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Abstract Application of Argon Plasma Technology to Hydrophobic and Hydrophilic Microdroplet Generation in PDMS Microfluidic Devices Brennan Graham Microfluidics has gained popularity over the last decade due to the ability to replace many large, expensive laboratory processes with small handheld chips with a higher throughput due to the small channel dimensions [1]. Droplet microfluidics is the field of fluid manipulation that takes advantage of two immiscible fluids to create droplets from the geometry of the microchannels. This project includes the design of a microfluidic device that applies the results of an argon plasma surface treatment to polydimethylsiloxane (PDMS) to successfully produce both hydrophobic and hydrophilic surfaces to create oil in water (O/W) and water in oil (W/O) microdroplets. If an argon plasma surface treatment renders the surface of PDMS hydrophilic, then O/W microdroplets can be created and integrated into a larger microdroplet emulsion device. The major aims of this project include: (1) validating previously established Cal Poly lab protocols to produce W/O droplets in hydrophobic PDMS microdroplet generators (2) creating hydrophilic PDMS microdroplet generators (3) making oil in water droplets in hydrophilic PDMS microdroplet generators (4) designing a multilayer microfluidic device to transfer W/O droplets to a second hydrophilic PDMS microdroplet generator v W/O droplets were successfully created and transferred to a second hydrophilic PDMS device. The hydrophilic PDMS device also produced O/W droplets in separate testing from the multilayered microfluidic PDMS device. The ultimate purpose of this project is to create a multilayer microdroplet generator that produces water in oil in water (W/O/W) microdroplet emulsions through a stacked device design that can be used in diagnostic microdroplet applications. Thesis Supervisor: Dave Clague Title: Professor of Biomedical Engineering

Dewatering fine coal is needed in many applications but has remained a great challenge. The hydrophobic-hydrophilic separation (HHS) method is a powerful technology to address this problem. However, organic solvents in solvent-coal slurries produced during HHS must be recovered for the method to be economically viable. Here, the experimental studies of recovering solvents from pentane-coal and hexane-coal slurries by combining liquid-solid filtration and in-situ vaporization and removing the solvent by a carrier gas (i.e., drying) are reported. The filtration behaviors are studied under different solid mass loading and filtration pressure. It is shown that using pressure filtration driven by 20 psig nitrogen, over 95% of solvents by mass in the slurries can be recovered, and filtration cakes can be formed in 60 s. The drying behavior was studied using nitrogen and steam at different temperatures and pressures. It is shown that residual solvents in filtration cakes can be reduced below 1400 ppm within 10 s by 15 psig steam superheated to 150C, while other parameter combinations are far less effective in removing solvents. Physical processes involved in drying and the structure of solvent-laden filtration cakes are analyzed in light of these results. / Master of Science / Coal particles below a certain size are discarded to waste tailing ponds as there is no economically viable method for processing them. However, a new process called hydrophobic-hydrophilic separation offers a solution to this problem. A hydrophobic solvent is used to displace water from a coal-water slurry, and it is then easier and cheaper to filter and dry this new coal-solvent slurry. In this work experimental studies of recovering solvents from pentane-coal and hexane-coal slurries by combining filtration and drying are reported. The filtration behaviors are studied under different solid mass loading and filtration pressures. It is shown that using pressure filtration driven by 20 psig nitrogen, over 95% of solvents by mass in the slurry can be recovered, and filtration cakes can be formed in 60 s. The drying behavior was studied using nitrogen and steam at different temperatures and pressures to evaporate any remaining solvents. It is shown that the remaining solvents in filtration cakes can be reduced below 1400 ppm within 10 s by using 15 psig steam superheated to 150C as a drying medium, while other parameter combinations are far less effective in removing solvents. Physical processes involved in drying and the structure of solvent-laden filtration cakes are analyzed in light of these results.

High performance engineering materials, poly(arylene ether)s, having very good mechanical properties, excellent oxidative and hydrolytic stability are promising candidates for alternative materials used in the field of Proton Exchange Membrane Fuel Cells (PEMFCs) and Reverse Osmosis (RO) applications. In particular, wholly aromatic sulfonated poly(arylene ether sulfone)s are of considerable interest in the field of PEMFCs and RO, due to their affordability, high Tg, and the ease of sulfonation. Proton exchange membrane fuels cells (PEMFCs) are one of the primary alternate source of energy. A Proton exchange membrane (PEM) is one of the key component in a PEMFC and it needs to have good proton conductivity under partially humidified conditions. One of the strategies to increase proton conductivity under partially RH conditions is to synthesize hydrophobic-hydrophilic multiblock copolymers with high Ion exchange capacity (IEC) values to ensure sufficient ion channel size. In this thesis two multiblock systems were synthesized incorporating trisulfonated hydrophilic oligomers and were characterized in the first two chapters of the thesis. The first multiblock system incorporated a non-fluorinated biphenol-based hydrophobic block. The second study was focused on synthesizing a fluorinated benzonitrile-based hydrophobic block. A fluorinated monomer was incorporated with the aim to improve phase separation which might lead to increased performance under partially humidified conditions. The third study featured synthesis and characterization of a novel hydroquinone-based random copolymer system precursor, which after post-sulfonation, shall form mono-sulfonated polysulfone materials with potential applications in reverse osmosis. The ratio of the amount of hydroquinone incorporated in the copolymer were varied during the synthesis of the precursor to facilitate control over the post-sulfonation process. The simple and low cost process of post-sulfonating the random copolymer enables the precursor to be a promising material to be used in the reverse osmosis application. / Master of Science

Froth flotation has long been regarded as the best available technology for ultrafine particles separation. However, froth flotation has extreme deficiencies for recovering ultrafine particles that are less than 30-50 μm in size for coal and 10-20 μm for minerals. Furthermore, dewatering of flotation products is difficult and costly using currently available technologies. Due to these problems, coal and mineral fines are either lost to tailings streams inadvertently or discarded purposely prior to flotation. In light of this, researchers at Virginia Tech have developed a process called hydrophobic-hydrophilic separation (HHS), which is based originally on a concept known as dewatering by displacement (DbD). The process uses non-polar solvents (usually short-chain alkanes) to selectively displace water from particle surfaces and to agglomerate fine coal particles. The resulting agglomerates are subsequently broken (or destabilized) mechanically in the next stage of the process, whereby hydrophobic particles are dispersed in the oil phase and water droplets entrapped within the agglomerates coalesce and exit by gravity along with the hydrophilic particles dispersed in them. In the present work, further laboratory-scale tests have been conducted on various coal samples with the objective of commercial deployment of the HHS process. Test work has also been conducted to explore the possibility of using this process for the recovery of ultrafine minerals such as copper and rare earth minerals. Ultrafine streams produced less than 10% ash and moisture consistently, while coarse coal feed had no observable degradation to the HHS process. Middling coal samples were upgraded to high-value coal products when micronized by grinding. All coal samples performed better with the HHS process than with flotation in terms of separation efficiency. High-grade rare earth mineral concentrates were produced with the HHS process ranging from 600-2100 ppm of total rare earth elements, depending on the method and reagent. Additionally, the HHS process produced copper concentrates assaying greater than 30% Cu for both artificial and real feed samples, as well as, between 10-20% Cu for waste samples, which all performed better than flotation. / Master of Science / Froth flotation has long been regarded as the best available technology for separating fine particles. Due to limitations in particle size with froth flotation, and high downstream dewatering costs, a new process has been developed called the hydrophobic-hydrophilic separation (HHS) process. This process was originally based on a concept known as dewatering by displacement (DbD) which was developed by researchers at Virginia Tech in 1995. The process uses hydrocarbon oils, like pentane or heptane, to selectively collect hydrophobic particles, such as coal, for which it was originally developed. In coal preparation plants, a common practice is to purposefully discard the ultrafine stream that flotation cannot recover and has an increased dewatering cost. The HHS process can effectively recovery this waste stream and produce highgrade salable product, with significantly reduced cost of dewatering. In the work presented, laboratory-scale tests have been conducted on various coal samples with the objective of commercial deployment of the HHS process. In this respect, several varying plant streams have been tested apart from the traditional discard stream. Additionally, test work has expanded into mineral commodities such as copper and rare earth minerals. In this work, salable high-value coal products were achievable with the HHS process. Ultrafine streams consistently produced less than 10% ash and moisture. Coarse coal feeds had no observable degradation to the HHS process and were able to produce single digit ash and moisture values. Middling coal samples were upgraded to high-value coal products when micronized by grinding. All coal samples performed better with the HHS process than with flotation in terms of separation efficiency. High-grade rare earth mineral concentrates were produced with the HHS process ranging from 600- 2100 ppm of total rare earth elements depending on the method and reagent. Additionally, the HHS process produced copper concentrates assaying greater than 30% Cu for an artificial and feed samples, as well as, between 10-20% Cu for waste samples, which all performed better than flotation.

This thesis pertains to the processing of ultra-fine mineral particles and low rank coal using the hydrophobic--hydrophilic separation (HHS) method. Several explorative experimental tests have been carried out to study the effect of the various physical and chemical parameters on the HHS process. In this study, the HHS process has been employed to upgrade a chalcopyrite ore. A systematic experimental study on the effects of various physical and chemical parameters such as particle size, reagent dosage and reaction time on the separation efficiencies have been performed. For this, a copper rougher concentrate (assaying 15.9 %Cu) was wet ground and treated with a reagent to selectively hydrophobize the copper-bearing mineral (chalcopyrite), leaving the siliceous gangue minerals hydrophilic. The slurry was subjected to a high-shear agitation to selectively agglomerate the chalcopyrite and to leave the siliceous gangue dispersed in aqueous phase. The agglomerates were then separated from dispersed gangue minerals by screening and the agglomerates dispersed in a hydrophobic liquid (n-pentane) to liberate the water trapped in the agglomerates. The chalcopyrite dispersed in the hydrophobic liquid was separated from the medium to obtain a concentrate substantially free of gangue minerals and moisture. The copper recoveries were substantially higher than those obtained by flotation. The HHS process was also tested on ultrafine mono-sized silica beads. The results were superior to those obtained by flotation, particularly with ultrafine particles. The HHS process has also been tested successfully for upgrading subbituminous coals. Low-rank coals are not as hydrophobic as high-rank coals such as bituminous and anthracite coals. In the present work, a low-rank coal from Wyoming was hydrophobized with appropriate reagents and subjected to the HHS in a similar manner as described for processing copper. The results showed that the HHS process reduced the moisture substantially and increased the heating value up to 50% without heating the coal. Laboratory-scale tests conducted under different conditions, e.g., particle size, reagent type, reaction time, and pretreatments, showed promising results. Implementation for the HHS process for upgrading low-rank coals should help reduce CO2 emissions by improving combustion efficiencies. / Master of Science

Proton exchange membrane fuel cells (PEMFCs) have been extensively studied as clean, sustainable and efficient power sources for electric vehicles, and portable and residential power sources. As one of the key components in PEMFC system, proton exchange membranes (PEMs) act as the electrolyte that transfers protons from the anode to the cathode. The state-of-art commercial PEM materials are typically based on perfluorinated sulfonic acid containing ionomers (PFSAs), represented by DuPont's Nafion®. Despite their good chemical stability and proton conductivity at high relative humidity (RH) and low temperature, several major drawbacks have been observed on PFSAs, such as high cost, high fuel permeability, insufficient thermo-mechanical properties above 80°C, and low proton conductivity at low RH levels. Therefore the challenge lies in developing alternative PEMs which feature associated ionic domains at low hydration levels. Nanophase separated hydrophilic-hydrophobic block copolymer ionomers are believed to be desirable for this purpose Three series of hydrophobic/hydrophillic, partially fluorinated/sulfonated multiblock copolymers were synthesized and characterized in this thesis. The hydrophilic blocks were based upon the nucleophilic step polymerization of 3, 3′-disulfonated, 4, 4′-dichlorodiphenyl sulfone (SDCDPS) with an excess 4, 4′-biphenol (BP) to afford phenoxide endgroups. The partially fluorinated hydrophobic blocks were largely based on 4, 4′-hexafluoroisopropylidenediphenol (6F-BPA) and various difluoro monomers (excess). These copolymers were obtained through moderate temperature (~130-150°C) coupling reactions, which minimize the ether-ether interchanges between hydrophobic and hydrophilic telechelic oligomers via a nucleophilic aromatic substitution mechanism. The copolymers were obtained in high molecular weights and were solvent cast into tough membranes, which had nanophase separated hydrophilic and hydrophobic regions. The performance and structure-property relationships of these materials were studied and compared to random copolymer systems. NMR results supported that the multiblock sequence had been achieved. They displayed superior proton conductivity, due to ionic, proton conducting channels formed through the self-assembly of the sulfonated blocks. The nano-phase separated morphologies of the copolymer membranes were studied and confirmed by transmission electron microscopy (TEM) and small angle X-ray scattering (SAXS). Through control of a variety of parameters, including ion exchange capacity and sequence lengths, performances as high, or even higher than those of the state-of-the-art PEM, Nafion®, were achieved. Another series of semi-crystalline hydrophobic poly(ether ether ketone)-hydrophilic sulfonated poly(arylene ether sulfone) (PEEK-BPSH100) multiblock copolymers was first synthesized and characterized. However due to their semi-crystalline structure, PEEK blocks are insoluble in most organic solvents at relatively low reaction temperatures, which prevents the coupling reaction between PEEK and BPS100. In order to facilitate the synthesis and processing, removable bulky ketimine was introduced to synthesize amorphous pre-oligomers poly(ether ether ketimine) (PEEKt). The synthetic procedure first involves the synthesis of hydrophobic poly(ether ether ketimine)-hydrophilic sulfonated poly(arylene ether sulfone) (PEEKt-BPS100) multiblock pre-copolymers via coupling reactions between phenoxide terminated hydrophilic BPS100 and fluorine terminated hydrophobic PEEKt blocks. The membranes cast from PEEKt-BPS100 were boiled in 0.5M sulfuric acid water solution to hydrolyze the amorphous PEEKt blocks to semi-crystalline PEEK blocks and acidify BPS100 blocks to BPSH100 blocks simultaneously. FT-IR spectra clearly showed the successful hydrolysis and acidification. The proton conductivity, water uptake and other membrane properties of the acidified semi-crystalline PEEK-BPSH100 membranes were then evaluated and compared with those of the state-of-the-art PEM, Nafion®. / Ph. D.

Dans ce travail, nous avons cherché à mettre en évidence l'influence de la méthode de préparation de films minces de polymère pour la biofonctionnalisation de surfaces planes. Dans un premier temps, un polymère réactif, le poly(méthacrylate de glycidyle) p(GMA) a été choisi et sa capacité de fixation vis-à-vis d'une biomolécule modèle l'albumine de sérum bovin a été étudiée. Deux stratégies principales de préparation du film polymère ont été utilisées : la technique du grafting onto et celle du grafting from avec deux voies de synthèse : la polymérisation radicalaire classique (PRC) avec l'amorceur en solution et la polymérisation initiée à partir de la surface avec un amorceur photochimique. Il a été montré que la méthode du « grafting from » permettait l'obtention de films d'épaisseur plus élevées que la technique du « grafting onto » avec une meilleure capacité de fixation de biomolécules de BSA. Ces films de p(GMA) se sont révélés relativement hydrophobes, ce qui nous incités à analyser l'influence de la balance hydrophobe/hydrophile des interfaces sur leurs propriétés, dans un second temps. Par la préparation de films copolymères poly(GMA-co-acrylamide) et poly(GMA-co-méthacrylate de glycérol) et la modification des films de poly(GMA) par de l'éthanolamine, l'influence de l'hydrophilie du film sur la capacité de fixation en molécules de BSA et l'activité de reconnaissance moléculaire de celles-ci ont été évaluées. Il a été démontré que par un choix judicieux de la méthode d'hydrophilisation du film polymère, il est possible de réduire considérablement l'adsorption non-spécifique de biomolécules d'où l'obtention de films polymères bioinertes. De plus, les résultats préliminaires ont montré qu'il est possible d'améliorer sensiblement la capacité de reconnaissance moléculaire entre la BSA et son anticorps l'anti-BSA / It is now well accepted that polymeric spacers permit to attach proteins to surfaces efficiently as they carry several binding sites. Moreover, direct attachment of proteins to surfaces might result in the decrease of bioactivity which is critical in the case of the development of biosensors. In this context, we modified gold substrates by polymer grafts via the so-called (i) grafting onto and (ii) grafting from strategies. In (i) preformed polymer chains were attached to the surface, whilst in (ii) surface-confined photopolymerization was performed on either acrylic monomer- or initiator-functionalized gold substrates. The polymer grafts were further biofunctionalized by covalent immobilization of an active protein (bovine serum albumin, BSA). Given the protein-polymer and polymer-gold covalent bonds, the gold/polymer/BSA hybrids permitted to design robust optical biosensors. The modified gold substrates were characterized in terms of chemical composition (X-ray photoelectron spectroscopy), hydrophobicity (contact angle measurements), and polymer coating thickness (surface plasmon resonance, SPR). SPR was also used to monitor in real time the interaction between the grafted antigen to the specific antibody (aBSA). Using unique reactive monomer, glycidyl methacrylate (GMA) in the present case, the implementation of these three methods is assumed to provide polymer films of similar chemical composition but varied interfacial chains conformation. In this respect, influence of polymer chains mobility on the performance of the immunosensing reaction was evaluated. In a further step, hydrophobic/hydrophilic balance of the polymer films was modulated through copolymerization of GMA with acrylamide, and with glycerol methacrylate. It was demonstrated that control over the surface chemical composition of the polymer grafts allows preparing bioinert films, i.e. resistant to non specific adsorption, with enhanced biospecific activity

Ce travail de thèse a porté sur l’étude des propriétés d’adsorption et de séparation en milieu humide par des polymères de coordination poreux. Nous avons utilisé les techniques de volumétrie d’adsorption, de séparation par chromatographie en phase gaz, de spectroscopie de fluorescence ainsi que et de la modélisation Monte Carlo afin de comprendre les mécanismes qui gouvernent l’adsorption de ces matériaux. Les molécules adsorbables étudiées sont l’eau et des vapeurs d’hydrocarbures linéaires (pentane et n-hexane), cycliques (cyclohexane et cyclohexène) et polycycliques (anthracène). Les adsorbants étudiés sont les Analogues de Bleu de Prusse (ABP) ainsi que deux matériaux organométalliques poreux de référence, le ZIF8 et le CuBTC. Parmi les résultats principaux apportés par ce travail, nous avons montré que les Analogues de Bleu de Prusse ont une stabilité hydrothermale remarquable leur conférant des propriétés d’adsorption en milieu humide très prometteuses. Parmi les ABP étudiés, le Co[CoIII(CN)6]0.66 ⊡0.33 .5.2H2O a montré des propriétés d’adsorption et de séparation très supérieures à celles d’autres ABPs. Par exemple, il a été possible de moduler sa balance hydrophobe/hydrophile par le contrôle de la coordination de l’eau sur le centre métallique insaturé, tout en préservant son caractère lipophile. Nous avons aussi montré que cet analogue de bleu de Prusse est capable de séparer des mélanges d’hydrocarbures secs ou très humides de façon répétée. Sur un aspect plus fondamental, nous avons montré que le confinement d’anthracène par le ZIF-8, qui est un composé organométallique poreux possédant des cavités de taille ajustée à celle du fluorophore, pouvait induire une extinction complète de certaines bandes d’absorption de celui-ci. / This PhD thesis is focused on the study of wet adsorption and separation properties by porous coordination polymers. We used adsorption volumetry, gas chromatography separation techniques, fluorescence spectroscopy techniques and Monte Carlo modeling to understand the mechanisms governing the adsorption of these materials. The adsorbable molecules studied are water and hydrocarbon vapors linear (pentane and n-hexane), cyclic (cyclohexane and cyclohexene) and polycyclic (anthracene). The adsorbents studied are the Prussian Blue Analogues (ABP) as well as two reference porous organometallic materials, ZIF8 and CuBTC. Among the main results provided by this work, we have shown that Prussian Blue Analogues have remarkable hydrothermal stability conferring them very promising wet adsorption properties. Among the ABPs studied, Co [CoIII (CN)6] 0.66 ⊡0.33 .5.2H2O showed adsorption and separation properties far superior to those of other ABPs. For example, it has been possible to modulate its hydrophobic / hydrophilic balance by controlling the coordination of water on the unsaturated metal center, while preserving its lipophilic character. We have also shown that this Prussian blue analogue is capable of separating mixtures of dry or very wet hydrocarbons repeatedly. On a more fundamental aspect, we have shown that the confinement of anthracene by ZIF-8, which is a porous organometallic compound with cavities of size adjusted to that of the fluorophore, could induce a complete extinction of certain absorption bands of this one.

Ce doctorat s’intègre dans le projet ANR BIOMET (JC05_59809) et a eu pour vocation d’améliorer les connaissances actuelles sur l’influence de la matière organique dissoute sur la spéciation et la biodisponibilité des métaux particulièrement dans le cas de système sous forte pression urbaine tel que la Seine. La compréhension de l’influence de la MOD sur la spéciation et la biodisponibilité des métaux suppose en outre que l’on connaisse précisément sa composition chimique ou plus exactement ses groupements fonctionnels. C’est pourquoi une partie de ces travaux est consacrée à l’extraction et la caractérisation de la matière organique dissoutes d’origine naturelle et urbaine. La caractérisation des fractions de MOD a été réalisée de manière originale par une approche multidimensionnelle à l’aide d’un véritable éventail de techniques analytiques (analyses élémentaires; isotopiques; fonctionnelles et moléculaires) permettant ainsi de mieux aborder l’influence de la MOD sur la spéciation et la biodisponibilité du cuivre et du plomb. Parallèlement à l’étude de caractérisation de la MOD, ce travail s’attache à mieux cerner le rôle de la MOD d’origine urbaine en particulier de la MOD hydrophile sur la spéciation et la biodisponibilité du cuivre et du plomb. Dans cet objectif, les techniques : potentiométrique à l’aide d’électrode ionique spécifique et la récente technique électrochimique AGNES ainsi que des biotests de toxicité aiguë (Daphnia magna) et de bioaccumulation (Fontinalis antipyretica) ont permis : - d’étudier la complexation du cuivre et du plomb par les fractions de MOD obtenues afin notamment de fournir des paramètres de complexations des MOD hydrophiles peu connues jusqu’à présent et ainsi mieux prédire le transport des métaux en milieu urbain. - d’évaluer le rôle protecteur de la MOD d’origine urbaine sur la biodisponibilité du cuivre. Les résultats ont montré que la MOD issue des effluents de station d’épuration et rejetée dans le milieu récepteur présente des caractéristiques originales à savoir un fort pourcentage de MOD hydrophile, c’est à dire une faible hydrophobicité et un degré d’aromaticité peu marqué soulignant le caractère peu réfractaire de cette MOD. En revanche une plus grande diversité fonctionnelle de la MOD d’origine urbaine par rapport à la MOD naturelle a été mise en évidence notamment par un taux de structures protéiques très important. Les résultats obtenus ici pour la première fois vis-à-vis des MOD hydrophiles d’origine urbaine ont montré une réactivité particulière vis-à-vis du cuivre. En effet les fractions HPI et plus généralement les fractions issues des effluents de station d’épuration ont montré une teneur en sites complexants plus élevée que ce qu’il avait été déjà observé pour des matières organiques naturelles. Les valeurs des constantes ne semblent pas varier d’une fraction à l’autre et assez peu suivant l’origine de la MOD. Le rôle particulier de l’azote dans la complexation du cuivre notamment sous la forme de groupements amines a été mis en évidence. Les approches utilisées pour évaluer la biodisponibilité du cuivre ont révélé pour l’ensemble des fractions de MOD un effet protecteur vis-à-vis du cuivre sur les organismes vivants. Néanmoins cet effet protecteur ne se trouve pas à la hauteur de celui pouvant être prédit par le modèle de l’ion libre principalement dû à une biodisponibilité de certains complexes organiques. Ceci peut être expliqué par l’important taux de structures protéiques dans ces MOD, les protéines étant connues pour jouer un rôle important dans les mécanismes de transports des métaux au sein des organismes. Par ailleurs, l’outil DGT (Diffusive gradient in thin films) a montré son efficacité pour évaluer la fraction biodisponible du cuivre / This thesis was carried out on the framework of the ANR BIOMET research project (JC05_59809). The main objectives were to improve the current knowledge on the influence of dissolved organic matter (DOM) on metal speciation and bioavailability in urban aquatic system. The understanding of the influence of DOM on trace metal speciation and bioavailability required that the chemical composition of DOM, more exactly its binding sites should be well understood. Therefore, one part of this research focused on the isolation and characterization of DOM. The characterization of DOM, with a multidimensional approach was realized with a lot of various techniques (such as elemental, isotopic, functional and molecular) that were of prime importance to better understand the influence of DOM on trace metal speciation and bioavailability. To accomplish these objectives, the techniques such as potentiometry with ionic selective electrode and the recent electroanalytical technique AGNES and bioassays such as a bioaccumulation test (Fontinalis antipyretica) and an acute toxicity test (Daphnia magna) were performed in order to: - study the copper and lead binding by isolated DOM fractions in order to give some binding parameters of hydrophilic DOM and to better predict the fate of the trace metal. - evaluate the protective role of urban DOM on copper bioavailability. Results showed some particular characteristics of DOM from wastewater effluent such as a high proportion of hydrophilic DOM, i.e. a low hydrophobicity and a low degree of aromaticity underlying the low refractory character of urban DOM. Nevertheless a higher content of various functional groups was determined in urban DOM than in natural DOM. A very high content of proteinaceous structures was particularly identified in urban DOM. Binding experiments revealed for the first time on urban hydrophilic DOM, a higher content of binding sites than in natural DOM and these sites are strongly correlated to amino groups. The values of binding constant seem to not vary according to the nature of the fraction or the origin. The different approaches used to evaluate copper bioavailability depicted a protective role of DOM to the organisms Daphnia magna and Fontinalis antipyretica. Nevertheless this protective effect is hardly explained by the free ion concentration due to a bioavailability of some organic complexes. This feature could be explain with the high content of proteinaceous structures in these DOM fractions, since proteins are well know to play an important role in the transport mechanisms of trace metals into the organisms. Moreover, the DGT device (diffusive gradient in thin films) shows a good efficiency to assess the bioavailable copper