Triclosan é um composto orgânico de baixa solubilidade que vem sendo utilizado em formulações de cremes dentais e faciais, xampu, sabonetes, embalagens de gêneros alimentícios e diversos tipos de materiais, tais como, adesivos, brinquedos, sapatos, selantes, tintas, colchão, roupas, pisos, toldos e rejuntes. O amplo uso deste composto deve-se à grande eficácia contra bactérias Gram negativas e Gram positivas. Por seu extenso uso, evidências da presença de Triclosan têm sido frequentemente relatadas em efluentes urbanos e industriais, águas superficiais e sedimentos de ambientes dulcícolas, estuarinos e marinhos, como também em organismos aquáticos como algas, peixes e mamíferos. Neste contexto, o presente estudo avaliou a toxicidade aguda e crônica de Triclosan para diferentes invertebrados marinhos de águas tropicais. Para tanto, ensaios de toxicidade aguda foram realizados com o copépodo Nitokra sp (mortalidade) e com o ouriço-do-mar Lytechinus variegatus (taxa de fertilização). Para a avaliação do efeito crônico, ensaios de toxicidade de curta duração (desenvolvimento embriolarval) foram realizados com o ouriço-do-mar L. variegatus e Perna perna. Além desses métodos, o ensaio do Tempo de Retenção do Corante Vermelho Neutro foi empregado com a finalidade de se avaliar os efeitos do Triclosan sobre a estabilidade da membrana lisossômica de hemócitos de P. perna. Na avaliação do efeito agudo, o valor médio da CL(I)50;96h encontrada para o copépodo foi de 0,20 mg.L-1 enquanto que o valor médio da CI(I)50;1h para ouriço-do-mar foi de 0,28 mg.L-1. Já na avaliação do efeito crônico, o valor médio da CI(I)50;24h para ouriço-do-mar foi de 0,14 mg.L-1 e para o molusco bivalve a média da CI(I)50;48h, foi de 0,13 mg.L-1. O efeito na estabilidade da membrana lisossômica de hemócitos de P. perna ocorreu em concentrações a partir de 12 ng.L-1. Estes resultados evidenciam o risco ecológico da introdução contínua desse composto em ambientes marinhos, e devem ser considerados para identificação de concentrações seguras e futura regulação do bactericida Triclosan na legislação ambiental nacional e internacional. / Triclosan is a low solubility organic compound that has been used in toothpastes, face cream, shampoos, soaps, food packages, and a variety of other materials such as stickers, toys, shoes, paints, clothes, tiles, awnings and grout. The reason for its intense use as biocide is its great efficacy against Gram-negative and Grampositive bacteria. Evidences of Triclosan presence in urban and industrial effluents, superficial waters and sediments from freshwater, estuarine, and marine environments, as well as aquatic organisms (algae, fishes, mammals) have been reported in the literature. In this context, the present study assessed the acute and chronic toxicity of Triclosan to different tropical marine invertebrates. Acute toxicity bioassays using the copepod Nitokra sp (mortality) and the sea-urchin Lytechinus variegatus (fertilization rate) were performed. Short-term chronic toxicity bioassays with Lytechinus variegatus and the bivalve mussel Perna perna were carried out in order to assess Triclosan chronic effects. Besides, the Neutral Red Retention Time assay was employed to evaluate the effect of Triclosan on the stability of lysosomal membrane of hemocytes of Perna perna. In the acute toxicity assays, the mean value of LC(I)50;96h obtained for the copepod was 0.20 mg L-1, whereas the mean value of IC(I)50;1h for the sea-urchin was 0.28 mg L-1. In the chronic toxicity assays, the mean value of IC(I)50;24h recorded for the seaurchin was 0.14 mg L-1, whilst for the bivalve mollusk the mean value of IC(I)50;48h was 0.13 mg L-1. The effect on the lysosomal membrane stability of Perna perna hemocytes started to occur from 12 ng L-1. The results evidence the ecological risk associated to the continuous introduction of Triclosan into marine aquatic environments and must be considered in the identification of safety concentrations and future regulation of this bactericide compound in national and international environmental legislation.

Cette thèse, intégrée dans le projet Cosmet’eau et la phase 4 du programme OPUR, apporte de nouveaux éclairages sur la dynamique des parabènes, du triclosan et du triclocarban en milieu urbain sous forte pression anthropique. Ces molécules sont des biocides couramment utilisés en tant que conservateur dans de très nombreux produits de consommation courante comme les cosmétiques, les produits alimentaires ou pharmaceutiques, les textiles, les produits d’entretien. En dépit de leur toxicité, ces molécules ne font l’objet d’aucun suivi réglementaire dans l’environnement. Aussi, ce travail porte sur la dynamique de ces micropolluants en milieu urbain : de leurs sources (à savoir les eaux grises et eaux vannes) jusqu’au milieu récepteur (amont-aval de l’agglomération parisienne par échantillonnage ponctuel et passif) en intégrant leur comportement en station d’épuration à l’échelle de l’ouvrage.Au niveau des sources, cinq types d’eau grise issus de douche, lavabo, lave-linge, lave-vaisselle et vaisselle manuelle ont été considérés. Une variabilité importante des concentrations au sein de chacune des eaux grises et entre les différents types d’eau grise a été observée. Cette variabilité est le reflet des pratiques de consommation des différents volontaires. Au final, les eaux des lave-linge et douche sont les plus contaminées. Pour les parabènes, le linge est la source majoritaire de contamination des eaux de lave-linge, dans le cas des eaux de douche, les personnes et les produits de soins corporels sont les principales sources de contamination.Depuis 2010, les flux quotidiens par habitant de ces molécules ont diminué significativement (d’un facteur compris entre 2 et 7 suivant la molécule). Cette diminution s’explique par (i) les changements de formulation des produits cosmétiques et (ii) l’apparition de nouvelles réglementations plus contraignantes.Le suivi du devenir en station d’épuration (sur Seine Centre), à l’échelle de l’ouvrage, a montré que le traitement primaire (décantation physico-chimique lamellaire) permet un abattement quasi-total du triclosan, les parabènes sont majoritairement éliminés lors de la biofiltration notamment au niveau du premier étage (Biofor). La généralisation d’un traitement tertiaire (du type Carboplus® suivi lors de ce travail) permettrait de réduire ces rejets.Les rejets urbains par temps de pluie (ici les déversoirs d’orage) constituent une source importante de contamination pour le milieu récepteur au regard des niveaux que nous avons observé. La combinaison des rejets des stations d’épuration, par temps sec et par temps de pluie, et des déversoirs d’orage contribue à augmenter les niveaux en parabènes, triclosan et triclocarban de la Seine comme le suivi de deux stations de mesure en amont et aval de l’agglomération parisienne l’a mis en évidence. Les échantillonneurs passifs sont des outils prometteurs pour déterminer la fraction biodisponible du TCS et du TCC. En effet, la méthode développée dans le cadre du projet Cosmet’eau a été appliquée avec succès à la Seine. / This Phd thesis is part of both the Cosmet’Eau project and the OPUR programme. It provides new insights into the dynamics of parabens, triclosan and triclocarban in urban areas under strong human pressure. These molecules are biocides commonly used as preservatives in a wide range of consumer products, such as cosmetics, food and pharmaceutical products, textiles and cleaning products. Despite their toxicity, these molecules are not subject to any regulatory monitoring in the environment. Also, this work deals with the dynamics of these micropollutants in urban areas: from their sources (namely gray water and sewage) to the receiving environment (upstream-downstream of the Parisian conurbation with punctual and passive sampling) along with their behavior at different stages of a wastewater treatment plant.At the source level, five types of greywater from shower, washbasin, washing machine, dishwasher and manual dish washing were considered. Significant variability in concentrations within each greywater and between different types of greywater was observed. This variability reflects the consumption practices of the different volunteers. Besides, the waters from washing machines and showers are the most contaminated. For parabens, clothes are the main source of contamination of washing machine waters, while in shower waters, people and personal care products are the main sources of contamination.Since 2010, per capita daily flows of these molecules have decreased significantly (by a factor between 2 and 7 depending on the molecule). This decrease can be explained by (i) changes in the formulation of cosmetic products and (ii) the emergence of new, more restrictive regulations. The monitoring of the fate in wastewater treatment plant (Seine Centre), at the scale of the device, showed that primary treatment (lamellar physico-chemical decantation) allows a quasi-total removal of triclosan, while parabens are predominantly eliminated during the biofiltration, at the level of the first stage (Biofor). The general use of a tertiary treatment (like Carboplus®, studied during this work) would reduce discharges to receiving waters of parabens, triclosan and triclocarban.Urban discharges during wet weather period (here combined sewer overfows) constitute a major source of contamination for the receiving waters with respect to the measured levels. The combination of sewage treatment plant discharges, during dry and wet weather periods, and combined sewer overfows contributes to increasing levels of parabens, triclosan and triclocarban in the Seine River, as the monitoring of two sites, upstream and downstream of the Paris conurbation, has highlighted it. Passive samplers are promising tools for determining the bioavailable fraction of TCS and TCC. Indeed, the method developed within the framework of the Cosmet'eau project has been successfully applied to the Seine

Triclosan (TCS) is a personal care product with many fields of application and is of public interest for several years now. Monitoring studies showed that TCS is a ubiquitous chemical in the aquatic environment. Aquatic organisms are exposed to TCS in a broad range of concentrations, from ng L-1 up to lower μg L-1. TCS has a bactericidal effect for various types of gram-positive and gram negative bacteria. TCS targets a specific bacterial fatty acid biosynthetic enzyme, enoyl-[acyl-carrier protein] reductase (Schweizer, 2001). Therefore the terminal reaction in the fatty acid elongation cycle is inhibited (Levy et al., 1999). Although effects on non-target organisms are reported, the Mode of Action (MoA) of TCS is not well examined for those organisms. The aim of this PhD thesis was to investigate effects of TCS on non-target autotrophic organisms at different levels of biological complexity in the aquatic environment. In this thesis microalgae have been found to be very sensitive to TCS. In some cases even higher sensitivities than in bacteria were observed, which is in accordance with published effect data (Harada et al., 2008; Orvos et al., 2002). Similarly to bacteria, high species sensitivity differences were observed for algae (Franz et al., 2008). In bacteria these sensitivity differences can be ascribed to several resistance mechanisms reported in Schweizer (2001). These findings lead to the question about the reasons for species sensitivity differences in algae. A mesocosm study was performed to detect effects of TCS across levels of biological organization and to investigate the impact of sensitivity differences on complex aquatic communities. For that purpose, structural and functional effects parameters were observed.

The concentration of triclosan in antiseptics, disinfectants and preservatives in products exceeds the minimal lethal levels. Extensive use of triclosan and antibiotics results in bacterial resistance to their active ingredients. The precise relationship between use and resistance, however, has been challenging to define. The aim of the study was to identify and determine antibiotic resistance profiles of triclosan tolerant heterotrophic plate count bacteria isolates from sewage influent and effluent. R2 agar supplemented with triclosan was utilised to isolate the triclosan resistant bacteria. To determine the minimum inhibitory concentration (MIC), organisms were incubated for 24 hours at selected concentrations of triclosan. Polymerase chain reaction (PCR) amplification of the 16S rRNA region was done to identify isolates. An assay for cross resistance to various antibiotics was performed. Determination of enhanced resistance to antibiotics by adding antimicrobials to the medium will be performed by using three antibiotics. High performance liquid chromatography was conducted to quantified levels of triclosan persistent in sewage water. Forty-four isolates were resistant to levels of triclosan ranging from 0.25 mg/l to 0.5 mg/l. Minimum inhibitory concentration values of these isolates ranged from 0.125 mg/l to >1 mg/l of triclosan. 16S rDNA methods were used and five main genera namely, Bacillus, Pseudomonas, Enterococcus, Brevibacillus and Paenibacillus were identified. Cell wall targeting antibiotics showed more pronounced relation with the triclosan concentration. Relation to triclosan concentration is not as apparent with the antibiotic targeting protein synthesis. Combination of antimicrobials indicated that at certain triclosan concentrations synergism or antagonism is observed. The importance of applying the correct concentration and combination of antimicrobials is observed. Levels of triclosan were found throughout the sewage water. HPLC values indicated the presence of triclosan at post-grid removal and effluent of the WWTP. The triclosan concentrations decrease through the WWTP but small concentrations enter our water bodies. The presence of bacterial species that are resistant to high concentrations of triclosan and multiple antibiotics enter our natural water bodies and is cause for concern. / MSc (Environmental Sciences), North-West University, Potchefstroom Campus, 2015

The concentration of triclosan in antiseptics, disinfectants and preservatives in products exceeds the minimal lethal levels. Extensive use of triclosan and antibiotics results in bacterial resistance to their active ingredients. The precise relationship between use and resistance, however, has been challenging to define. The aim of the study was to identify and determine antibiotic resistance profiles of triclosan tolerant heterotrophic plate count bacteria isolates from sewage influent and effluent. R2 agar supplemented with triclosan was utilised to isolate the triclosan resistant bacteria. To determine the minimum inhibitory concentration (MIC), organisms were incubated for 24 hours at selected concentrations of triclosan. Polymerase chain reaction (PCR) amplification of the 16S rRNA region was done to identify isolates. An assay for cross resistance to various antibiotics was performed. Determination of enhanced resistance to antibiotics by adding antimicrobials to the medium will be performed by using three antibiotics. High performance liquid chromatography was conducted to quantified levels of triclosan persistent in sewage water. Forty-four isolates were resistant to levels of triclosan ranging from 0.25 mg/l to 0.5 mg/l. Minimum inhibitory concentration values of these isolates ranged from 0.125 mg/l to >1 mg/l of triclosan. 16S rDNA methods were used and five main genera namely, Bacillus, Pseudomonas, Enterococcus, Brevibacillus and Paenibacillus were identified. Cell wall targeting antibiotics showed more pronounced relation with the triclosan concentration. Relation to triclosan concentration is not as apparent with the antibiotic targeting protein synthesis. Combination of antimicrobials indicated that at certain triclosan concentrations synergism or antagonism is observed. The importance of applying the correct concentration and combination of antimicrobials is observed. Levels of triclosan were found throughout the sewage water. HPLC values indicated the presence of triclosan at post-grid removal and effluent of the WWTP. The triclosan concentrations decrease through the WWTP but small concentrations enter our water bodies. The presence of bacterial species that are resistant to high concentrations of triclosan and multiple antibiotics enter our natural water bodies and is cause for concern. / MSc (Environmental Sciences), North-West University, Potchefstroom Campus, 2015

<p>More than 50% of wastewater biosolids are applied to agricultural fields as fertilizer in the U.S. This technique has been used for decades as a widely accepted beneficial reclamation method for biosolids, which meet the established regulatory levels for nutrients, metals, and pathogens. A major drawback to land application is the potential environmental release of non-regulated organic contaminants, which accumulate in biosolids during the wastewater treatment process. Recent studies have been performed to identify and quantify the presence of emerging contaminants in biosolids, and others have investigated the effects of compounds already identified as `priority pollutants' and whose use is waning. However, there is limited research on the effect of emerging organic contaminants on soil microbial ecology and nutrient cycling. Because many of the compounds found in biosolids are specifically designed to elicit biological modifications (e.g., antimicrobials), there is a risk that these compounds will disrupt microbial soil functions, decrease soil productivity, and ultimately affect the long term viability of these ecosystems, resulting in unforeseen economic and social costs. Therefore, there is a clear need to characterize the effects of novel contaminants on soil health.</p><p>This dissertation was divided into three distinct parts examining the impacts of emerging organic contaminants on soil microbial ecology with increasing complexity to better reflect environmental conditions. To assess the ecological impacts, the functional endpoint of denitrification was selected because it provides a vital indication of soil health. Denitrifying bacteria play a critical role in this process, and thus, were used as indicator organisms for determining contaminant ecotoxicological potential. Furthermore, antimicrobial agents (a.k.a., bactericides or biocides) were selected as model contaminants because they are designed specifically to deactivate microorganisms, are heavily used in the U.S with over $1 billion in yearly sales, and have been measured in biosolids.</p><p>Overall, the objectives of this dissertation were to: 1) develop a rapid, high-throughput functional assay that measured denitrification inhibition for screening potential ecological impacts of biosolids-derived antimicrobial agents, 2) determine the potential effects of common and emerging biosolids-derived antimicrobial agents on denitrification by a model soil denitrifier, Paracoccus denitrificans PD1222, 3) examine the impacts of the most commonly used antimicrobial, triclosan (TCS), on wastewater treatment efficiency in bench scale sequencing batch reactors (SBRs) coupled with anaerobic digesters, 4) examine the impacts of biosolids aged and spiked with TCS on denitrification under simulated agricultural soil conditions, and 5) evaluate potential impacts of TCS in `traditional' biosolids on denitrification in agricultural soil under field conditions.</p><p>The first phase of research pertaining to Objectives 1 and 2 examined the baseline interactions between biosolids-derived antimicrobial agents and soil microbial ecology. However, to isolate the effect of an individual contaminant from the myriad of contaminants found in biosolids, there was a need for developing a rapid, high-throughput method to evaluate general ecotoxicity. In the first part of this dissertation, we developed a novel assay that measured denitrification inhibition in a model soil denitrifier, Paracoccus denitrificans Pd1222. Two common (TCS and triclocarban) and four emerging (2,4,5 trichlorophenol, 2-benzyl-4-chlorophenol, 2-chloro-4-phenylphenol, and bis(5-chloro-2-hydroxyphenyl)methane) antimicrobial agents found in biosolids were analyzed as model contaminants. Overall, the assay was reproducible and measured impacts on denitrification over three orders of magnitude exposure. The lowest observable adverse effect concentrations (LOAECs) were 1.04 &mu;M for TCS, 3.17 &mu;M for triclocarban, 0.372 &mu;M for bis-(5-chloro-2-hydroxyphenyl)methane, 4.89 &mu;M for 2-chloro-4-phenyl phenol, 45.7 &mu;M for 2-benzyl-4-chorophenol, and 50.6 &mu;M for 2,4,5-trichlorophenol. Compared with gene expression and cell viability based methods, the denitrification assay was more sensitive and resulted in lower LOAECs. Of the six compounds examined, four resulted in LOAECs that were below or within an order of magnitude of concentrations that were measured in the environment, indicating potential ecological impacts.</p><p>In the second part of the dissertation, the impacts of emerging contaminants were examined first under laboratory conditions mimicking wastewater treatment processes (Objective 3) and then agricultural fields (Objective 4). For this phase, TCS, which is the most widely used antimicrobial agent and identified in the first phase for potential ecological impacts, was used as the model contaminant. To mimic wastewater treatment processes, bench scale SBRs coupled with anaerobic digesters were set up and operated. The SBRS and digesters were seeded with activated and anaerobically digested sludge from the North Durham Water Reclamation Facility (NDWRF, Durham, NC). Reactors were fed synthetic wastewater with or without 0.73 &muM of TCS. Samples were taken periodically to monitor chemical oxygen demand (COD), ammonium (NH₄<super>+</super>), nitrate (NO₃<super>-</super>), nitrite (NO<sub>2</super>-</super>), total suspended solids (TSS), volatile suspended solids (VSS), dissolved oxygen (DO), and phosphate (PO₄<super>3-</super>) and pH. In addition, biomass samples were collected for DNA extraction and microbial community analysis using terminal restriction fragment length polymorphism (T-RFLP) of 16S SSU rDNA. Methane production was also monitored for the anaerobic digesters. In addition, the final digested biosolids that were generated from the SBRs fed with and without TCS were analyzed for TCS concentration, TSS, VSS, TKN, phosphorus (as P₂O₅), potassium (as K₂O), and pH. Overall, biological processes associated with nitrogen removal (nitrification and denitrification), were impacted by TCS entering the SBRs regardless of the starting microbial community. Both of the SBRs that were not receiving TCS reached steady-state at greater than 92% NH₄<super>+</super>, removal within the first week of operation, whereas the SBRs receiving TCS took 42 and 63 days to reach steady-state removal at that level. However, while NH₄<super>+</super> removal was temporarily inhibited, elevated levels of NO₃<super>-</super> and NO₂<super>-</super> in the effluent of the TCS fed SBRs, suggested longer-term impacts on nitrite oxidizing bacteria (NOB) and denitrifiers. After Day 58, the NO₃<super>-</super> effluent concentration for the SBRs receiving TCS was 3.9 ± 0.16 mg/L, which was 2.4 times greater than the NO₃<super>-</super> effluent of the SBRs not receiving TCS (1.7 ± 0.08 mg/L). Similarly, after Day 58, the NO₂<super>-</super> effluent of the SBRs receiving TCS reached a steady-state concentration of 8.7 ± 0.75 mg/L. The mean NO₂<super>-</super> concentration in the controls after Day 58 was 7.7 times lower at 1.1 ± 0.78 mg/L, but was still trending towards 0 when the reactors were stopped. No inhibition was observed for COD and PO₄<super>3-</super> removal. In addition, non-metric multidimensional scaling (NMS) ordination analysis showed that the microbial communities between SBRS fed with and without TCS were similar on Day 0, but increased in difference to Day 41, around when the major changes in nitrification were observed. After a slight increase in similarity between the control and TCS SBR microbial communities on Day 41, the communities increased in difference to Day 63.</p><p>To mimic agricultural field conditions, containers of soil were amended with the biosolids generated from the SBRs. The containers were maintained in a growth-chamber to simulate field lighting and watering conditions. Three biosolids treatments were examined: 1) biosolids generated from the SBRs not fed TCS, but that still had low backgrounds of TCS (a.k.a., Control Biosolids); 2) biosolids generated from the SBRs fed with TCS (a.k.a., Aged TCS Biosolids); and 3) biosolids that were generated by the SBRs not fed TCS, but spiked with TCS 24 h before application (a.k.a., Spiked TCS Biosolids). Alfalfa was planted in half of the containers receiving the Control and Aged TCS Biosolids to assess differences due to vegetation. To assess the overall ecotoxicity of biosolids aged and spiked with TCS, the function, abundance, and diversity of the soil denitrifying communities were examined. The impacts on total bacteria abundance and diversity were also examined for comparison. Specifically, the denitrifying enzyme activity (DEA) assay was used to measure functional impacts, quantitative polymerase chain reaction (qPCR) was used to measure impacts on abundance, and T-RFLP was used to measure impacts on diversity. Correlations between these methods were also examined for possible interactions between denitrifier function and community structure and to provide insight into targets of inhibition. Lastly, a denitrification inhibition score was developed to quantify global impacts of TCS on denitrification. The containers with plants that received biosolids aged with and spiked with TCS showed potential long-term inhibition based on measurement of soil denitrification at 26.9 ± 4.6 &mu;g/kg and 68.6 ± 26.9 &mu;g/kg of TCS, respectively. Denitrifier abundance and diversity, however, were more sensitive to TCS in biosolids and inhibition was observed throughout the experiment, with maximum inhibition on Days 7 and 28. Inhibition of denitrifier abundance and diversity was observed at TCS concentrations as low as 17.9 ± 1.93 &mu;g/L, which was about 10 to 3000 times lower than concentrations reported by other studies that showed impacts on other functional endpoints (i.e., respiration, phosphatase activity, NO₃<super>-</super> and NO₂<super>-</super> production, and Cy17 stress biomarker abundance), even after taking pH into account. Five significant correlations were developed, three of which related qPCR and the DEA assay, or abundance and activity. However, the analyses that were correlated did not yield the same results as far as significant inhibition in the presence of TCS. Thus, while the results suggested some relatedness between activity, abundance, and diversity, the results generally support the use of multiple methods to determine the ecotoxicity of biosolids-derived organic contaminants. As a result, a denitrification inhibition score was developed that took into account all three methods to determine the overall ecotoxicity of TCS in biosolids. Overall, the denitrification inhibition score showed that denitrification was inhibited by both biosolids that were aged and spiked with TCS over the extent of the 84 day experiment, but maximum inhibition occurred after a week to about a month. While the denitrification inhibition score indicated that the TCS in the biosolids aged with TCS was less bioavailable than in the spiked biosolids, the impacts of the aged and spiked biosolids could have also been due to differences in TCS concentrations.</p><p>Objective 5 consisted of a long-term soil sampling campaign on four agricultural fields receiving Class B municipal biosolids. Soil samples were taken before and after biosolids application and were analyzed to elucidate potential impacts of TCS in the biosolids on denitrification. Again, to assess the overall impacts of TCS on the soil denitrifying community, the DEA assay, qPCR, and T-RFLP were used to measure impacts on function, abundance, and diversity, respectively. Similar to Objective 4, the analysis included an examination of potential correlations between denitrifying community structure and function, and quantification of global impacts using the denitrification inhibition score. As expected, the results in this pilot-study reflected the complexity of the system that was analyzed and many more samples, which account for variables including, but not limited to soil characteristics, biosolids characteristics, biosolids application rates, and chemical composition and quantities, would be needed to show any statistically significant differences. Nevertheless, several key results were obtained. Again potential long-term inhibition of denitrification was observed using the DEA assay, however the effects of exhaustion of resources, such as NO₃<super>-</super>, or significant changes in the local environment were suspected, but could not be verified. Inhibition was also observed for denitrifier abundance, but little to no inhibition was observed when examining the relative number of denitrifying species. Thus, while the abundance of denitrifiers was reduced, and denitrification was eventually depressed, the number of species in the soil remained constant. When looking at the denitrification inhibition score, which took all three measurements into account, increased inhibition over time was observed with the exception of the measurements on Days 30 and 103, which indicated overall, but weak inhibition of denitrification by the application of biosolids. NMS ordinations showed no correlation between the shift in denitrifying microbial community and TCS. Because of the complexity of the soil and biosolids and because of the myriad of contaminants likely in the biosolids, the results may not be significant and a more in-depth study was recommended.</p><p>Overall, the results presented in this dissertation provide a systematic evaluation of the effects of biosolids-derived TCS on agricultural soil microbial ecology. First, it was demonstrated that statistically significant inhibition of denitrification could be used as a potential indicator of biosolids-derived emerging organic contaminant ecotoxicity. The denitrification assay that was developed was then used to analyze ecotoxicological potential of six emerging biosolids-derived antimicrobial agents, and found inhibition of denitrification at environmentally relevant concentrations. The most widely used antimicrobial agent, TCS, was further shown to inhibit wastewater treatment processes, as well as, denitrification in simulated agricultural conditions after being aged with and spiked into biosolids. In addition, evidence showing potential inhibition of denitrification by TCS in `traditional' biosolids under field conditions was also obtained. Based on these results, this dissertation asserts that biosolids-derived emerging organic contaminants pose a potential risk to agricultural soil microbial ecology and overall soil health. Future studies, however, are needed to examine the impacts of other contaminants that might be flagged with the assay developed in this dissertation under more complex conditions mimicking the environment. Furthermore, other research is needed to examine the role microbial communities play in the bioavailability of emerging contaminants, especially TCS, and a more extensive, in-depth study is needed to characterize the individual impacts of emerging contaminants on soil microbial communities under field conditions.</p> / Dissertation

Amphibians are particularly sensitive to environmental degradation and, therefore, serve as effective environmental quality indicators. Research has suggested that amphibian declines are exacerbated by manmade environmental toxicants, especially those found in high concentrations in urban areas. The NIH has pinpointed genotoxicity as a major route of cancer causation, and has since developed stringent testing procedures for potentially hazardous chemicals. One such method, recognized for its simplicity and economy, is the micronucleus assay. A study was conducted assessing the genotoxicity of the widely used antimicrobial agent Triclosan to American Bullfrog tadpoles. Lithobates catesbeianus tadpoles were reared in glass aquaria containing ultra-high purity water and were dosed with nominal concentrations of 2.3 µg/L, 23 µg/L, and 230 µg/L Triclosan, reflecting 1x, 10x, and 100x concentrations of the compound as found in US surface waters. Eight replicates of each of the three levels of Triclosan contamination were prepared, as well as eight replicates per control group. Each replicate contained three tadpoles in a glass aquarium, from which one tadpole per tank was sampled after 1, 8, or 15 days following initial exposure to test compounds. Erythrocytes were prepared on slides and scored for micronucleus presence under 1000x magnification. Triclosan induced significant micronucleus formation after only 24 hours in all treatments relative to the negative control and exhibited a maximum of 15 micronuclei per 2,000 erythrocytes scored. Modeling of MN induction dynamics by treatment suggested that the best predictor of micronucleus induction was the acute TCS exposure level, as described by a linear mixed effects model including a binomial term of time exposed. Micronucleus induction was TCS concentration dose-dependent. This study supports that Triclosan induces significant genetic damage at environmentally relevant concentrations. It is clear that the effects of genotoxic agents must be certified so proper regulatory protocols can be developed and enforced, in order to conserve wildlife and promote human health.

O objetivo da presente tese foi desenvolver um adesivo com nanocápsulas (NCs), contendo indometacina, e um sistema adesivo com nanocápsulas (NC), contendo indometacina e triclosan, e avaliar suas propriedades. As NCs foram produzidas por meio do método de deposição de polímero, secas e caracterizadas quanto ao tamanho de partículas, à forma, quantidade de fármaco encapsulado e citotoxicidade. Uma resina adesiva foi formulada. Foram adicionadas ao adesivo 1%, 2%, 5% e 10% de NCs em massa, e um grupo permaneceu sem NC. As NCs, contendo indometacina e triclosan, foram também incorporadas no primer comercial a 2% em peso e um grupo permaneceu sem NCs. Os adesivos foram avaliados quanto ao GC imediato e tardio, à degradação em solvente, liberação dos fármacos, difusão de indometacina pela dentina e resistência de união. O adesivo com 10% de NCs, contendo indometacina, foi também avaliado quanto à ação anti-inflamatória em modelo animal. O primer e o adesivo com as diferentes concentrações de NCs, contendo indometacina e triclosan, foram avaliados quanto à liberação dos fármacos, difusão de indometacina pela dentina, ao efeito antimicrobiano, grau de conversão (GC) in situ, ângulo de contato e à resistência de união à microtração. Os dados foram analisados por ANOVA, Tukey e teste t. As NCs apresentaram forma esférica e viabilidade celular acima de 80%. As NCs, contendo indometacina, apresentaram diâmetro médio de 165 nm e as NCs, contendo indometacina e triclosan, 159 nm. O adesivo, contendo 10% de NCs com indometacina, apresentou efeito anti-inflamatório. A incorporação de NCs não alterou o GC, que variou de 63,63 ± 1,01% a 70,50 ± 2,08%. A degradação em solvente não foi alterada com 2% de NCs. Tanto os adesivos quanto o primer apresentaram liberação controlada. A indometacina permeou através da dentina. O adesivo e primer também apresentaram efeito antimicrobiano. A incorporação de NCs no primer e no adesivo não influenciou o GC in situ nem a resistência de união imediata, em comparação aos materiais sem NCs. O uso concomitante do primer e adesivo com NCs aumentou o ângulo de contato e diminuiu a resistência de união longitudinal. Conclui-se que o uso do adesivo com a incorporação de NCs tem potencial para proporcionar ações terapêuticas à adesão dentinária. / The aim of the present thesis was to develop an adhesive with nanocapsules (NCs) containing indomethacin and an adhesive system with nanocapsules containing indomethacin and triclosan and to evaluate their properties. NCs were prepared by the interfacial deposition of preformed polymer technique, dried and characterized regarding particle size, encapsulated drug content and cytotoxicity. Adhesive resin was produced. Concentrations of 1%, 2%, 5% and 10% of NCs were added in the adhesive and a group was maintained with no NCs. Indomethacin and triclosan-loaded NCs were also incorporated into a commercial primer in a concentration of 2% and a group was maintained with no NCs. Adhesives were evaluated regarding immediate and late degree of conversion (DC), degradation in solvent, drug release, indomethacin diffusion through dentin and bond strength. The adhesive with 10% of NCs containing indomethacin was also evaluated regarding the anti-inflammatory effect in an animal model. Primer and adhesive with different concentrations of NCs containing indomethacin and triclosan were evaluated regarding drug release, indomethacin diffusion through dentin, antimicrobial effect, in situ degree of conversion, contact angle and bond strength. Data were analyzed through ANOVA, Tukey post-hoc and t-test.NCs presented a spherical shape and cell viability higher than 80%. NCs containing indomethacin presented an averaged size of 165 nm and NCs containing indomethacin and triclosan, 159 nm. The adhesive with 10% of NCs containing indomethacin presented anti-inflammatory effect. The incorporation of NCs presented no alteration of DC, varying from 63.63 ± 1.01% a 70.50 ± 2.08%. Degradation in solvent suffers no influence of NCs with 2% of NCs. Adhesives and primer presented controlled drug release. Indomethacin diffused through dentin. Adhesive and primer also presented an antimicrobial effect. The incorporation of NCs in adhesive and primer showed no influence on in situ DC and immediate bond strength compared to materials with no NCs. The use in combination of primer and adhesive with NCs resulted in higher contact angle and lower longitudinal bond strength. It is possible to conclude that the use adhesive with incorporation of NCs has potential to provide therapeutic effects on dentin adhesion.

I avloppsvattnet förekommer både naturliga och antropogena ämnen som kan orsaka en negativ miljöpåverkan, såsom eutrofiering och en syrefattig recipient. Förekomsten av näringsämnen och organiskt material kan också vara en värdefull resurs, för det slam som återstår efter reningsstegen i avloppsreningsverket kan användas för att höja näringshalten i till exempel skogar och på åkermark. För att minska risken för skador på reningsprocessen, en negativ miljöpåverkan på recipienten genom skadliga ämnen som passerar genom avloppsreningsverket samt en negativ miljöpåverkan på grund av ett förorenat slam är det betydelsefullt att fokusera på mängden skadliga ämnen som förs till anläggningen. Syftet med examensarbetet är att kartlägga förekomsten av hushållskemikalier hos sex hushåll i Västerås som är anslutna till det kommunala reningsverket, studera befintlig litteratur som beskriver vad som sker med utvalda kemikalier då de når avloppsreningsverk i allmänhet, och sedan jämföra detta med Kungsängsverket. De sex hushållens sammanlagda antal produkter var 293 stycken, varav 66 var rengöringsprodukter, 16 tvättprodukter, 112 badrumsprodukter och 99 stycken var garageprodukter. Vid kommunala reningsverk är det främst kväveavskiljningen och den biologiska reningen som riskerar att ta skada vid förekomst av kemikalierna. Beståndsdelarna i färg visar en toxicitet för vattenlevande organismer, vilket även parabener och zinkpyrition gör. Triclosan är toxisk för vattenlevande organismer, akutgiftig för däggdjur och kan leda till mikrobiell resistens. Fosforföreningar omvandlas till fosfater i vattenmiljöer och ökar risken för eutrofiering.

A series of small molecules was synthesized to probe three protein targets in order to elucidate the key small molecule-protein interactions required for potency. Triclosan is an antibacterial compound that has surfaced as a potential environmental hazard and is hypothesized to cause perturbations in the thyroid hormone response of frogs. Using a C-fin assay and a GH3 cell line, our work suggests that triclosan itself may not in fact be the cause of the observed endocrine disruptions. Instead, methyl triclosan (a result of biological methylation during waste water treatment) was shown to disrupt the thyroid hormone response in tadpoles. Secondly, a set of probes was designed based on a cyclopentane scaffold derived from the known neuraminidase inhibitor peramivir. Kinetic assays using both a recombinant neuraminidase protein and an inactivated sample of influenza virus showed that the guanidine group contributes a 10 fold increase in potency while the α-hydroxyl group was observed to have little to no effect. This result suggests that future neuraminidase drug design based on a cyclopentane scaffold may forgo the use of both the guanidinium group and the hydroxyl group to potentially increase the oral availability of these drugs while sacrificing little in the way of potency. Finally, a series of truncated analogues related to the western half of the natural product didemnaketal A was synthesized. These compounds will be used as probes to better understand the mechanism of didemnaketal-mediated protease inhibition. It is hypothesized that a more rigid structure (due to molecular gearing enforced by the presence of additional methyl groups, relative to previously examined analogues) will increase the potency of these molecules toward HIV-1 protease and may lead to new information for designing next-generation dissociative inhibitors. Work was also begun toward the total synthesis of the natural product itself. / Graduate