradou 200 mg/L de paracetamol, abaixo dos níveis de deteção, sem formação aparente de metabolitos secundários tóxicos e removeu cerca de 95 ± 5% de 500 mg/L do fármaco, após 144 h de incubação a 28 ºC. Durante o processo de degradação do paracetamol foram detetados os metabolitos 4-aminofenol, hidroquinona e ácido 2-hexenóico. Realizaram-se estudos de biodegradação com uma comunidade bacteriana enriquecida a partir de uma amostra de lama recolhida num sistema de lagunagem em que foram favorecidas as BSR. As culturas foram efectuadas na presença de fluoxetina e lactato e usando o fármaco como única fonte de carbono, em condições redutoras e anaerobiose. O consórcio obtido revelou ter capacidade de degradar 20 mg/L e 50 mg/L de fluoxetina para valores abaixo do limite de deteção, após 28 e 31 dias, respetivamente. Utilizando fluoxetina como única fonte de carbono, verificou-se que o consórcio removeu 20 mg/L de fluoxetina abaixo dos limites de deteção e 66 ± 9% de 50 mg/L do fármaco, após 31 dias. Norfluoxetina foi identificada na degradação de 50 mg/L fluoxetina, enquanto na biodegradação de 20 mg/L de fluoxetina, foi detetado outro possível metabolito, o 2,3-dimetil-5-(trifluorometil)benzeno-1,4-diol, após 28 dias de ensaio. A população bacteriana inicial era constituída principalmente por Desulfomicrobium e Desulfovibrio, enquanto que no decurso das experiências usando fluoxetina como única fonte de carbono, verificou-se a ocorrência de um claro “shift” na comunidade com o aumento do grupo Wastewater-sludge group vadinBC27, Macellibacteroidetes, Dethiosulfovibrio, Bacteroides, Tolumonas, Sulfuricurvum, f_Enterobacteriaceae, bactérias identificadas pela primeira vez como possíveis degradadoras de fluoxetina. Em aerobiose, foram preparadas culturas inoculando o efluente bruto com a lama, simulando as condições da ETAR. Neste ensaio a remoção de fluoxetina foi de aproximadamente 89%, aparentemente com uma contribuição mínima de adsorção à lama. Durante este ensaio, a população inicial sofreu um “shift" com o aumento de estirpes dos géneros Aeromonadales_OTU_14, Acinetobacter, Rheinheimera, Shewanella, Pseudomonas, f_Pseudomonadaceae_OTU_4914; OTU_24; f_JI49D030_OTU_6171, Methylobacillus e Piscinobacter, sendo apontados pela primeira vez como os prováveis responsáveis pela biodegradação da fluoxetina. Da lama onde se obteve a comunidade anterior foram selecionados seis isolados com capacidade de utilizar fluoxetina como única fonte de carbono que foram identificados como Pseudomonas putida, Enterobacter ludwigii, Pseudomonas nitritireducens, Alcaligenes faecalis, Pseudomonas aeruginosa e Pseudomonas nitroreducens. Pseudomonas nitroreducens foi a mais eficiente, com remoções de 55 ± 1% e 21 ± 1% a 20 mg/L e 50 mg/L de fluoxetina, respetivamente, após 168 h. Os isolados obtidos não apresentaram elevadas percentagens de remoção deste fármaco, contudo estas foram significativas quando comparadas com o controle negativo. Embora o principal mecanismo de remoção de fluoxetina descrito na literatura seja por adsorção, neste estudo a biodegradação parece desempenhar o papel principal na degradação do fármaco. Das lamas ativadas foram obtidos isolados bacterianos com capacidade de crescimento na presença de 50 mg/L de 17α-etinilestradiol a partir de uma lama recolhida na vala de oxidação da ETAR Noroeste de Faro, sendo estes identificadas como Acinetobacter bouvetii, Acinetobacter kookii, Pantoea agglomerans e Shinella zoogloeoides. Os resultados revelaram que Acinetobacter bouvetii, Acinetobacter kookii, Pantoea agglomerans e Shinella zoogloeoides, degradaram 47 ± 4%, 55 ± 3%, 64 ± 4% e 35 ± 4%, respetivamente de 13 mg/L 17α-etinilestradiol, após 168 h a 28 ºC. A degradação de 17α-etinilestradiol por estes isolados resultou em vários intermediários/vias possíveis que são mais facilmente degradados e menos tóxicos do que o composto original. Os compostos detetados foram: ácido manónico 1,4-lactona pertencente às estruturas “gama lactona”, ácido 4-hidroxi-2-quinolinacarboxílico, ácido 2,3-di-hidroxibenzóico, ácido D-glucónico e ácido Z-aconítico, ácido 2-pentenodióico (ácido insaturado) e o ácido 2-butenodióico após possível meta-clivagem do anel A. O ácido 2-butenodióico (ácido fumárico), é um ácido dicarboxílico e um metabolito intermediário no ciclo dos ácidos tricarboxílicos/ciclo de Krebs. Em relação ao sistema granular realizaram-se diferentes abordagens a nível experimental visando optimizar o sistema e a degradação do ibuprofeno. Este processo ainda está em curso.

The discovery of antibiotics is considered as one of the most significant scientific achievements of the 20th century – lives of millions of people and animals have been saved. Thenceforth, substantial amounts of administered antibiotics and their metabo-lites have been excreted into waste stream via urine and faeces. In this dissertation, primary focus is the qualitative balance of 14 antibiotics and one metabolite in urban water management and in urban waters, respectively. In particular, antibiotics pre-scribed to human beings are drained in the urban sewer system and finally enter the environment: (i) Continuously via the effluent of the wastewater treatment plant after a partially effective removal or degradation or (ii) Intermittent via combined sewer overflow structures due to capacity limitations of the urban drainage system. The fate and the potential effects and risks of these substances on ecosystems and hu-man health are of major concern – their direct toxic effect to all trophic levels as well as the global spread of antibiotic resistance genes are challenging. Hence, an assessment of microbial community activity due to antibiotic exposure is presented. In particular, systematic work has been carried out to study the presence and character-istics of 14 antibiotics in urban waters. In detail, investigations were conducted to gain scientific knowledge with respect to adsorption, desorption, abiotic, biotic and photolyt-ic degradation as well as activity-inhibition of microorganism communities in sewage and of natural freshwater biofilm communities, respectively, due to inevitable urban drainage overflows. In order to provide information to assist potential management strategies, which miti-gate surface water pollution and minimize the adverse impacts of antibiotics on activity of microorganism communities, the following specific topics were addressed: ⑴ The occurrence of 14 antibiotics and one metabolite were determined in sewag-es at three sampling sites in the city of Dresden, Germany. ⑵ The adsorption affinities of 14 antibiotics and one metabolite to size dependent sewer sediments were determined in experimental investigations, three sam-pling campaigns and subsequently an antibiotic-specific adsorption coefficient, normalized to organic content, was quantified. ⑶ The desorption affinity and -dynamics of 14 antibiotics and one metabolite were quantified in size dependent sewer sediments in experimental investigation and with statistical analysis. ⑷ The abiotic, biotic and photolytic degradation affinity of 14 antibiotics and one metabolite were quantified based on batch experiments with three different sewages at 7°C and 22°C, with artificial irradiation and different dilution ratios of the sewage at 30°C and subsequently a model framework decrypted ranges of abiotic, biotic and photolytic degradation coefficients. ⑸ The occurrence of three antibiotics, namely ciprofloxacin, clarithromycin and doxycycline was determined in sewage sampled during dry weather conditions in a small catchment of Dresden, which spills intermittently combined sewage (a mixture of sewage and storm water) to an adjacent brook in the case of capacity limitations of the urban drainage system during periods of intense rainfall and subsequently the three antibiotics were determined in the adjacent brook water. ⑹ Then, the activity-inhibition of microorganism community in sewage of this small catchment was quantified due to an exposition with three different antibiotics and three different antibiotic concentrations. ⑺ Last but not least, the activity-inhibition of natural freshwater biofilm communities in the adjacent brook was quantified via exposure to three antibiotics, which were individually dosed in three different concentrations, and also in mixture. ⑻ Finally, a two-dimensional hierarchical cluster analysis with dendrogram and heat map based on before mentioned activity inhibition of natural freshwater biofilm communities were conducted to identify hot spots of antibiotic tolerant and resistant bacterial subpopulations due to inevitable urban drainage system overflows.:List of Figures IV List of Tables VIII Symbols and Abbreviations XII List of Publications on the Ph.D. topic XIX 1 General Introduction 2 1.1 Background 2 1.2 Aims and Objectives 3 1.3 Innovation and Contribution to the Knowledge 4 1.4 Outline of this Thesis 4 1.5 References 6 2 Adsorption and Desorption Affinity of 14 Antibiotics and One Metabolite for particulate components in urban drainage systems 10 2.1 Introduction 11 2.2 Materials and Methods 12 2.2.1 Study area 12 2.2.2 Sewer sediment and sewage sample collection 12 2.2.3 Sediment fractionation 13 2.2.4 Antibiotic determination in sewage and sediment 13 2.3 Results and Discussion 18 2.3.1 Antibiotics in composite sewage samples 18 2.3.2 Antibiotics adsorbed to sewer sediments 19 2.3.3 Organic-bound antibiotic load as a linear function of liquid concentration 20 2.3.4 Adsorption dynamics and adsorption coefficient determined by bath experiments 20 2.3.5 Mineral composition of sewer sediment SED#1B 23 2.3.6 Initial characteristics of sediment SED#1B 23 2.3.7 Desorption dynamics and desorption coefficient of SED#1B 24 2.4 Conclusions 25 2.5 References 26 3 Abiotic, Biotic and Photolytic Degradation Coefficients of 14 Antibiotics and One Metabolite 32 3.1 Introduction 34 3.2 Materials and Methods 35 3.2.1 Study area and sample collection 35 3.2.2 Experimental set up 35 3.2.3 Modelling framework 38 3.2.4 Procedure of model calibration 40 3.3 Results and Discussion 43 3.3.1 Primary metabolic parameter 43 3.3.2 Secondary metabolic parameter 44 3.4 Conclusions 50 3.5 References 50 4 Activity-Inhibition of Microorganisms due to an Exposition with different Antibiotics and Concentrations 56 4.1 Assessing Antibiotic Resistance of Microorganisms in Sanitary Sewage 56 4.1.1 Introduction 57 4.1.2 Material and Methods 58 4.1.2.1 Sampling Site and Antibiotic Agents 58 4.1.2.2 Analyzing Antibiotics 60 4.1.2.3 Respiration Rate 60 4.1.3 Results and Discussion 60 4.1.3.1 Concentration Range of Antibiotics and Typical Sewage Parameters 60 4.1.3.2 Oxygen Uptake Rate 62 4.1.4 Summary and Conclusions 63 4.1.5 References 64 4.2 Hot Spots of Antibiotic Tolerant and Resistant Bacterial Subpopulations in Natural Freshwater Biofilm Communities due to Inevitable Urban Drainage System Overflows 66 4.2.1 Introduction 68 4.2.2 Material and Methods 69 4.2.3 Results and Discussion 72 4.2.4 Conclusions 76 4.2.5 References 76 5 Summery and General Coclusions 82 5.1 Adsorption and Desorption Affinity 82 5.2 Abiotic, Biotic and Photolytic Degradation 83 5.3 Activity-Inhibition of Microorganism Communities due to Antibiotic Exposure 84 5.4 Enhancement of the Stockholm County Council (2014) assessment of antibiotics 84 5.5 References 87 6 Proposed Directions of Future Research 90 7 Appendixes 94 7.1 Chapters 94 7.2 Figures 95 7.3 Tables 115 7.4 References 139