Tesi sul tema "Antifouling"

Les polyanhydrides sont des polymères biodégradables largement utilisés principalement dans le milieu biomédical. La biocompatibilité des produits de dégradation ainsi que leur érosion de surface en font de bons candidats pour la formulation de peintures antifouling. Mais, leur faible solubilité et leur haute température de fusion limite les applications dans le domaine environnemental. Une nouvelle stratégie de formulation à partir d’oligomères a donc été mise au point. Cette étude a consisté en la conception et la caractérisation de films à partir d’oligomères de polyanhydrides. La cohésion des films est maintenue à partir de liaisons faibles. De plus, une influence de la polarité et de la volatilité du solvant de formulation sur la nature et la force des liaisons faibles a été constatée. La mise en évidence de ces interactions a été réalisées à partir de techniques complémentaires telles que la viscosimetrie capillaire, la spectroscopie infra-rouge, la microscopie à lumière polarisée ainsi que la diffraction de rayons X. L’effet de ces interactions, ainsi que l’influence de facteurs environnementaux (pH, salinité), sur la dégradation et l’érosion des films, à également été étudié. Leur comportement a été analysé en immersion à l’aide de la coulométrie Karl Fisher, chromatographie liquide haute performance, microscopie électronique à balayage, microscopie confocale à balayage laser. Bien qu’un effet des liaisons sur la cinétique d’érosion ait été constaté, les polyanhydrides sont trop erodables pour être utilisé comme liant des peintures antifouling
Polyanhydrides are well-known biodegradable polymers. Their surface-eroding properties in aqueous media make them desirable for the controlled release of bioactive molecules as drugs and functional tissue substitutes. Their main advantages are the adjustability of degradation and release rates, the zero order kinetics of release and biocompatibility. However, their low solubility in common organic solvents and their high melting points limit their potential applications, especially in environmental fields. This is the reason why new strategies of formulation need to be considered to enlarge the use of polyanhydrides and overcome their main disadvantages. The purpose of this study was to evaluate the potential of anhydride oligomers as film-forming matrices. The use of oligomers cleared up many difficulties of synthesis (which is long, extended and tedious), stability and solubilization. Covalent bonds were replaced by weak interactions to preserve cohesion between molecules and obtain film properties. Coating characterization was carried out by nuclear magnetic resonance and electrospray ionization to obtain chemical structures of the products. The formation of weak interactions, which confer cohesion between chains, allowed film properties to be observed. The impact of the solvent polarity on the specific organization was investigated by a combination of focused methods: capillary viscosimetry, IR spectroscopy, polarized light microscopy and x-ray diffraction. Oligomer coating being designed to using in marine environment, hydration and erosion studies was performed. Water uptake was carried out from Karl Fischer Coulometry with different water condition (pH and salinity). Hydration was also studied from CLSM and SEM to evidence the erosion zone. Finally degradation was carried out from weight loss, HPLC with the monomer release and from SEM with the observation of coating surface. Although an effect of interaction was observed on erosion kinetic, polyanhydrides are so erodible to antifouling paint application

Biofouling of marine surfaces is an age-old problem that affects natural and man-made surfaces exposed to the aquatic environment. The tenacious attachment of seaweed and invertebrates to man-made surfaces, notably on ship hulls, has incurred undesirable economic losses. The initial stage of the biofouling process has been attributed to the attachment of marine bacteria and their subsequent formation of biofilm which attract the settlement of larger sessile organisms including barnacles and seaweed. Silver nanostructured materials have a well-documented history as antimicrobial agents against pathogenic bacteria due to their ability to penetrate cell walls and interfere with crucial cellular processes. However, there is a surprising lack of information on their activity against marine biofilm bacteria that have critical roles in the initiation of marine fouling processes. This PhD project explores the antifouling properties of novel silver nanocomposite materials as potent antifouling agents against targeted organisms present in marine environments. The study consists of the syntheses of novel silver nanocomposite materials using various templates/matrices such as ion-exchange polymeric microspheres, zeolites, TiO2 nanotubes and graphene nanosheets. These materials were characterized through various instrumentation techniques including scanning electron microscopy (SEM), energy dispersive X-ray spectrometry (EDX), X-ray powder diffraction (XRD), UV-visible (UV-vis) spectrophotometry, transmission electron microscopy (TEM), accelerated surface area porosimetry (ASAP), thermal gravimetric analysis (TGA), and Raman spectroscopy to elucidate their physical properties. Their antifouling effects were evaluated on Halomonas pacifica, a model marine microfouling bacterium, through an established static biofilm assay. In addition, the biological effects of these silver nanocomposites were also studied on marine microalgae Dunaliella tertiolecta and Isochrysis sp. Silver-polymer nanocomposite (Ag-PNC) microspheres were formed through a rapid chemical synthesis procedure at room temperature via the reduction of silver nitrate by sodium borohydride. The introduction of Ag nanoparticles (AgNPs) enhanced the thermal stability of the Dowex microspheres by shifting the glass transition temperature to above 300°C and the material decomposition occurred above 460°C. XRD analysis confirmed the presence of metallic Ag, while UV-vis absorption studies showed the characteristic surface plasmon resonance (SPR) for AgNPs ranging from 406 – 422 nm maximum absorption wavelengths. SEM imaging revealed the uniform distribution of AgNPs with diameters between 20 – 60 nm on the surface of the microbeads. The Ag-PNC materials, diluted to a concentration of 1 mg/mL in marine broth, showed a potent inhibitory effect on H. pacifica biofilm formation, with up to 76% decrease of biofilm when contrasted with the polymeric microspheres without Ag. Ag-PNCs also caused significant growth inhibition of D. tertiolecta and Isochrysis sp. Silver-zeolite nanocomposite clusters (AgZ) were formed through a low temperature chemical reduction method using the environmentally friendly trisodium citrate. The stable and porous inner structure of ZSM-5 zeolites performed a dual role as a stable size-control template and a reservoir of antimicrobial nanosilver. SEM revealed the globular and cluster-like morphology of the AgZ composites, with a homogenous distribution of silver particles on the surface of the clusters. EDX results displayed an increasing Ag loading with higher concentrations of Ag precursor, up to 10 wt% Ag. The UV-visible absorption displayed the characteristic SPR absorption maximum ranging from 408 – 500 nm. The AgZ clusters with metallic silver loading of up to 10 wt% Ag, diluted to a concentration of 1 mg/mL, reduced H. pacifica biofilm attachment of up to 81% compared to pure zeolite alone. XRD analysis clearly indicated the presence of metallic Ag while the ZSM-5 zeolite crystalline framework remained largely intact after the Ag crystal growth process. Brunauer-Emmett-Teller (BET) analysis showed a reduction in surface area of up to 44% with the incorporation of AgNPs into the zeolite, indicating the formation and growth of Ag within the internal pores and channels of the zeolite. Although the introduction and crystal growth of silver nanoparticles within the porous structure of the zeolite caused a change from a mesoporous to a largely macroporous structure, the integrity of the zeolite template was preserved. Silver-titania nanotube (Ag/TNT) composite material was prepared through a novel 2-step hydrothermal synthesis method. Titania nanotubes were chosen as a support material for the AgNPs as its greater specific surface area on the inner and outer surfaces of its tubular structure lead to enhanced properties. The morphology, particle size, chemical content, crystal structure, optical properties and surface area were systematically characterized. Determination of biofilm inhibitory properties revealed that Ag/TNT (concentration of 0.1 mg/mL) with the lowest silver content (0.95 wt% Ag) decorated with AgNPs of approximately 3 nm reduced biofilm formation of H. pacifica by 98% compared to pure titania nanotubes and bulk silver alone. Growth inhibition of D. tertiolecta and Isochrysis sp. were also observed. Interestingly, the antifouling properties were improved with a size decrease of AgNPs. The work shows that titania nanotubes are a stable and effective support for the anchoring and growth of AgNPs. The addition of very low amounts of Ag enhanced the antifouling property of pure TiO2 to produce an extremely potent antifouling effect on the targeted organisms. Graphene-Ag (GAg) nanocomposites were prepared from a novel and mild hydrothermal synthesis method which bypasses the formation of graphene oxide. The GAg nanocomposite combines the antimicrobial property of silver nanoparticles and the unique structure of graphene as a support material, with potent marine antifouling properties. The results show that GAg nanocomposites displayed significant biofilm inhibition property on H. pacifica and antiproliferative effects on D. tertiolecta and Isochrysis sp. As low as 1.3 wt% of Ag loading on a GAg sample, diluted to a concentration of 0.1 mg/mL, inhibited biofilm formation from H. pacifica. The GAg sample with 4.9 wt% Ag loading was associated with a biofilm inhibition of 99.6%. The marine antifouling properties of GAg nanocomposites were a synergy of the biocidal AgNPs anchored on the flexible graphene sheets, thereby providing maximum active contact surface areas to the target organisms. The GAg material was characterized with SEM, EDX, TEM, XRD and Raman spectroscopy. In addition, the GAg material exhibited the surface-enhanced Raman scattering (SERS) effect. The AgNPs were estimated to be between 72-86 nm, observed supported on micron-scaled graphene flakes. These results strongly suggest that the 4 types of silver-based nanocomposite materials are promising marine antifouling agents. The addition of very low amounts of Ag enhanced the antifouling property of the support structure, and the nanocomposites were shown to be more effective on the targeted organisms compared to the matrix material or bulk silver alone. In addition, the precursor materials used in the syntheses are affordable and easily available, whilst the synthetic methods and conditions are facile, environmentally friendly, and capable of producing high yields.

Industries (food, beverage, petrochemical, etc.) normally use various gravitational separation echniques in their processes. Such separation processes often suffer from the deposition of undesirable material on the active surfaces of the process equipment, e.g. a high-speed separator or decanter, causing a slew of problems with the process or product quality. To restore operational efficiencies, additional cleaning steps using both water and chemicals are required, making the process more expensive and less environmentally friendly. Other than operating time and concentration of the process fluid there are several factors such as surface nature, surface roughness, type of material, surface charge, etc which influence the fouling deposition of surfaces. Fouling on the surfaces can grow following different mechanisms. The goal of this research work is to learn more about the nature of foulant interactions with stainless steel surfaces and eventually design some antifouling methodology. It is too difficult to study foulingfor all kinds of solutions and industries, so we tried to investigate the organic deposition in dairy and brewery industries by using lab-scale synthesized milk and beer solutions, For quantitative and statistical examination of these characteristics, several experimental approaches (FTIR, percent weight change, surface roughness, surface energy) were used. It was confirmed that fouling grows on the surfaces in a non-linear fashion irrespective of the time and concentration of the solution. The fouling of surfaces can be improved by producing more hydrophilic surfaces or by reducing surface roughness. Steric hindrance, electrostatic charge, and water barrier or hydration layer theories can be used to modify the surface nature and hence the fouling deposition. For antifouling purposes, PMMA (organic) and tungsten oxide (inorganic) coatings were employed. The PMMA was deposited using a dip-coating technique using (6%,10%, and 12%) PMMA solution, and the tungsten oxide coating was carried out by using a standard two electrode electrochemical system under different voltage (3.5V and 4.5V) and time (5min, 10 min, and 20 min) conditions. The coatings were characterized by using different techniques and their antifouling effects were studied in model milk and model beer solutions
Vid industriella processer (livsmedel, petrokemisk etc.) används ofta olika tekniker för separation med hjälp av gravitation. Sådana separationsprocesser drabbas ofta av oönskade beläggningar och påväxt på processutrustningens aktiva ytor så som t.ex. i en separator eller en dekanter, vilket orsakar problem med processen eller produktkvaliteten. För att återställa driftseffektivitet krävs särskilda rengöringssteg med både vatten och kemikalier vilket gör processen dyrare och mindre miljövänlig. Förutom drifttid och processvätskans sammansättning finns det flera faktorer såsom ytbeskaffenhet, ytjämnhet, materialtyp, ytladdning m.m. som påverkar mängden oönskade beläggningar på ytor. Föroreningarna på ytor kan tillväxa med olika mekanismer. Målet med detta forskningsarbete är att studera interaktionen mellan olika former av påväxt och ytan på rostfritt stål och senare utforma metoder för att förhindra bildandet av sådana oönskade beläggningar. Det är en stor utmaning att studera olika typer av påväxt för alla typer av flöden och industrier. I studien undersöktes organisk påväxt inom mejeri- och bryggeriindustrin genom att använda syntetiserade mjölk- och ölprodukter i laboratorieskala, för kvantitativa och statistiska undersökningar av dessa egenskaper. Flera olika experimentella metoder användes (FTIR, viktförändring, ytjämnhet, ytenergi). Det bekräftades att tillväxten på ytorna var olinjärt oavsett tid och lösningens koncentration. Bildandet och tillväxt av oönskade beläggningar kan minskas med hjälp av mera hydrofila ytor eller genom att minska ytans ojämnhet. Steriska hinder, elektrostatisk laddning och vattenbarriär eller hydratiseringsskal kan användas för att modifiera ytan och därmed fördröja bildandet av oönskade beläggningar. För att förhindra påväxt belades ytan med PMMA (organisk) och volframoxid (oorganisk). PMMA deponerades genom en doppbeläggningsteknik med användning av (6%, 10% och 12%) PMMA-lösning och volframoxidbeläggningen utfördes med ett elektrokemiskt tvåelektrodssystem med olika spänningar (3,5V och 4,5V) och tider (5min, 10min och 20min). Ytbeläggningarna karakteriserades genom att använda olika tekniker och deras förmåga att förhindra snabb påväxt studerades i modellösningar av mjölk och öl.

The technique of tin-119m Mossbauer and NMR spectroscopy has been used to study the chemical and structural changes undergone by a series of triorganotin biocides when dispersed in an antifouling paint and a polyurethane system. In the antifouling paint, based on the Hypalon polymer, tri-n-butyltin chloride, tri-n-butyltin acetate and to-(triphenyltin) oxide are essentially unmodified. Bis-(tri-n-butyltin) oxide, in contrast, was entirely converted into two new species, one of which was identified as tri-n-butyltin chloride. The second component was thought to be a sulphonate ester. However, synthesis of such compounds and subsequent spectroscopic analysis did not support this theory. Triphenyltin acetate experienced some dephenylation when incorporated into the paint, with a diphenyltin species detected by Mossbauer spectroscopy and GC analysis. Triphenyltin chloride suffered more extensive dephenylation, the dried paint containing the mono- and diphenyltin species. The organotin release-rate of eight Hypalon paint samples has been determined using a flow system and analysis of the effluent by graphite furnace atomic absorption spectrometry. All eight samples were shown to have release-rates significantly less than that required for an effective antifouling coating. Incorporation of triorganotin biocides into the polyurethane resulted in physical changes to the polymer and in certain cases, chemical transformations in the organotin. to-(tri-n-butyltin) oxide is entirely converted into new species in the polyurethane. From work carried out on model systems, the likely products are N-stannylcarbamate derivatives, which are thought to be catalysts in polyurethane formation. It is also suggested that the extensive and undesirable foaming observed in such polyurethanes, is due to the formation of isocyanate oligomers which act as branch points for polymer cross-linking. Tri-n-butyltin chloride, to-(triphenyltin) oxide and triphenyltin chloride all catalyze the polyurethane reaction, the latter increasing the setting time rather than reducing it. Mossbauer and infrared evidence from model studies suggests that dative bonding between triorganotins and isocyanates is occuring, via the oxygen of the isocyanate group. It is these complexes which are proposed to be the catalytic species. The relative merits of the triorganotins are discussed with respect to their potential as effective antifouling agents in the polyurethane system.

The technique of tin-119m Mossbauer spectroscopy has been used to study the chemical and structural changes undergone by a series of triorganotin biocides when dispersed in neoprene-based marine antifouling elastomers. Chemical speciation using tin-119 nuclear magnetic resonance spectroscopy and gas chromatography, indicate that the majority of the tri-n-butyltin (TBT) biocides are converted to TBT-chloride, TBT-stearate and di-n-butyltin distearate during the polymer processing and curing conditions. Similar studies of triphenyltin (TPT)-containing elastomers demonstrate the extensive chemical degradation suffered by these compounds upon incorporation into the elastomer matrix. Spectroscopic and chromatographic analyses of a marine-exposed elastomer, originally containing bis(TBT)oxide (TBTO), revealed the presence of monobutyl- and dibutyltin compounds as degradation products. A "backscatter"Mossbauer technique showed that the ultimate degradation product, distributed in the near surface regions of the sample, was stannic oxide. The possible binding of organotin biocides with components of the coating formulation was investigated by variable temperature Mossbauer measurements on TBT-chloride systems. At temperatures below 60K, the organotin was shown to exist as a polymeric compound in which the tin atom is penta-coordinate. Application of the Debye model of solids yielded a value of the recoilless fraction at 80K, f(80K), of 0.30 and the vibrational freedom of the tin atom was isotropic. This associated structure was observed to break down upon dispersion into uncured neoprene. The Mossbauer parameters and f(80K) = 0.17 were consistent with the presence of discrete, teracoordinate molecules. In the authentic coating matrix, the Mossbauer parameters indicated a tetracoordinate geometry for the biocide. However, the large f(80K) = 0.34 is more typical of the pentacoordinate structure exhibited by pure TBT-chloride. The presence of TBT and TPT additives on the curing behaviour of the basic coating formulation was investigated and it was found that TBTO effectively delayed the onset of curing in this system. This effect was attributed to interfering reactions between the organotin and the accelerated zinc oxide cure system, and to the existence of a competing crosslinking reaction directly involving TBTO. The TPT biocides were seen to promote the onset of curing and this was attributed to the formation of tin-based Lewis acid catalysts during the chemical breakdown of the original biocide.

Unspecific interactions, at the interface between a synthetic material and an aqueous biological environment, leading to irreversible protein adsorption can cause to undesired consequences. These include fouling of a boat hull or a triggered immune response. Thus, stealthy materials are a topic that has generated a great deal of interest in the scientific community. This work deals with the design of networks, nanoparticles, and surfaces containing poly(ethylene glycol) (PEG), known for its resistance to protein adsorption and non-toxic nature. Initially, PEG-based networks, hydrogels, were synthesized using photoinduced thiol-ene chemistry in order to afford coatings targeted for marine antifouling applications. By varying the length of the PEG chain, curing chemistry, cross-linker as well as hydrolytical stability, a library of hydrogel coatings was produced. The coatings were subsequently characterized with respect to curing efficiency, thermal and mechanical properties, and aqueous stability. Furthermore, the antifouling properties of coatings were evaluated using in vitro tests with proteins, marine bacteria, and diatoms. As a final test the coatings were evaluated in a four month field test. It was found that coatings comprising longer PEG chains displayed enhanced antifouling performance, compared to shorter PEGs. In addition, the choice of cross-linker, curing chemistry, and hydrolytical stability also affected the properties to a great extent. This thesis further deals with the design of amphiphilic linear dendritic hybrids, with PEG as the hydrophilic block. Using non-toxic 2,2-bis(methylol) propionic acid (bis-MPA) based dendrons, bearing click functional cores (alkyne or allyl) and peripheral hydroxyl groups, as macrointitiators for ring-opening polymerization of ε-caprolactone, a library of star branched materials was afforded. As a final step, click functional (azide or thiol) PEGs were attached using copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) or thiol-ene click chemistry. The size of the dendrons was varied from generation 0-4, along with variation of both poly(ε-caprolactone) (PCL) length and PEG length. The materials were designed in order to allow a study of the impact of the dendron generation. Finally, the hybrid materials were used for the preparation of micelles, as well as for the formation of honeycomb membranes. The micelles critical micelle concentration, size and drug loading capacity were shown to be highly dependent on the generation of the dendron. The generation of the dendron also had a profound effect on the ability of the hybrid materials to form ordered honeycomb membranes, and hybrid materials of the 3rd generation yielded the most highly ordered membranes.
Ickespecifika interaktioner vid gränsytan, mellan ett syntetiskt material och en vattenbaserad biologisk miljö, kan leda till irreversibel adsorption av proteiner. Detta kan i sin tur leda till oönskade följdeffekter, såsom beväxning på båtskrov eller trigga en immunologisk reaktion. För att motverka dessa effekter har forskare utvecklat så kallade smygmaterial. Denna avhandling behandlar design av nätverk, nanopartiklar och ytor innehållande poly(etylenglykol) (PEG), som är känt för sina smygegenskaper och för att vara icke-toxiskt. Initialt behandlar avhandlingen PEG-baserade nätverk, hydrogeler, syntetiserade med fotoinitierad tiol-enekemi, för användning som beväxningsavvisande beläggningar för marina applikationer. Genom att variera olika parametrar, såsom längden på PEG-kedjan, härdningskemin, tvärbindaren samt den hydrolytiska stabiliteten, byggdes ett bibliotek av hydrogelbeläggningar upp. Hydrogelbeläggningarna karaktäriserades sedan med avseende på härdningseffektivitet, termiska och mekaniska egenskaper, samt hydrolytisk stabilitet. Vidare studerades beläggningarnas avvisande förmåga mot proteiner, bakterier samt kiselalger. Slutligen studerades ytbeläggningarna i ett fyra månader långt fälttest. Av testerna framgick att längre PEG-kedjor gav beläggningar med bättre avvisande förmåga. Dessutom framgick att valet av tvärbindare, härdningskemi samt hydrolytisk stabilitet var av betydelse för beläggningarnas effektivitet. Denna avhandling behandlar vidare design av amfifila linjära dendritiska hybridmaterial, med PEG som den hydrofila delen. Genom att använda icke-toxiska 2,2-bis(metylol)propionsyrabaserade dendroner, med en klickfunktionalitet i kärnan (alkyne eller allyl) och perifera hydroxylgrupper, som makroinitiatorer för ringöppningspolymerisation av ε-kaprolakton byggdes ett bibliotek av material upp. För att göra materialen amfifila, kopplades klickfunktionella PEG-kedjor (azid eller tiol) till kärnan med koppar(I)-katalyserad azid-alkyn cykloadditionskemi alternativt tiol-enekemi. Storleken på dendronerna varierades från generation 0-4, dessutom varierades längden på både poly(ε-kaprolakton)- och PEG-kedjorna. Materialen designades så att inverkan av dendrongenerationen kunde studeras. Slutligen användes dessa hybridmaterial för att framställa miceller samt isoporösa filmer. Micellernas kritiska micellbildningskoncentration, storlek samt förmåga att laddas med läkemedel visade sig vara mycket beroende av dendrongenerationen. Dendrongenerationen visade sig vidare även ha stor inverkan i hybridmaterialens förmåga att självorganisera sig till en isoporös struktur och material av tredje generationen gav de mest välordnade filmerna.
QC 20101125

Biofouling, through undesirable colonisation of surfaces by marine organisms, is responsible for significant economic and environmental impacts that justify the use of antifouling (AF) coatings. Extracts from cultivable microalgae were investigated as a sustainable and eco-friendly source of AF compounds. A multi-functional bioassay was developed to test microalgal extracts against biofilm-forming bacteria and diatoms (behaviour, growth, adhesion, toxicity, species interactions). The AF activity of extracts from seven microalgae was investigated against microfoulers, macroalgae and barnacles, and, the impact of stresses was assessed. Inhibitory activities were recorded against a wide spectrum of organisms between 0.01 and 100 μg.mL-1, and complex doseresponses with stimulatory effects were highlighted. The AF activity was improved using competition and dark stress on marine microalgae, and was repressed using predation on freshwater species. Replicate experiments conducted using dark stress demonstrated the stability of stress responses, when comparing AF activity and metabolic profiles, for Cylindrotheca closterium and Thalassiosira pseudonana. These strains, which showed high biomass and extract yields (5%-10%), were further studied for fractionation and field-tests. Fractions of intermediate polarity from C. closterium demonstrated the most potential in the laboratory. In the field tests, Cc5 still showed moderate activity against early formation of algal slime in Portsmouth, and Cc6 reduced invertebrates’ settlement in Toulon. The microalgal culture-associated bacteria studied showed high diversity, microalgal proximity, and multifaceted chemical interactions. A comparative study showed that microalgal and bacterial extracts exhibited different patterns of AF activity and distinct chemical signatures, confirming the microalgal origin of extracts studied throughout. This work proposed a multi-functional AF screening of microalgal compounds for a better understanding of the mode of action, it demonstrated the tangible potential of microalgal extracts for AF applications, and it addressed the limits of an AF strategy based solely on chemical interactions - encouraging future use of combinatory approaches.

Biofouling can occur everywhere water is present and is a major problem for a wide variety of materials and devices such as biomedical devices and implants, as well as industrial and marine equipment. After the ban of the efficient but environmentally harmful antifouling paints based on tributyltin, the creation of new, effective and low-toxic antifouling paints is a major challenge. The aim of this thesis is to develop novel modified nanoparticles (NPs) as functional fillers for low toxic and environmentally friendly antibacterial/antifouling coatings for mobile and stationary applications in maritime, hospitals, industries, etc. Application of mesoporous silica nanoparticles (MSNs) as antifouling/antibacterial carriers attracted a few attention so far, specifically with a dual synergetic effect. In the present work, MSNs modified with quaternary ammonium salts (QASs) and loaded with the active agent DCOIT were synthesized as functional fillers for antifouling/antibacterial coatings. From the family of the MSNs, MCM-48 was selected as a carrier because of its cubic pore structure, high surface area, and high specific pore volume. Chapter 3 details the synthesis and characterisation of spherical MCM-48 MSNs with two different average sizes; 400 nm and 120 nm. As revealed from the X-ray diffraction and nitrogen isotherms, both types of MSNs showed highly ordered cubic mesostructure, high BET surface area, high pore volume and narrow pore size distribution. From the morphological studies using scanning electron microscopy and transmission electron microscopy, the MCM-48 with average size of 400 nm exhibited spherical morphology and some aggregates or fused together particles were present, while the MCM-48 with average size of 120 nm showed spherical morphology and all the NPs were monodispersed. Chapter 4 documents the surface modification of MCM-48 NPs with two different types of QASs; dimethyloctadecyl[3-(trimethoxysilyl)propyl]ammonium chloride and dimethyltetradecyl[3-(triethoxysilyl)propyl]ammonium chloride. The QAS modified MCM-48 revealed strong covalent bonds between the QAS and the surface of the NPs. The surface functionalization was confirmed by diffuse reflectance infrared Fourier transform spectroscopy, thermogravimetric analysis, elemental analysis, and ζ-potential measurements. Additional loading of the QAS modified MCM-48 with a commercially available biocide (Parmetol S15) resulted in a synergetic dual antibacterial/antifouling effect. Either loaded or unloaded QAS-modified MSNs exhibited high antibacterial performance confirming their dual activity. In the case of the modified NPs with average size of 400 nm, the loaded QAS-modified MCM-48 (dual functionalised) killed all exposed bacteria after 3 h of incubation and presented 100% reduction at the antibacterial tests against Gram-negative and Gram-positive bacteria. Furthermore, the QAS-modified MCM-48 (single functionalised) presented 77−89% reduction against the exposed Gram-negative bacteria and 78−94% reduction against the exposed Gram-positive bacteria. Furthermore, spherical MCM-48 NPs with smaller average size of 120 nm were surface modified with the two types of QASs and tested against Gram-negative and Gram-positive bacteria in order to investigate if there is size-dependency at the antibacterial properties of silica-QAS core-shell NPs. The smaller sized modified Marios Michailidis - April 2018 8 | P a g e NPs showed increased antibacterial properties (83-94% reduction against Gram-negative bacteria and 87-97% reduction against Gram-positive bacteria) compared to their counterparts with average size of 400 nm implying that smaller size of modified NPs could provide better antibacterial properties. In chapter 5, the modified NPs were homogeneously added in coating formulations. The nanocontainer-doped paints showed good distribution of the functionalised nanocontainers in the coating matrix, increased hydrophobicity and similar roughness values with the pristine coating formulation. In the antibacterial tests, the formulated paints revealed excellent antibacterial properties where all the bacteria were dead after 16 hours of exposure. Additionally, the nanocontainer-doped paints presented high anti-macrofouling properties against mussels. The paints containing the dual functionalised NPs presented 100% inhibition of mussels' attachment after 72 hours exposure of mussels in culture plates coated with the nanocontainer-doped paints. Furthermore, all of the nanocontainer-treated paints illustrated low toxicity against the Red Sea mussels Brachidontes pharaonis and the brine shrimps Artemia salina. Finally, PVC panels were coated with the formulated paints (containing either 2 or 5 wt % modified nanoparticles) and immersed in Red Sea (Eilat, Israel) for field test. After six months of exposure, the paints containing the NPs with dual effect showed significantly lower biofouling coverage (below 10%) compared to the biofouling coverage of pristine paint (50%) indicating their high antifouling properties in real sea water conditions. The results of this work show that (i) the dual functionalised MCM-48 can be used as functional fillers for coating formulations providing excellent antibacterial/antifouling properties to the coatings, (ii) the dual functionalised MCM-48 can be used as environmental-friendly low-toxic alternatives for tin-based paints and (iii) the covalently attached QASs on the surface of the nanoparticles remain active even after complete release of the biocide, which considerably increases their functional lifetime in the coating formulations.

Les traitements thermiques des produits laitiers induisent des phénomènes d’encrassement des échangeurs thermiques, et donc des nettoyages réguliers, qui alourdissent les coûts de production ainsi que l’impact environnemental des procédés. Il est donc important de comprendre ces phénomènes d’encrassement et de développer des stratégies pour les limiter. Cette thèse vise à étudier l’impact des variations des propriétés de surface sur l’encrassement laitier, mettre au point des surfaces anti-encrassantes bioinspirées innovantes et comprendre le mode d’action de ces surfaces. . Il a été démontré que les propriétés de surface d’un substrat sont cruciales pour contrôler l’encrassement : la diminution conjointe de la rugosité et de l’énergie de surface sont favorables à la réduction de l’encrassement. Suivant ce constat, d’excellentes propriétés encrassantes ont été obtenues suite à la mise au point de trois surfaces bioinspirées (surfaces lubrifiées « SLIPS », revêtements par plasma atmosphérique et revêtements amphiphiles). Les revêtements amphiphiles ont obtenu sans conteste les meilleurs résultats. Ils préviennent non seulement totalement l’encrassement mais également l’adhésion de bactéries pathogènes. Ce type de revêtement pourrait donc permettre de réaliser des économies non négligeables, non seulement en termes de coût de nettoyage des installations industrielles, mais également en termes d’impact environnemental des procédés. Afin de quantifier l’impact de la modification de surface sur l’empreinte environnementale de la pasteurisation, une étude d’Analyse du Cycle de Vie a été menée et a permis d’établir que l’utilisation d’un revêtement anti-encrassant permettrait de réduire l’impact environnemental d’un procédé de pasteurisation de plus de 70%
Fouling is an ongoing issue which burdens the cost of dairy thermal processes as well as their environmental impact. Understanding the fouling phenomena and finding mitigation solutions is therefore of high interest. Consequently, this works aims at: (i) studying the impact of surface properties variation on dairy fouling and (ii) designing and characterizing the mechanisms of action of novel biomimetic antifouling surfaces. It was demonstrated that surface properties were crucial for fouling mitigation, low roughness and low surface energy being the most favorable conditions for fouling reduction. In a second time, three types of biomimetic surfaces, namely slippery liquid infused surfaces (SLIPS), nano-rough atmospheric plasma coatings and amphiphilic environment-responsive coatings were proven efficient against isothermal dairy fouling. The amphiphilic coatings unquestionably presented the best antifouling performances as they totally prevented fouling development as well as pathogenic bacteria adhesion. Such surfaces should allow for significant savings in cleaning costs and environmental impact through the adaptation of the cleaning procedures. In order to assess the real effect of the antifouling coatings on the footprint of the pasteurization process, a Life Cycle Assessment study was carried out. It was demonstrated that the use of such an antifouling coating could lead to the reduction of the environmental impact of a pasteurization process by more than 70%

Doutoramento em Biologia
Antifouling (AF) systems are used worldwide to prevent biofouling, a natural phenomenon characterized by the attachment and growth of living organisms onto submerged surfaces. Biofouling may lead to elevated economic losses not only due to the deterioration of submerged structures but also, in the case of vessels, to the increase of frictional drag that ultimately results in the rise of fuel consumption and the reduction of the vessel´s operational time. The hulls chemical protection against this phenomenon has been a common practice in the naval industry but the leaching of biocides from AF systems into the aquatic environment has frequently caused severe adverse ecological impacts. Therefore, it is critical to search for more environment-friendly AF biocides, valuing the compromise of having a good AF performance to target organisms and the lowest ecological impact. Organotin compounds (OTs), especially tributyltin (TBT) and to a lesser extent triphenyltin (TPT), have been used as biocides in AF paints from the 1960's until their ban in 2008. As a consequence, a number of other compounds emerged as substitutes. There was a return to predominantly copper based AF paints with one or more booster biocides added to the formulations to achieve a maximum performance. These alternatives comprised diuron, irgarol 1051, zinc pyrithione, tolylfluanid and dichlofluanid among others. However, some of them have also been banned or phased out from the market due to their toxic effects to aquatic organisms. One objective of the present thesis was to assess the current levels of these AF biocides along the Portuguese coast. For this purpose, a survey was performed in 2011 where both sediments and tissue samples from the mediterranean mussel Mytillus galloprovincialis were collected and analysed to determine the extent of the above mentioned biocides contamination. It was also assessed the imposex levels in the gastropod Nucella lapillus as a proxy to evaluate the status of TBT pollution. The tissues of this bioindicator were also analysed for OTs due to the proven cause-effect relationship between imposex and TBT/TPT body burden. The results reveal that despite the OT global ban in 2008, TBT was still ubiquitous being measured in all assessed sediment samples (3.0 – 850 ng Sn g-1 dry weight (dw)) and in both mussel (1.1 – 420 ng Sn g-1 dw) and dogwhelk tissues (1.4 – 30.4 ng Sn g-1 dw). TPT was less abundant as it was detected in approximately 50 % of the samples: concentration in sediments varied between the detection limit (dl) of 0.1 and 43 ng Sn g-1 dw; in mussel tissues ranged between the dl of 0.2 and 14 ng Sn g-1 dw; in dogwhelk tissues varied from the dl of 0.2 to 1.6 ng Sn g-1 dw. The percentage of N. lapillus females affected by imposex (%I), the vas deferens sequence index (VDSI) and the relative penis size index (RPSI) were used to measure the intensity of imposex and varied between 36.7 – 100%, 0.39 – 7.75 and 0 – 1.22, respectively. Sterile females were only found at one site. Despite the TBT ubiquity in the Portuguese coast, with major incidence close to naval harbours, a decreasing temporal trend of organotin pollution has been observed; in fact, the comparison of 2011 results with those from previous years denote an evident decline of TBT contamination of sediments and biota (namely, mussels and dogwhelks) and N. lapillus imposex. This survey also detected Cu, Zn and diuron in both sediments and mussel tissues. Although the metals have natural and wide anthropogenic sources and diuron may also come from pesticide use in agriculture, the results may point towards their ubiquitous use in AF-paints in a recent past. In the sediments, Cu, Zn and diuron varied between 1.7 and 240 mg Kg-1 dw, the dl of 10 and 620 mg Kg-1 dw and 7.7 and 67 ng g-1 dw, respectively. In mussel tissues Cu varied between 4 and 11 mg Kg-1 dw, Zn between 94 and 640 mg Kg-1 dw and diuron ranged from the dl of 3 to 20 ng g-1 dw. Seldom it was also detected irgarol - measured in 6 (out of the 13) sampled sediment stations (6.8 – 43 ng g-1 dw) and in 5 (out of the 37) mussel tissue samples (18.7 – 183 ng g- 1 dw) - and its metabolite GS26575 measured in only one sediment sample (41 ng g-1 dw) and varying between 4.1 and 188 ng g-1 dw in 4 (out of 37) mussel tissue samples. Tolylfluanid was only measured in 3 (out of 13) sediment samples (9.4 – 21.9 ng g-1 dw) and in 2 (out of 37) mussel tissues samples (5.3 and 26.8 ng g-1 dw). Dichlofluanid content in mussel tissues varied between 2.3 - 160 ng g-1 dw occurring in 5 (out of 37) stations whilst in sediments was always below the detection limit. Nonetheless, contrasting with TBT spatial distribution, no specific pattern could be clearly recognized on the distribution of these AF biocides. With the implementation of the new Biocidal Product Regulation (EU Regulation 528/2012), emerging substances aiming to present a high AF potential to target organisms and a low environmental impact arose. A low toxicity to non-target species as well as a low tendency to bioaccumulate and a rapid transformation to less toxic products are required characteristics of environment-friendly biocides. For this thesis a bibliographic research was carried out in order to select emerging biocides with promising properties. Three biocides were chosen: tralopyril, triphenylborane pyridine (TPBP) and capsaicin. Tralopyril is the main active substance of ECONEA®, being marketed as a non-persistent and biodegradable copper-free biocide, effective in controlling fouling by barnacles, hydroids, mussels and polychaetas. TPBP, also known as Borocide®, is an organoborane compound used as a broadspectrum biocide mainly in Japan. Capsaicin is a natural occurring substance, derived from the chili pepper plant commonly used as animal repellent and claimed to improve the paint efficiency. Therefore, most of the work of this thesis comprehended the characterization of some aspects of the PBT (Persistence, Bioaccumulation and Toxicity) criteria of these biocides in order to perform a proper ecological risk assessment. Giving the scarcity of data in the literature, the major objective of this thesis was to gather new data about the toxicity of tralopyril, TPBP and capsaicin to freshwater and marine organisms, in order to provide novel insights regarding the potential impact of these chemicals to aquatic ecosystems. Regarding the marine environment, a study with early life stages of target and non-target species was carried out. It showed that the tested biocides impaired larval development in the mussel M. galloprovincialis (EC50_tralopyril = 3.1 μg L-1 and EC50_capsaicin = 3868 μg L-1) inhibited larval growth in the sea urchin P. lividus (EC50_tralopyril = 3.0 μg L-1 and EC50_capsaicin = 5248 μg L-1) and caused mortality to the copepod T. battagliai (EC50_tralopyril = 0.9 μg L-1, EC50_capsaicin = 1252 μg L-1 and EC50_TPBP = 14 μg L-1) in a dose-dependent way. When comparing the risk posed by these three emerging biocides with TBT, a reference AF substance, in a seawater marina model, it was concluded that the three tested compounds pose less risk than TBT but, nevertheless, tralopyril and TPBP might represent a considerable threat to the marine ecosystems. Capsaicin was described as the least toxic compound, apparently with no risk but also not presenting a high biocide performance. The next step was to characterize the toxicity of tralopyril, TPBP and capsaicin to three freshwater model species: the algae Chlamydomonas reinhardtii, the crustacean Daphnia magna and the fish Danio rerio. The lethal effects of the three biocides were studied for all the aforementioned species whilst sub-lethal effects were only assessed for the algae and zebrafish. Tralopyril was the only biocide that caused mortality to C. reihardhtii (LC50 = 71 μg L-1). In the D. magna acute immobilization test, tralopyril and TPBP revealed similar toxicities but only if the concentrations were mantained constant by means of solution renewal. To the zebrafish, tralopyril was undoubetly the most lethal (LC50 = 5.0 μg L-1) followed by TPBP (LC50 = 447.5 μg L-1). Capsaicin did not exert any lethal effects to any of the species tested. Regarding sub-lethal toxicity, both tralopyril and TPBP caused significant effects on C. reihardhtii effective quantum yield and ATP content, however TPBP was much less toxic. Concerning zebrafish, TPBP inhibited the swim bladder inflation, caused heart edema, reduced the blood flow and, at the highest concentrations, led to a diminish of the heart beat rate, whilst tralopyril only caused an inhibition of the swim bladder inflation though at much lower concentrations. Capsaicin caused an increase on the zebrafish embryo heart beat rate although not in a doseresponsive way. The results herein provided data on the effects of the three emerging biocides on freshwater ecosystems but also, as these species belong to different trophic levels, it helped to reduce the assessment factor used in risk assessment, decreasing the uncertainty of such prediction. Thus, the risk assessment performed for a freshwater marina corroborated previous investigations where capsaicin was identified as an environment-friendly compound whilst tralopyril and TPBP were identified as possible threats to the ecossystem. In order to have some insights into the mode of action of the two most toxic compounds referred above - tralopyril and TPBP - further investigations using a differential proteomic analysis were carried out. Sub-lethal tralopyril and TPBP concentrations regulated both general stress-related and compoundspecific proteins on the zebrafish embryo proteome. The common protein regulations between compounds are thought to be stress-related changes and comprehended the altered expression of proteins involved in energy metabolism, eye structure and in cell differentiation processes. Sub-lethal tralopyril exposure specifically upregulated six other proteins involved in energy metabolism, cytoskeleton, cell division and mRNA splicing whilst exposure to TPBP comprised the regulation of three proteins belonging to the cytoskeleton, cell growth and protein folding. Moreover, calculations on the toxic ratio of both compounds identified TPBP as a baseline toxicant whilst tralopyril was found to have a specific toxicity mode of action. As tralopyril became an important candidate to be used in European waters (due to its inclusion on the Biocidal Product Regulation) and also because this biocide revealed the highest and more specific overal toxicity, additional studies were undertaken to better understand the persistence and bioaccumulation potential of this compound. For that, it was essential to develop a sensitive analytical method to measure tralopyril in aqueous matrices. Therefore, a targeted analytical LC-MS/MS method was established to quantify tralopyril in DMSO (the solvent used in all studies portrayed in this thesis) and water samples (natural river and seawater, and medium used for zebrafish exposures - E3 medium). The limits of quantitation achieved for the different media are sufficiently low to detect tralopyril without a preconcentration step (0.05 μg L-1 for river water and 0.025 μg L-1 for the other media analysed). Estimated tralopyril half-lives were 6.1 h for seawater, 8.1 h for river water and 7.4 h for E3 medium at 18º C. In order to study the bioaccumulation potential, tralopyril concentrations were measured in the whole tissues of M. galloprovincialis following acute and chronic exposure, and after a 10-day depuration period. This biocide rapidly accumulated in the mussel tissues, reaching the steady-state condition within 13 days. Nevertheless, mussels seemed to be able to eliminate the compound since 10 days of depuration resulted in 80% elimination of the accumulated biocide. In order to further understand the sub-lethal toxicity of tralopyril to M. galloprovincialis and the possible mode of action, differential proteomics analysis was performed on the gill proteome of this species following acute and chronic exposure, and after a 10-day depuration period. Interestingly, not only tralopyril but also DMSO (used as solvent) significantly modulated the protein expression in mussel gils after acute and chronic exposure. Altogether, 46 proteins involved in bioenergetics, immune system, active efflux and oxidative stress were found to be regulated in the different exposure scenarios. Notably, after the depuration period, alterations of several proteins were still observed possibly reflecting either the continued effect or the incomplete elimination of this chemical. In conclusion, this thesis shows a change in the paradigm regarding AFbiocides, i.e., moving from older generation biocides with high ecological risk, to new ones that should desirably present low persistence and no impact to nontarget species, a difficult task that involves perseverance and hard work.
Os sistemas anti-vegetativos (AV) são usados mundialmente para evitar a incrustação biológica, um fenómeno natural caracterizado pela fixação e crescimento de organismos vivos em superfícies submersas. A bioincrustação pode levar a elevadas perdas económicas, não só devido à deterioração de estruturas submersas, mas também, no caso de navios, ao aumento da resistência à deslocação que resulta num acréscimo do consumo de combustível e na redução do tempo de vida operacional do navio. A proteção química dos cascos dos navios, contra este fenómeno, tem sido uma prática comum na indústria naval, mas a lixiviação de biocidas dos sistemas AV no meio aquático tem frequentemente causado impactos ecológicos adversos graves. Portanto, é fundamental procurar biocidas AV, mais amigos do ambiente, valorizando o compromisso entre ter um bom desempenho AV para atingir organismos alvo mas com um menor impacto ecológico. Os compostos organoestânicos (OTS), especialmente o tributil-estanho (TBT) e, em menor medida o trifenil-estanho (TPT), têm sido utilizados como biocidas em tintas AV desde a década de 1960 até à sua proibição em 2008. Como consequência, uma série de outros compostos surgiram como substitutos. Houve um retorno predominante ao uso de tintas AV à base de cobre com um ou mais biocidas de reforço adicionados às formulações, para alcançar um desempenho máximo. Estas alternativas incluíram diuron, Irgarol 1051, piritiona de zinco, tolilfluanida e diclofluanida entre outros. No entanto, alguns deles também foram proibidos ou retirados do mercado devido aos seus efeitos tóxicos para os organismos aquáticos. Um dos objectivos da presente tese foi avaliar os níveis destes biocidas AV presentes ao longo da costa Portuguesa. Com esta finalidade, foi realizado um estudo científico, em 2011, onde foram recolhidas e analisadas amostras de sedimentos e de tecido de mexilhão (Mytillus galloprovincialis) para determinar a extensão da contaminação pelos biocidas acima mencionados. Foram também avaliados os níveis de imposex no gastrópode Nucella lapillus como forma de verificar o estado da poluição por TBT. Os tecidos deste bioindicador foram somente analisados para o conteúdo de organoestânicos devido à relação causa-efeito já comprovada entre imposex e o conteúdo de TBT / TPT nos tecidos. Os resultados revelaram que, apesar da proibição mundial de OTs, em 2008, o TBT continua disseminado por toda a costa, sendo detetado em todas as amostras de sedimento (3.0 - 850 ng Sn g-1 peso seco (ps)) e também em amostras de tecidos de mexilhão (1.1 - 420 ng Sn g-1 ps) e do gastrópode N. lapillus (1.4 - 30.4 ng Sn g-1 ps). O TPT é menos abundante, uma vez que apenas foi detectado em cerca de 50% das amostras: a concentração nos sedimentos variou entre o limite de detecção (ld) de 0.1 e 43 ng Sn g-1 ps; a concentração nos tecidos de mexilhão variaram entre o ld de 0.2 e 14 ng Sn g-1 ps; a concentração em tecidos do gastrópode variaram entre o ld de 0.2 e 1.6 ng Sn g-1 ps. A percentagem de fêmeas do gastrópode N. lapillus afectadas por imposex (% I), o índice de sequência de vaso deferente (VDSI) e o índice de tamanho relativo do pénis (RPSI) foram usadas para medir a intensidade do imposex e variaram entre 36.7 - 100%, 0.39 - 7.75 e 0 - 1.22, respectivamente. Fêmeas estéreis foram encontradas apenas numa estação de amostragem. Apesar da ubiquidade do TBT no litoral Português, com maior incidência perto de portos navais, foi observada uma tendência temporal decrescente da poluição por organoestanhos; efetivamente, a comparação dos resultados de 2011 com os de anos anteriores denotam uma diminuição evidente da contaminação por TBT não só nos sedimentos e biota (tanto em tecidos de mexilhão como de gastrópodes) mas também no imposex de N. lapillus. Nesta pesquisa também foi detectada a presença de Cu, Zn e diuron em ambas amostras de sedimento e tecidos de mexilhão. Embora os metais possam provir de várias fontes, tanto naturais como antropogénicas, e o diuron possa advir da utilização de pesticidas na agricultura, estes resultados sugerem o uso ubíquo destes biocidas em tintas AV num passado recente. Nos sedimentos, Cu, Zn e diuron variaram entre 1.7 e 240 mg kg-1 ps, entre o ld de 10 e 620 mg kg-1 ps e 7.7 e 67 ng g-1 ps, respectivamente. Em tecidos de mexilhão o Cu variou entre 4 e 11 mg kg-1 ps, Zn entre 94 e 640 mg kg-1 ps e diuron variou desde o ld de 3 a 20 ng g-1 ps. Raramente foram ainda detectados Irgarol – medido em 6 (das 13) estações de sedimentos amostrados (6.8 - 43 ng g-1 ps) e em 5 (das 37) amostras de tecido de mexilhão (18.7 - 183 ng g-1 ps) – e o seu metabolito GS26575 medido em apenas uma amostra de sedimento (41 ng g-1 ps) e variando entre 4.1 e 188 ng g-1 ps em 4 (das 37) amostras de tecido de mexilhão. Tolilfluanida só foi medido em 3 (de 13) amostras de sedimentos (9.4 - 21.9 ng g-1 ps) e em 2 (de 37) amostras de tecido de mexilhão (5.3 e 26.8 ng g-1 ps). O conteúdo de diclofluanida em tecidos de mexilhão variaram entre 2.3 e 160 ng g-1 ps ocorrendo em 5 (de um total de 37) estações, enquanto nos sedimentos esteve sempre abaixo do limite de detecção. No entanto, contrariamente à evidente distribuição espacial do TBT, a distribuição destes biocidas na costa portuguesa não apresentou um padrão específico. Com a implementação do novo Regulamento dos Produtos Biocidas (Regulamento da UE 528/2012), surgiram algumas substâncias emergentes cujo propósito é apresentar um elevado potencial AV para organismos alvo e baixo impacto ambiental. A baixa toxicidade para espécies não-alvo, bem como uma baixa tendência de bioacumulação e uma rápida transformação para produtos menos tóxicos são características necessárias de biocidas mais amigos do ambiente. Para esta tese foi realizada uma pesquisa bibliográfica, a fim de selecionar compostos emergentes com propriedades promissoras. Três biocidas foram escolhidos: tralopyril, trifenilborano piridina (TPBP) e capsaicina. Tralopyril é a principal substância ativa do produto ECONEA®, comercializado como um biocida livre de cobre, não persistente e biodegradável, eficaz no controle da proliferação de cracas, hidrozoários, mexilhões e poliquetas. TPBP, também conhecido como Borocide®, é um composto organoborano usado como biocida de largo espectro, principalmente, no Japão. A capsaicina é uma substância natural, derivada da planta da pimenta utilizada como repelente de animais e que adicionada às tintas AVs alegadamente melhora a sua eficácia. Assim, a maior parte do trabalho desta tese compreendeu a caracterização de alguns aspectos dos critérios PBT (Persistência, Bioacumulação e Toxicidade) destes biocidas, a fim de realizar uma avaliação adequada do risco ecológico. Devido à escassez de dados na literatura, o principal objetivo desta tese foi reunir novos dados sobre a toxicidade do tralopyril, TPBP e capsaicina tanto em organismos marinhos como de água doce, a fim de proporcionar novos conhecimentos sobre o potencial impacto desses produtos químicos nos ecossistemas aquáticos. Relativamente ao ambiente marinho, foi realizado um estudo com estádios iniciais de vida de espécies-alvo e não-alvo. Observou-se que os biocidas testados comprometeram o desenvolvimento larvar do mexilhão M. galloprovincialis (EC50_tralopyril = 3.1 μg L-1 e EC50_capsaicina = 3868 μg L-1) inibiram o crescimento larvar do ouriço do mar P. lividus (EC50_tralopyril = 3,0 μg L-1 e EC50_capsaicina = 5248 μg L-1) e causaram mortalidade no copépode T. battagliai (EC50_tralopyril = 0,9 μg L-1, EC50_capsaicina = 1252 μg L-1 e EC50_TPBP = 14 μg L-1) em função da dose usada. Ao comparar o risco ambiental apresentado pelos três biocidas emergentes com o do TBT, uma substância AV de referência, num modelo de marina de água salgada, concluiu-se que os três compostos testados apresentam menor risco que o TBT. No entanto, tanto o tralopyril como o TPBP podem representar uma ameaça considerável para os ecossistemas marinhos. A capsaicina parece ser como o composto menos tóxico, aparentemente, sem risco mas também sem um alto desempenho biocida. O passo seguinte foi caracterizar a toxicidade do tralopyril, TPBP e capsaicina em três espécies modelo de água doce: a alga Chlamydomonas reinhardtii, o crustáceo Daphnia magna e peixe-zebra Danio rerio. Os efeitos letais dos três biocidas foram estudados para todas as espécies acima mencionadas, enquanto que os efeitos sub- letais foram avaliados apenas para as algas e peixe-zebra. Tralopyril foi o único biocida que causou mortalidade na alga C. reihardhtii (LC50 = 71 μg L-1). No teste agudo de imobilização da D. magna, tralopyril e TPBP revelaram toxicidades semelhantes, mas somente se as concentrações fossem mantidas constantes por meio de renovação da solução. Para o peixe-zebra, tralopyril foi sem dúvida o composto mais letal (LC50 = 5.0 μg L-1) seguido pelo TPBP (LC50 = 447.5 μg L-1). A capsaicina não exerceu quaisquer efeitos letais em nenhuma das espécies testadas. Em relação à toxicidade sub-letal, tanto o tralopyril como o TPBP causaram efeitos significativos no rendimento quântico efetivo e no conteúdo de ATP em C. reihardhtii, no entanto o TPBP apresentou uma toxicidade bastante mais baixa. Em relação ao peixe-zebra, o TPBP inibiu a inflação de bexiga natatória, causou edema no coração, reduziu o fluxo sanguíneo e, nas concentrações mais elevadas, levou a uma diminuição da taxa de batimento cardíaco, enquanto que o tralopyril causou apenas uma inibição da inflação de bexiga natatória embora numa concentração bastante menor. A capsaicina causou um aumento na taxa de batimentos cardíacos do embrião de peixe-zebra, embora de uma forma não dependente da concentração do composto. Os resultados aqui descritos não só proporcionam informação sobre os efeitos dos três biocidas emergentes sobre os ecossistemas de água doce, mas também, uma vez que estas espécies pertencem a níveis tróficos diferentes, ajudou a reduzir os factores de avaliação utilizados na avaliação de risco, diminuindo a incerteza de tal previsão. Assim, a avaliação de risco realizada para uma marina de água doce corroborou investigações anteriores, em que a capsaicina foi identificada como um composto amigo do ambiente, enquanto que o tralopyril e o TPBP foram identificados como possíveis ameaças ao ecossistema. Com a finalidade de compreender o modo de ação dos dois compostos mais tóxicos, acima referidos - tralopyril e TPBP - novas investigações foram efectuadas usando uma analise proteómica diferencial. Concentrações subletais de tralopyril e TPBP regularam tanto proteínas relacionadas com o stress geral do organismo, como também outras proteínas mais especificas da ação do composto, no proteoma do embrião de peixe-zebra. Pensa-se que a regulação de proteínas comuns entre compostos possa ser relacionada com o stress e abrangeram a alteração de proteínas envolvidas no metabolismo energético, estrutura do olho e processos de diferenciação celular. A exposição a concentrações sub-letais de tralopyril regularam positivamente seis proteínas específicas envolvidas no metabolismo da energia, do citoesqueleto, da divisão celular e no splicing do mRNA, enquanto que a exposição a TPBP levou à regulação de três proteínas pertencentes ao citoesqueleto, crescimento celular e protein folding. Foram ainda efectuados cálculos sobre o Toxic Ratio de ambos os compostos que identificaram o TPBP como um químico responsável por uma toxicidade mais geral, enquanto que o tralopyril parece ter um modo de ação mais específico.Uma vez que o tralopyril se tornou num candidato importante a ser utilizado em águas europeias (devido à sua inclusão no Regulamento dos Produtos Biocida) e também porque este composto revelou a mais alta e específica toxicidade, de entre os 3 biocidas, foram realizados estudos adicionais para compreender melhor a sua persistência e potencial de bioacumulação. Para isso, foi essencial desenvolver um método analítico sensível para medir este composto em matrizes aquosas. Assim, um método analítico por LC-MS/MS foi estabelecido para quantificar tralopyril em DMSO (o solvente utilizado em todos os estudos realizados nesta tese) e em amostras aquosas (águas naturais de rio e do mar, bem como o meio utilizado nas exposições de embriões de peixezebra - Medio E3). Os limites de quantificação do tralopyril nos vários meios utilizados foram suficientemente baixos para detectar tralopyril sem um passo de pré-concentração (0.05 μg L-1 para águas do rio e 0.025 μg L-1 para os outros meios de analisados). O tempo de meia-vida estimado para este composto foi de 6.1 h na água do mar, 8.1 h na água de rio e 7.4 h no meio E3 a 18º C. A fim de estudar o potencial de bioacumulação, foram medidas as concentrações de tralopyril em tecidos de M. galloprovincialis após exposição aguda e crónica, e após um período de depuração de 10 dias. Este biocida acumulou rapidamente nos tecidos de mexilhão, atingindo o estado estacionário em 13 dias. No entanto, pensa-se que os animais sejam capazes de eliminar o composto uma vez que 10 dias de depuração resultaram na eliminação de 80% do biocida acumulado. Com o objectivo de melhor compreender a toxicidade sub-letal do tralopyril em M. galloprovincialis e o modo de ação deste composto, foi realizada uma análise proteómica diferencial no proteoma das brânquias de organismos desta espécie após exposições aguda e crónica e depois de um período de depuração de 10 dias. Curiosamente, não só o tralopyril mas também o DMSO (utilizado como solvente), modularam significativamente a expressão de proteínas nas brânquias de mexilhão após exposições aguda e crónica. No total, 46 proteínas envolvidas na bioenergética, no sistema imunológico, active efflux e no stress oxidativo foram reguladas nos diferentes cenários de exposição. Alterações de várias proteínas foram ainda observadas, depois do período de depuração, possivelmente refletindo quer o efeito contínuo do tralopyril ou da sua eliminação incompleta. Em conclusão, este trabalho mostra uma mudança de paradigma em relação aos biocidas AV, isto é, há uma passagem do uso de biocidas mais antigos com alto risco ecológico, para biocidas mais novos que devem, desejavelmente, apresentar uma baixa persistência e nenhum impacto para as espécies não alvo, uma tarefa difícil que envolve perseverança e trabalho árduo.

Les biosalissures marines représentent l’accumulation indésirable d’organismes biologiques sur les surfaces de structures immergées dans l’eau de mer. Malheureusement, ce phénomène naturel a de sérieuses conséquences sur les plans économiques, environnementaux et matériels. Depuis l’interdiction de certains biocides dans les peintures antisalissures (en particulier le TBT en janvier 2008) à cause de leur toxicité envers des espèces marines non-ciblées et de leur accumulation dans l’environnement marin, la recherche a mis l’accent sur le développement de nouveaux revêtements antifouling efficaces,durables et respectueux de l’environnement sans relargage d’espèces toxiques. Ainsi, les travaux de cette thèse portent sur la fonctionnalisation de carbone vitreux par des glucides reliés à des systèmes conjugués électrostimulables via un lien triazole pour développer des surfaces à activité antifouling. En effet, ce type de revêtement a été conçu pour intervenir dans les premières étapes du biofouling. Tout d’abord, le glucide, très hydrophile, devrait lutter contre la formation du film conditionnant en s’entourant d’une barrière aqueuse résistante aux protéines. D’autre part, la modification de l’état de charge de la surface en continu par application d’un courant électrique sur le système conjugué électro-actif devrait perturber la colonisation bactérienneretardant l’installation du biofilm marin. Notre étude repose donc sur la synthèse et l’immobilisation d’un ensemble de glucides électrostimulables sur une surface de carbone vitreux par oxydation de l’amine aromatique en milieux organiques et aqueux. Un test microbiologique a été réalisé sur un des revêtements glucidiques en présence de la souche bactérienne TC8 dans les puits d’une microplaque contenant des cellules électrochimiques reliées à un potentiostat. La stimulation électrique de ce revêtement a permis d’améliorer ses propriétés antibactériennes
Marine biofouling represents the undesirable accumulation of biological organisms on the surfaces of structures submerged in seawater. Unfortunately, this natural phenomenon has serious economic, environmental and material consequences. Since the ban of some biocides in antifouling paints (TBT in January 2008) because of their toxicity on the nontargeted marine species and their accumulation in the marine environment, research has focused on the development of new efficient, durable and environmentally friendly antifouling coatings without releasing toxic species. Thus, the work of this thesis deal with the functionalization of glassy carbon surface by carbohydrates linked to an electrostimulable conjugated system via a triazole link in order to develop surfaces with antifouling activity. Indeed, this kind of coating was designed to intervene in the first steps of biofouling. First, the carbohydrate, which is very hydrophilic, should fight against the formation of the conditioning film by surronding itself with an aqueous barrier resistant to proteins. On the other hand, the continuous modification of charge state by applying an electric current to the electroactive conjugated system is expected to disrupt the bacterial colonization delaying the installation of marine biofilm. Our study is therefore based on the synthesis and the immobilization of electrostimulable carbohydrates on a glassy carbon surface by aromatic amine oxidation in organic and aqueous media. A microbiological test was carried out on one of the carbohydrate coatings in the presence of the TC8 bacterial strain in the wells of a microplate containing electrochemical cells connected to a potentiostat. Electrical stimulation of this coating allowed to improve its antibacterial properties

Surfaces submerged in marine environments rapidly get colonized by marine organisms, a process known as biofouling. Fouling costs maritime industries billions of dollars annually. The most common methods of combating marine biofouling are toxin containing antifouling coatings which often have detrimental non-target environmental effects. These effects and proposed bans on harmful substances in antifouling coatings, mandates development of more environmentally friendly antifouling technologies. Of these, foul-release coatings, which minimize attachment and adhesion of fouling organisms (rather than killing them) are promising alternatives. Here I explored the utility of petroleum waxes as novel antifouling/foul-release coatings. I first investigated the responses of propagules (larvae or spores) of six common fouling organisms to wax coatings in the laboratory. A wide variation in the response of these different organisms, and in the different types of response (settlement, adhesion, etc.) by the same organism, was observed, but the most inhibitory coatings were those made from microcrystalline wax and silicone oil. However, in field trials in Sydney Harbour, paraffin waxes had the strongest antifouling performance, with activity up to one year (the trial duration). These waxes also had strong foul-release effects, with fouling that did attach mostly removed by a low pressure water jet. Composition of fouling communities on paraffin waxes differed significantly from other waxes or controls, with little or no hard fouling organisms (barnacles, bivalves) on paraffin. The mechanisms of antifouling and foul-release actions of paraffin waxes appear to be due to changes in surface properties. The surfaces of the paraffin waxes changed noticeably after 4 - 8 weeks immersion in the sea or in seawater aquaria. Antibiotic treatments showed that this change in surface appearance was due to biological (microbial) activity. Bacteria appear to remove the amorphous phase from the surface of the paraffin waxes, revealing an underlying crystalline phase, which is less affected by bacterial action. I suggest that these crystals form a microstructured ?bed of nails? of crystals of varying shapes and sizes which inhibit settlement and reduce adhesion strength of those organisms which do settle.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2011.
Cataloged from PDF version of thesis.
Includes bibliographical references (p. 152-160).
This thesis work focuses on designing thin polyelectrolyte multilayer (PEM) films via layer-bylayer (LbL) deposition technique with the ability to kill pathogenic bacteria and inactivate human viruses, especially the influenza (flu) virus on contact. This work builds on four years of research at the Institute for Soldier Nanotechnologies (ISN) focusing on creating new, nonleaching microbicidal material; this film is envisioned to be used as permanent surface coatings for weapons, equipments, uniforms, personal items, etc. because a small reduction in the rate of infection will greatly enhance the readiness and performance of soldiers and other military personnel. Extending this application to everyday life, commonly handled objects such as doorknobs, computer keyboards, and touch screens can also be made sterile by coating them with these highly effective microbicidal PEM films. These films can also be used to prevent infections and long-term bacterial biofilms on implant surfaces. The ultimate aim of this thesis work is to create a broadly applicable multifunctional platform film technology that will satisfy various thin film surface coating applications; this film will impart a surface with long term antimicrobial / antifouling functionality via a permanent microbicidal base, and controlled delivery of a therapeutic agent via a hydrolytically degradable top film as needed. Efforts were focused on maximizing and understanding the factors that influence the microbicidal / antifouling property of the film; thus far, we successfully designed a set of contact-killing ionically cross-linked polymeric thin films; a hydrophobic polycation, linear NNdodecyl, methyl- poly(ethylenimine) (DMLPEI) with microbicidal activity was layered with a hydrophilic polyanion, such as poly(acrylic acid) (PAA), to create LbL films highly effective against Escherichia coli and Staphylococcus aureus (Gram negative and positive bacteria, respectively), as well as the influenza A/WSN (H1N1) virus. The microbicidal film was also demonstrated to significantly resist adsorption of protein from blood plasma relative to an uncoated substrate. By generating PEM films assembled with the hydrophobic N-alkylated poly(ethylenimine) and the hydrophilic poly(acrylic acid), an ultrathin film that exhibited antifouling and antimicrobial properties was created. Results showed that a fine balance of hydrophobicity and hydrophilicity on the surface of the films was needed to create molecularlevel heterogeneities unfavorable to protein adsorption; due to the contrasting nature of the polymer making up the film, nanoscale segregation of the polymer segments into hydrophobic and hydrophilic moieties could occur on the surface. We then moved on to design a dual functional LbL film construct combining the permanent microbicidal / antifouling base film with a hydrolytically degradable PEM top film offering controlled and localized delivery of therapeutics (e.g. antibiotic, anti-inflammatory drug, etc.). When the degradable top film is completely eroded, the surface will be left with the permanent microbicidal film for long-term prevention of fouling by biomolecules and microorganisms (e.g., proteins and bacteria).
by Sze Yinn (Jessie) Wong.
Ph.D.

Les polymeres possedant des groupements pendants perfluoroalkyles presentent des proprietes de surface remarquables. De plus l'incorporation de segments fluores dans un materiau polymere induit un caractere amphiphile tres marque. Nous avons pense pouvoir utiliser ces deux proprietes apportees par les groupements perfluores pour: * d'une part mettre au point de nouveaux revetements antifouling compatibles avec la protection de l'environnement, * d'autre part developper de nouveaux epaississants associatifs plus performants. Dans la premiere partie de ce memoire, divers reseaux polyurethanes a chaines laterales perfluoroalkylees (p. U. F. ) ont ete synthetises et caracterises par dsc et par tensiometrie. Independamment du pourcentage de fluor, les p. U. F. Presentent une faible tension superficielle (9 a 13 mn. M#-#1). Des tests en milieu marin ont montre que ces p. U. F. Permettent un nettoyage aise des surfaces immergees. Dans la deuxieme partie de ce travail, plusieurs poly(oxyde d'ethylene)s modifies par des segments fluores (f-heur) ont ete synthetises. Les etudes viscosimetriques et rheologiques en milieu aqueux montrent que les meilleures proprietes viscosifiantes sont obtenues avec une chaine poe longue (n = 454), des segments fluores c#8f#1#7(ch#2)#1#1- et un seul segment hydrophobe aux extremites de la chaine hydrophile

Biocide-based antifouling (AF) paints are the most common method for preventing biofouling, i.e. the growth of algae, barnacles and other organisms on boat hulls. AF paints for leisure boats are predominantly based on copper (Cu) as the main biocide, with zinc (Zn) present as a pigment and stabilizer. Both metals are released from the paint matrix into the water column, leading to contamination of marinas which typically have only limited water exchange. Thus, the aim of this PhD thesis was to describe the use of AF paints in different regions in Sweden, as well as the associated environmental consequences with regard to contamination of the environment and toxicity to non-target aquatic snails. Using a recently developed X-ray fluorescence application, high levels of Cu were detected on boats moored in freshwaters, despite a more than 20-year-old ban, as well as high levels of tin (Sn) on 10 % of the boats, indicating the presence of (old) tributyltin paints (TBT), which might pose an environmental risk and a health hazard for people performing the paint scraping (paper 1). In addition, very high levels of Cu and Zn were measured in the biofouling material collected from the boat hulls, and this is problematic because the biofouling is commonly disposed of on the soil in boatyards at the end of each season. No difference was found in the amount of biofouling on boats coated with Cu or biocide-free paints, which implies that Cu might be currently overused in areas of low salinity and low barnacle density (paper 2). This work also introduces the use of a new species for ecotoxicological field experiments, the snail Theodoxus fluviatilis. Chronic field experiments (paper 3) revealed 6-fold increases in snail mortality, negative growth and up to 67-fold decreased reproduction in marinas, compared to areas not impacted by boating (‘reference areas’). Moreover, a higher prevalence of snails with histopathological alterations (e.g. necrosis of gills, gonads, midgut gland and parasite infestation, among others) was observed in the marinas, compared to the reference areas (paper 4). Statistical modelling indicated that the majority of the toxic effects were best predicted by the metals, most likely originating from AF paints. The results presented in this thesis depict some important aspects of AF paint use in brackish water and highlight the necessity of implementing a suitable management practice for the heavily contaminated biofouling waste in order to minimize the risk to soils. In addition, the evidence of toxicity to snails in marinas can be used as a basis to increase the public understanding of the impact of recreational boating and encourage people to choose less toxic alternatives to AF paints.

At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript.

Marine biofouling can be defined as the undesirable adhesion to surfaces and further growth of organisms, mainly biofilm-forming microbes, macroalgae and invertebrates. The aim of this project was the prevention of adhesion and growth of organisms by using anti-biofilm molecules of natural origin. The antifouling properties of molecules isolated from an Artic marine sponge Stryphnus fortis, a sub-Artic ascidian Synoicum pulmonaria and micropeptides derived from the innate defence protein lactoferrin were assessed. The bromotyrosine derivative ianthelline isolated from S.fortis is shown to inhibit both marine micro- and macrofoulers with a pronounced effect on marine bacteria (MIC values: 0.1–10 μg/mL) and barnacle larval settlement (IC₅₀=3.0 μg/mL). Four molecules belonging to the recently discovered synoxazolidinone and pulmonarin families-both isolated from S.pulmonaria and four simplified synthetic analogs displayed minimum inhibition concentration (MIC) values in the micro- to nanomolar range against 16 relevant marine species involved in both the micro- and macrofouling processes. Synoxazolidinone C displayed selected antifouling properties comparable to the commercial antifouling product SEA-NINE. Among the 13 micropeptides derived from the innate defence protein lactoferrin, two peptides were particularly active against the microfoulers with MIC-values ranging from 1-0.01 μg/mL and comparable to the antifouling activities of the commercial biocide SEA-NINE.The contamination of tropical marine environments (water column and sediment) by leachate from antifouling paints has led to concern regarding the effects on corals and their symbionts. Therefore, it is of high importance to evaluate the impact of antifouling compounds on key coral reef organisms. The potential toxicity of two commercial biocides (SEA-NINE and Irgarol), one synthetic biocide (thiram) and two biocides of natural origin (myristic acid and Totarol) were assessed at environmental concentrations, toward the survival of two species of Symbiodinum sp.: Symbiodinium microadriaticum and Symbiodinium voratum. SEA-NINE-and Irgarol affect the growth and survival rate of the two species of symbiont but myristic acid, thiram and Totarol have shown no long term effect on Symbiodinium spp. Application of thermal stress (+2°C) increased the toxicity of SEA-NINE-and Irgarol to these species. The effect of increased mortality of these symbionts on coral bleaching as sea temperatures rise is unclear.

The detrimental effects of tributyltin (TBT) have been recorded on many marine organisms. As a result the UK Government imposed a partial ban on the use of organotin antifouling paints on boats less than 25 m in length, in 1987. In 1988 the Isle of Man Government followed suit introducing a licensing procedure restricting all uses of organotins. At concentrations less than 0.5 ng Sn/I female Nucella lap/Nus develop imposex - the superimposition of male sexual characteristics. To date there have been few studies measuring the recovery of Nucella populations after the introduction of restrictions. This study produces evidence of the extent of recovery in Nucella populations from sites in the south-west of England and on the Isle of Man. The recovery observed was measured by decreasing values of relative penis size, vas deferens sequence and the percentage of sterile adult females in the population. Following the 1987 ban the recovery of Nucella populations in the south-west has shown a linear response allowing predictions to be made for the time scale of complete recovery. In addition concentrations of TBT in the water and tissues of selected indicator organisms also showed decreases. Around the Isle of Man the illegal use of TBT paints was identified and later discouraged by the Marine Administration which was followed by a reduction in TBT concentrations in the water at sites around the Isle of Man. Levels of imposex in dogwhelk populations around the Isle of Man have decreased. Although effects of TBT on Nuce/la have been well documented at the cellular and individual level, the knock on effects on the community have not been investigated. Manipulative field experiments were used to demonstrate the role of Nucella lapd/us in structuring shore communities to allow predictions of the effect of TBT to be made. Rather than using the traditional approach of fences and cages, dogwhelks were removed by hand on regular visits to experimental sites creating treatments with reduced abundances of dogwhelks akin to shores affected by TBT. The role of Nucel/a was examined at different stages of a cycle existing on moderately exposed Manx shores where Fucus vesiculosus and Semibe/anus balanoides fluctuate in abundance. The removal of dogwhelks increased the abundance of Semibalanus ba/anoides on the shore and as a result likelihood of algal escapes from grazing by Patella vulgate also increased. In addition the removal of Nucela increased the size and longevity of newly established Fucus vesiculosus clumps. In a factorial experiment the role of Patella vulgate and Nucella lapillus were examined simultaneously. Nuce/la was found to have an significant effect but less than that of Patella. The presence of Nucella did, however, mediate the effect of Patella. In addition Nucella was found to have a direct effect on the level of Semibalanus balanoides settlement in the field with the number of barnacles settling in cleared areas being reduced on areas which had been previously occupied by Nucella.

Båtbottenfärger, eller antifoulingfärger, används för att motverka påväxt i form avexempelvis havstulpaner och alger på båtskrov. Färgerna kan antingen ha en kemisk,biologisk, fysikalisk eller mekanisk verkan på de vattenlevande organismerna. De kemisktverkande biocidfärgerna räknas som bekämpningsmedel och måste godkännas av Kemikalieinspektionen innan de får säljas och användas i Sverige. På grund av den lågasalthalten i Mälaren är problemet med påväxt mindre än på exempelvis Västkusten, och avden anledningen kan behovet av att måla båtar som endast färdas i Mälaren medbiocidfärger ifrågasättas. Initiativtagaren och uppdragsgivaren för examensarbetet är Bo Olsson på IKEM, InnovationsochKemiindustrierna i Sverige. Målet med examensarbetet var att analysera kopparläckagetfrån båtbottnar målade med biocidfärger innehållande koppar, att undersöka om läckagetgår att minska med olika täckningstekniker samt att diskutera för- och nackdelar medblästring av båtbottnar för att avlägsna biocidfärgerna helt. För att kunna svara på hur mycket koppar som läcker ut från biocidfärger och om läckaget går att minska med olika täckningstekniker, målades glasburkar på insidan med olika färgkombinationer och fylldes sedan med vatten från Mälaren. Efter en månad analyserades vattenproverna i en atomabsorptionsspektrometer med flamma. Analysresultaten visade ettläckage på 271 mg/m2 för VC 17 och 24,3 mg/m2 för Antifouling Copper Plus för en månad. Från en medelstor fritidsbåt, med en bottenyta på 24 m2, läcker mellan 580 och 6500 mg koppar ut till Mälarens sötvatten på en månad. Från analysen kunde en tydlig minskning av kopparläckaget för de båda studerade täckningsteknikerna ses med ca 60–80 % för Antifouling Sealer och 94–96 % för lamineringsepoxin. Trots att epoxilagret visade på en god reducering av kopparläckaget, rekommenderas Antifouling Sealer då epoxiplaster innehållerdet hormonstörande ämnet bisfenol A. Kostnaden för den mängd Antifouling Sealer som behövs till en medelstor fritidsbåt beräknades till ca 1700 SEK. Förutom att täcka över biociderna är ett annat tänkbart alternativ att blästra bort gamla biocidfärger för att sedan applicera exempelvis en foulingreleasefärg och därefter spola av båtbotten vid behov. Blästringen tar bort problemet med biociderna på båten för gott, men då avfallet läggs på deponi uppkommer ett nytt miljöproblem. Den ekonomiska kostnaden för blästring blir dock högre än om båten täcks med Antifouling Sealer, ca 16 000 SEK exklusive moms, men skulle kunna minskas för båtägarna med hjälp av LOVA-bidrag som delas ut av länsstyrelsen. En annan intressant lösning vore att använda färger som innehåller specifika enzymer som bryter ned de bindemedel organismerna använder för att kunna fästa. Den slutsats som kan dras av detta examensarbete är att det är svårt att utifrån ett miljöperspektiv bestämma den bäst lämpade metoden för att minska kopparläckaget till Mälaren. Varje metod kan medföra nya problem som också behöver utredas och därför bör försiktighetsprincipen följas för att undvika ökande påfrestningar på miljön. Intresset för att förbättra vattenmiljön är dock stort och utbudet på miljövänliga alternativ ökar ständigt.
Antifouling coats are used to prevent fouling, such as barnacles and algae, on boat hulls. With the use of antifouling coatings, fouling can be prevented chemically (with biocides),biologically, physically or mechanically. Coatings containing biocides are counted as pesticides and have to be approved by the Swedish Chemicals Agency before they can besold or used in Sweden. Due to the low concentration of salt, there are fewer problems withfouling in Lake Mälaren than in the water on the west coast of Sweden. This means that antifouling products containing biocides used on leisure boats in Lake Mälaren should be questioned. Bo Olson at Innovation and Chemical Industries in Sweden created the idea for this thesis. The goal was to analyze the copper leakage from boat hulls covered with antifouling coatings containing copper, to research if using different biocide-free coatings can reduce the leakage and to discuss the pros and cons of blasting as a method for removing the biocides. In order to answer how much copper the antifouling colors release into the freshwater and whether the leakages can be reduced by antifouling sealers, glass jars were painted on the inside with different color combinations and was filled with water from Lake Mälaren. After a month, the water samples were analyzed with a flame atomic absorption spectrometer. The results showed a leakage of 271 mg/m2, month for the color VC 17 and 24,3 mg/m2,month for the color Antifouling Copper Plus. From a medium sized leisure boat, with a wetted surface area of 24 m2, between 580 and 6500 mg copper leaks into the freshwater of lake Malaren in one month. The results from the analysis also showed a significant decrease of the copper leakage for both of the studied covering techniques, with 60–80 % for Antifouling Sealer and 94–96 % for the laminating epoxy. Even though the epoxy greatly reduced the copper leakage, Antifouling Sealer is recommended because of the presence of the endocrine disruptor bisphenol A in epoxy plastics. The cost of the amount of Antifouling Sealer needed for a medium sized leisure boat was estimated at SEK 1700. Another possible alternative to antifouling sealers is to blast the boat free from antifouling coatings, cover the boat with, for example, a fouling release coating and then clean it with a pressure washer when needed. Although blasting eliminates the problem with biocides for good, another environmental issue is created when the hazardous waste has to be deposited. The economical cost that comes with blasting is approximately SEK 16 000 excluding VAT for a 24m2 boat, and is significantly higher than if the Antifouling Sealer is applied instead. With financial aids from the County Administrative Board, the cost for the boat owners could however be lowered. Another interesting solution is to use coatings containing enzymes that break down the adhesives the organisms need when attaching to a surface. The conclusion of this thesis is that it is difficult to determine the most suitable method forreducing the copper leakage to Lake Mälaren from an environmental point of view. Each method may result in new problems that need to be investigated further. Therefore, the precautionary method is vital to avoid the increasing stress on the environment. The interest to improve the marine environment is however big and the range of environmental friendly alternatives are constantly increasing.

Thesis (MSc)--Stellenbosch University, 2015.
ENGLISH ABSTRACT: Biofouling in cooling water systems lead to several problems resulting in reduced efficiency and financial losses. Antifouling coatings present an environmental friendly solution to prevent biofouling alternatively to the current use of toxic chemicals in cooling water systems. In this study biofilm growth in a cooling water system was simulated in a modified flow cell system to evaluate industrial antifouling coatings and biocide-enriched coatings as potential antifouling coatings for metal surfaces. The design of a novel antifouling coating was also attempted. Firstly, analytical methods for biofilm monitoring to evaluate selected antifouling coatings and biocides were optimised. Pseudomonas sp. strain CT07 was selected to grow biofilms in the biofilm studies. A metal alloy of stainless steel and mild steel (3CR12) showed no corrosion after a 24 h biofilm growth and was selected as metal surface for the biofilm growth discs. Sonification for 5 min was determined as the optimum biofilm removal method from the growth discs. After biofilm removal the metal growth discs were stained with the LIVE/DEAD® BaclightTM Bacterial Viability kit. Visualisation by confocal laser scanning microscopy and flow cytometry revealed auto fluorescence signals from metal discs that hindered quantitative and qualitative analysis of the metal substrate. The use of Pseudomonas sp. strain CT07::gfp to grow biofilms on the metal growth discs and the exclusion of the stain SYTO9 from the LIVE/DEAD® BaclightTM Bacterial Viability kit reduced auto fluorescence signals from the metal discs. The industrial coatings containing quaternary ammonium salt (QAC), triclosan (TC) and copper oxide (CUO) respectively, showed the highest antimicrobial activity in the disc diffusion test. The minimum inhibition concentrations for silver nitrate (SN) and copper sulphate (CS) were 432 ppm and 160 ppm respectively. A minimum of 6.25 % of furanone solution (FR) was biocidal in the dilution susceptibility test. Secondly, the metal growth discs were coated respectively with the three selected industrial coatings QAC, TC and CUO and the epoxy biocide-enriched coatings SN, CS and FR and chemically characterised before and after exposure to biofilm formation. The antifouling activity of these coatings was also characterized. Growth media inoculated with Pseudomonas sp strain CT07::gfp was circulated through the modified flow cell system via a multichannel peristaltic pump for 48 h before the coated metal discs were removed and washed to perform chemical or antifouling analysis. All the industrial coatings and biocide enriched epoxy coatings complied with the thermal stability requirements of a cooling water system. Scanning electron microscopy (SEM) imaging and Energy dispersive X-ray spectroscopy (EDX) analysis confirmed that the adhesion properties of industrial coatings TC and QAC in aqueous environments were insufficient and that the copper and silver ions leached out of the biocide-enriched epoxy coatings. The qualitative analyses of the attachment of bacteria on the surfaces of both the industrial and biocide enriched epoxy coatings was confirmed by SEM, CLSM. The attached bacteria were removed and analysed quantitatively through plate counts and flow cytometry. None of the industrial coatings or the biocide incorporated epoxy coatings that were used in this study would therefore be efficient for the use on metal surfaces in cooling water systems. Thirdly, several approaches were followed to synthesise a poly(styrene-alt-maleic anhydride) (SMA) coating, chemically bind a furanone derivative, 2,5-dimethyl-4-hydroxy-3-(2H)-furanone, to the polymer back bone of the SMA coating for the application as an antifouling coating for cooling water systems. The synthesis of SMA was confirmed through 1H NMR and SEC and the synthesis of tert-butyl 2-(2-hydroxyethoxy) ethylcarbamate and 4-(2-(2-(tert-butoxycarbonyl)ethoxy)ethoxy)-4- oxobutanoic acid was confirmed through 1H NMR and ES-MS+. The synthesis of the end furanone derivative product could however not be achieved.
AFRIKAANSE OPSOMMING: Bio-aanpaksels in waterverkoelingsisteme veroorsaak talle probleme wat lei tot verminderde doeltreffendheid en finansiële verliese. Antimikrobiese oppervlakbedekkings verskaf ‘n omgewingsvriendelike oplossing om bio-aanpaksels te voorkom en ‘n alternatief vir die huidige gebruik van giftige chemikalieë in waterverkoelingsisteme. Biofilm groei in waterverkoelingsisteme was nageboots in ‘n gewysigde vloeiselsisteem om industriële aanpakwerende en biopsied bevattende antimikrobiese oppervlakbedekkings as potensiële aanpakwerende beskermingslae vir metaaloppervlaktes te evalueer. Die ontwerp van ‘n nuwe aanpakwerende beskermingslaag is ook ondersoek. Eerstens is analitiese moniteringsmetodes vir bio-aanpaksels op geselekteerde aanpakwerende antimikrobiese oppervlakbedekkings en biosiedes geoptimiseer. Pseudomonas sp. stam CT07 was verkies om bio-aanpaksels te simuleer gedurende hierdie studie. ‘n Metaalalooi van vlekvrye staal en sagte staal (3R12) het geen korrosie getoon na 24 uur se groei van bio-aanpaksels nie en is vir hierdie rede gebruik as metaal vir die bio-aanpaksel groeiplate. Dit was vasgestel dat sonifisering die optimale verwyderingsmetode vir groeiplate was. Na verwydering van bio-aanpaksels was die metaal groeiplate bedek met die LIVE/DEAD® BaclightTM bakteriële lewensvatbaarheid-toestel. Visualisering deur middel van konfokale mikroskopie en vloeisitrometrie het outofluoreserende seine vanaf die metaal groeiplate onthul wat kwantitatiewe en kwalitatiewe analise van die metaal substraat verhinder het. Die gebruik van Pseudomonas sp. stam CT07:gfp om bio-aanpaksels te kweek op metal plate en die uitsluiting van SYT09 van die LIVE/DEAD® BaclightTM bakteriële lewensvatbaarheid-toestel, het die outofluoreserende seine van die metaalskywe verminder. Industriële beskerminglae, wat onderskeidelik Kwaternêre ammonium sout (QAC), triclosan (TC) en koperoksied (CUO) bevat, het die hoogste antimikrobiese aktiwiteit in die skyf-diffusie toets getoon. Die minimum inhibisiekonsentrasies vir silwernitraat (SN) en kopersulfaat (CS) was onderskeidelik 432 dpm en 160 dpm. ‘n Minimum konsentrasie van 6.25% van die furanoonoplossing (FO) is geklassifiseer as ‘n biosied in die oplossingstoets.Tweedens was die metaal groei-skywe bedek met drie industriële beskermingslae QAC, TC en CUO en die epoksie-biosied-verrykte lae SN, CS en FR en chemiesgekarakteriseerd voor en na die vorming van bio-aanpaksel. Die karaktereienskappe van die aktiwiteit van die beskermingslae was ook vasgestel. Opgeloste triptiese soja sop vermeng met Pseudomonas sp strain CT07: gfp was gesirkuleer in die gemodifiseerde vloeisel deur ‘n multikanaal peristaltiese pomp vir 48 uur voordat die beskermde metaalskywe verwyder en gewas is om chemiese en aanpakwerende analise uit te voer. Al die industriële beskermingslae en biosied-verrykte epoksie-beskermingslae het aan die vereistes van termiese stabiliteit van ‘n waterverkoelingsisteem voldoen. Skandeer elektronmikroskopie (SEM) en X-straal spektroskopie (EDX) analise het aangetoon dat die aantrekkingseienskappe van industriële beskermingslae TC en QAC in waterige oplossings onvoldoende was en dat die koper- en silwerione uit die biosiedverrykte epoksie-resin beskermingslae diffundeer. Die kwalitatiewe analise van die aanpaksel van bakterieë op die oppervlaktes van beide industriële en biosied -verrykte epoksie-beskermingslae was bevestig deur SEM en CLSM. Die aangepakte bakterieë was verwyder en kwantitatief geanaliseer deur middel van plaattellings en vloeisitrometrie. Nie een van die industriële beskermingslae of die biosied-bevattende epoksie beskermingslae wat in hierdie studie gebruik is, is dus gepas vir gebruik op metaaloppervlaktes in waterverkoelingsisteme nie. Derdens was verskeie pogings aangewend om ‘n poli(stireen-alt-maleic anhidried) (SMA) beskermingslaag chemies te bind tot ‘n furanoon afgeleide 2.5-demitiel-4-hidroksie-3-(2H)- furanoon, tot die polimeer-ruggraat van die SMA beskermingslaag vir aanwending as ‘n aanpakwerende beskermingslaag vir waterverkoelingsisteme. Die sintese van SMA was bevestig deur 1H NMR en SEC en die sintese van tert-butyl 2-(2-hirdoksie-etoksie) etielkarbamaat en 4-(2- (2-(tert-butoksiekarboniel)etoksie)etoksie)-4-oksobutanoiesesuur was bevestig deur 1H NMR en ES-MS+. Die sintese van die uiteindelike afgeleide furanoon kon egter nie behaal word nie.

Autor no autoriza el acceso a texto completo de su documento hasta el 20/1/2021.
Magíster en Ciencias de la Ingeniería, Mención Química
Ingeniero Civil Químico
La acuicultura en Chile es el cuarto rubro exportador más importante, lo que ha hecho necesario el desarrollo de tecnologías que hagan más eficientes sus procesos. En particular, la proliferación de organismos sobre las mallas de cultivo (biofouling) es uno de los problemas relevantes que la tecnología tiene el desafío de resolver. Se trata de una dificultad tanto ambiental como económica, por lo que las estrategias de mitigación se vuelven fundamentales. El objetivo de este trabajo es desarrollar un material polimérico, con partículas de cobre incorporadas, que tenga acción antifouling en las mallas de cultivo producto de la liberación de iones cúpricos. Para llevar a cabo dicho objetivo, se procedió a adquirir y caracterizar tres partículas de cobre metálico con morfología y tamaño diferentes. Por otra parte, se sintetizaron tres partículas mediante la inmersión de algunos soportes inorgánicos (zeolita, óxido de silicio y nanotubos de carbono) en una solución de acetato de cobre. Las partículas obtenidas con el mencionado método, resultaron tener sales de cobre sobre los soportes inorgánicos utilizados. Para cuantificar el potencial anti-biofouling de las partículas adquiridas y sintetizadas, se midió el proceso de liberación de iones cúpricos en agua desionizada. El resultado de esta cuantificación es que las partículas adquiridas liberan iones cúpricos de forma más lenta y, por otra parte, que la cantidad de iones cúpricos liberados al final del ensayo, puede ser explicada mediante un modelo que supone una fase heterogénea en equilibrio, con especies iónicas presenten en la solución. Con las partículas adquiridas y sintetizadas, posteriormente se elaboraron compósitos poliméricos de poliamida y polietileno con 10% en peso de partículas. A partir de lo anterior, se procedió a medir la tasa de liberación de los materiales elaborados en agua de mar, para evaluar si estos superan el umbral de liberación de iones cúpricos, para obtener acción antifouling. El resultado es que en el mejor de los casos, solo superan dicho valor crítico hasta el quinceavo día y la tasa de liberación de iones cúpricos, mejora con el aumento de la polaridad de la matriz polimérica, el aumento del porcentaje de cobre de los rellenos y su disminución de la densidad. A partir de los resultados obtenidos de los ensayos de liberación desde los compósitos con partículas de cobre, y considerando que el polímero más utilizado en la industria acuícola es el polietileno, se evaluó la acción antifouling de algunos de ellos durante dos meses, midiendo las unidades formadoras de colonias por unidad de superficie al final del ensayo. Se encontró que las diferencias entre los blancos y las muestras, no son significativas, como es de esperarse tomando en consideración que estos compósitos solo superan el umbral de acción antifouling hasta el octavo día. Finalmente, la prolongación del tiempo en que la tasa de liberación está sobre el umbral, ayudará a que los materiales poliméricos con partículas de cobre, tengan una acción antifouling significativa. Frente a esto, la incorporación de un relleno como la celulosa, que se caracteriza por mejorar la captación de agua por compósitos poliméricos, ayudaría a prolongar el tiempo en que la tasa de liberación se encuentra sobre el umbral de acción antifouling.

Prevention of the accumulation of undesirable biological material i.e. biofouling upon a solid surface requires the use of antifouling systems. The solid surface may be a contact lens, an off shore oil rig or a living organism. When chemicals are employed as a mechanism of defense against biofouling, the agents involved are known as antifouling agents. Marine algae must protect themselves from fouling organisms and it is thought that one of the mechanisms used by these organisms is the production of secondary metabolites with an array of biological activities. In vitro studies have shown numerous compounds isolated from marine algae to possess antibacterial, antifungal and antimacrofouling activity. The aim of this study was to evaluate the secondary metabolite extracts of selected Southern African marine macro-algae as a potential source of compounds that inhibit biofilm formation and that could be used as antifouling agents. In this project, marine macro-algae were collected from various sites along the South African coastline. Their extracts were screened for antimicrobial activity against four ubiquitous microorganisms, Staphylococcus aureus, Klebsiella pneumoniae, Mycobacterium aurm and Candida albicans. Results of screening assays guided the fractionation of two Rhodophyta, Plocamium corallorhiza and Laurencia flexuosa. The algae were fractionated using silica gel column chromatography and compounds were isolated by semi-preparative normal phase HPLC. Compound characterization was performed using UV, IR and advanced one- and two-dimensional NMR (¹H, ¹³C NMR, COSY, HSQC, HMBC and NOESY) spectroscopy and mass spectrometry. Ten halogenated monoterpenes including four members of the small class of halogenated monoterpene aldehydes were isolated from extracts of P. corallorhiza. The compounds isolated included the known compounds 3,4,6,7-tetrachloro-3,7-dimethyl-1-octene; 4,6-dibromo-1, 1-dichloro-3,7 -dimethyl-2E,7 octadiene; 4,8-d ibromo-1,1,7 -trichloro-3, 7-dimethyl-2,5Eoctadiene;1 ,4,8-tribromo-3, 7 -dichloro-3,7-dimethyl-1 E,5E-octadiene; 8-bremo-6, 7-dichloro-3,7-dimethyl-octa-2E,4E-dienal; 4-Bromo-8-chloro-3,7-dimethyl-octa-2E,6E-dienal; 4,6- Dibromo-3,7-dimethyl-octa-2E,7-dienal; 2,4-dichloro-1-(2-chlorovinyl)-1-methyl-5-methylidene-cyclohexane and two new metabolites 4,8-chloro-3,7-dimethyl-2Z,4,6Z-octatrien-1-al and Compound 3.47. Methodology was developed for the chemical derivatization and mass spectrometric analysis of the aldehydic compounds, The aldehyde trapping reagent 0-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine hydrochloride was used to derivatize the molecules, stabilizing them and allowing for their complete characterization. From Laurencia flexuosa a new cuparene sesquiterpene 4-bremo-2-(5-hydroxy-1,2,2- trimethylcyclopent-3-enyl)-5-methylphenol was isolated along with two geometric isomers of the vinyl acetylene bromofucin , An halogenated monoterpene 3S*,4R*-1-bromo-3,4,8-trichloro-9-dichloromethyl-1-E,5-E,7-Z-octatriene was also isolated but was suspected to be a contaminant and an investigation into its biological source revealed that it originated from Plocamium suhrii. A third alga, Martensia elegans was extracted based on published reports of antimicrobial compounds in related species. A new a-alkyl malate derivative was isolated and characterized. Selected compounds isolated during the course of the study were employed in preliminary assays that tested their ability to inhibit biofilm formation by Pseudomonas aeruginosa. The halogenated monoterpenes isolated from the Plocamium species were the only active compounds. 3S*,4R*-1-bromo-3,4,S-trichloro-g-dichloromethyl-1-E,5-E,7-octatriene from P. suhrii inhibited biofilm formation through antibacterial activity on planktonic cells but could not prevent biofilm formation when employed as a film on the surface of microtitre plate wells. 1,4,8-tribromo-3,7-dichloro-3,7-dimethyl-1E,5E-octadiene and 4,6-dibromo-1,1-dichloro-3,7-dimethyl-2E,7-octadiene inhibited biofilm formation when applied as a film to the microtitre plate wells but had no significant antibacterial activity. No potential antifouling agents were identified in this project but the antimicrobial activity exhibited by the crude algal extracts was highly encouraging and a number of new research areas have been identified.
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Dans un contexte où les revêtements antifouling à base de biocides toxiques sont de plus en plus réglementés, il est indispensable de développer des solutions plus écologiques telles que les Fouling Release Coatings (FRCs) principalement à base d'élastomères silicones. Les FRCs peuvent relarguer facilement la salissure marine grâce à leurs propriétés physico-chimiques telles que leur faible énergie libre de surface et leur faible module élastique qui minimisent les interactions entre la salissure et la surface et diminuent les forces d'adhérence. Une autre catégorie de revêtements antifouling nommée Self-Polishing Coating, allie une érosion contrôlée de la surface à un relargage de biocides, permettant une protection efficace des coques de bateau contre la salissure marine. Cette thèse a pour objectif de créer de nouveaux FRCs dit « hybrides » car ils combinent différents composants et mécanismes d'action. Pour cela, deux stratégies ont été développées :1)des élastomères silicones renfermant des additifs hydrolysables avec des quantités allant de 5 à 20 %mass.2) des réseaux réticulés PDMS/polyester contenant de 12 à 27 % massique en polyester hydrolysable.La finalité de ces nouveaux revêtements était d'obtenir des surfaces chimiquement et/ou physiquement ambiguës vis-à-vis des organismes marins : (1) soit par la migration d'additifs hydrolysables en surface, par exemple, grâce à l'ajout de poly(méthacrylate de bis(triméthylsilyloxy)méthylsilyl), (2) soit par l'érosion du réseau hybride PDMS/polyester , par exemple, grâce à la réticulation de la poly(E-caprolactone) ou du poly(D,L-lactide-co-glycolide) avec les chaînes PDMS. Les propriétés physico-chimiques telles que la mouillabilité, l'énergie de surface, le module élastique et les propriétés d'hydrolyse/érosion des revêtements ont été étudiées avant et pendant leur immersion en milieu aqueux. L'efficacité antisalissure marine des revêtements a été évaluée lors de leur immersion in situ en mer Méditerranée et lors de tests biologiques ciblant des organismes marins spécifiques
Ln a context where biocidal antifouling coatings are more and more regulated, it is essential to develop more environmentally friendly systems, such as the Fouling Release Coatings (FRCs), mainly based on silicone elastomers. FRCs can release the marine biofouling thanks to their physico-chemical properties i.e. low surface free energy and low elastic modulus that minimize interactions between the foulant and the surface, and reduce the adhesion strength. Another category of antifouling coatings, named Self-Polishing Coatings, combines a controlled surface erosion as well as the release of biocides, and thus efficiently prevents the ·marine fouling from settling on ship hulls. The objective of this PhD work was to design navel « hybrid » FRCs that combine different components and mechanisms of action. Two strategies were thus developed:1) Silicone elastomers containing hydrolyzable additives from 5 to 20 wt.%2) Crosslinked PDMS/polyester networks with 12 to 27 wt.% of hydrolyzable polyesters.The aim of these navel coatings was to obtain surfaces chemically and physically ambiguous towards marine organisms: either (1) by the migration of hydrolyzable additives towards the surface, for example, thanks to the addition of poly(bis(trimethylsilyloxy)methylsilyl methacrylate) or (2) by the erosion of PDMS/polyester hybrid networks, for instance, thanks to the covalent crosslinking of poly(E-caprolactone) or poly(D,L-lactide-co-glycolide) segments with PDMS chains. Physico-chemical properties such as the wettability, the surface free energy, the elastic modulus, and the hydrolysis/erosion properties of the coatings have been studied before and during immersion in aqueous medium. The antifouling efficiency of the coatings has been evaluated during field immersion in the Mediterranean Sea, and bioassays targeting specific marine species

Titanium dental implants are a commonly used solution for the replacement of lost teeth. Even though the success rate is high, the number of infections related to the placement of the implant is still remarkable and may impair the proper function of the device, leading to health and economic costs. The infections related to medical devices start with a bacterial adhesion and proliferation on the material surface, leading to the formation of a complex biofilm able to protect the bacteria from the host immune response and the treatment with antibiotic. Due to the difficulty of treatment of the implant site one the biofilm is settled, one of the strategies to avoid the infection is to deal with the initial bacterial adhesion. This PhD thesis deals with the development of polymeric antibacterial coatings on titanium for dental implants, focusing on the achievement of fast and cost-effective procedure. With this aim, different coating strategies have been developed, tested and compared. A pre-treatment of the titanium surface was optimized in the first part of the thesis in order to achieve a clean surface and to enhance the chemical reactivity of the titanium oxide. With this aim, low pressure plasma activation was the selected method. The use of plasma activation allows for the removal of organic contaminants while increasing the surface energy of the treated surfaces. For the preparation of the polymeric antibacterial coatings, two different antifouling polymers have been used, namely, polyethylene glycol (PEG) and poly-2-hydroxyethylmetacrylate (PHEMA). PEG coatings were prepared by three different techniques, a wet chemical technique (silanization), a plasma enhanced chemical vapor deposition and an electrochemical process (electrodeposition). The three methods rendered an ultra-thin coating able to resist the bacterial adhesion. On the other hand, PHEMA-like coatings were prepared in a novel set-up by treating the liquid monomer by a plasma jet. Moreover, the different coatings were biofunctionalized in order to achieve multifunctionality and enhance the performance of the coating. For instance, the combination of PEG with a cell adhesion peptide (RGD) reported a better human fibroblast adhesion while maintaining the antifouling properties of the coating. PEG was also used as a platform for the immobilization of antimicrobial peptides (AMP). The bonding of the polymer with the AMP was optimized, achieving a surface able to reduce the bacterial adhesion and to kill the bacteria still able to adhere to the surface. Finally, the combination of two different plasma polymerized coatings with antibiotics (either Doxycycline or Vancomycin) was used as a drug delivery system.
Els implants dentals de titani són la solució més estesa per substituir peces dentals. Tot i que les taxes d'èxit són elevades, el nombre d'infeccions relacionades amb la col·locació de l'implant és elevat, i influeix en el mal funcionament de l'implant, amb un elevat cost tan a nivell econòmic com de salut. Les infeccions associades als dispositius sanitaris comencen amb una adhesió i proliferació dels bacteris a la superfície del material, que comporta la formació d'un biofilm capaç de protegir els bacteris de l'acció del sistema immunitari de l'hoste i del tractament amb antibiòtics. Aquesta tesi doctoral es basa en el desenvolupament de recobriments polimèrics antibacterians en titani per aplicacions dentals, buscant aconseguir mètodes ràpids i econòmics. Per tal d'assolir aquest objectiu, s'han desenvolupat, provat i comparat diferents estratègies per obtenir els recobriments. En la primera part de la tesi s'ha optimitzat un pretractament de la superfície del titani, per tal d'obtenir una superfície neta i millorar la reactivitat química de l'òxid de titani. El mètode seleccionat per l'activació ha estat l'activació per plasma, que permet eliminar els contaminants orgànics i augmentar l'energia superficial de les mostres tractades. Els polímers seleccionats per als recobriments han estat el polietilenglicol (PEG) i el 2-hidroxietilmetacrilat (PHEMA), que tenen propietats antifouling. Per preparar els recobriments de PEG s'han utilitzat tres mètodes diferents, la silanització, la polimerització per plasma i l'electrodeposició. Els tres mètodes han donat com a resultat una capa fina capaç de resistir l'adhesió bacteriana. Per altra banda, els recobriments amb PHEMA s'han preparat amb una nova metodologia, tractant el líquid amb un plasma jet. Els diversos recobriments s'han biofuncionalitzat per tal d'aconseguir una multifuncionalitat i millorar el seu funcionament. La combinació del PEG amb un pèptid d'adhesió cel·lular ha permès millorar l'adhesió de fibroblasts i mantenir les propietats antifouling del recobriment. La immobilització de pèptids antibacterians al PEG permet obtenir una superfície resistent a l'adhesió bacteriana i amb efecte antibacterià sobre els bacteris capaços d'adherir-se al recobriment. Per últim, la combinació de dos recobriments preparats per polimerització per plasma amb dos antibiòtics (vancomicina o doxiciclina) permet obtenir un sistema d'alliberació de fàrmacs a la superfície del titani.

Anthropogenic inputs of copper into the marine environment have increased in recent decades due to a ban on tributyl tin as an antifoulant and its subsequent replacement by copper. Dose-response experiments were conducted to assess the impact of copper on environmentally relevant biota: phytoplankton, brown alga, mussels, and amphipods.  Both alga and amphipods assimilated dose-dependent copper concentrations, while mussels did not.  Effects of copper exposure included reduced growth of algae, increased superoxide dismutase activity (SOD; an indicator of stress) in mussels and increased mortality in amphipods.  Reduced carbon and nitrogen assimilation and a change in diversity were measured in phytoplankton. Environmental surveys were employed to assess the dispersal of copper into water, sediment and biota from fish farm nets.  Alga and mussels transplanted to waters adjacent to fish farms were employed to assess copper uptake and impact.  A zone of increased copper concentration was found around farms with concentrations decreasing over distance.  Within the contaminated zone, or footprint, decreased growth in algae and increased stress in mussels were recorded. By incorporating different trophic levels into an ecotoxicological study this project provided a more detailed view of copper in the coastal ecosystem than those based on one species.  Of the methods employed, algae were the most consistent in showing a farm based impact.  The different methods and species used together showed the complexity of fish farm copper in the coastal environment.  Copper interacts with other farm outputs such as organic matter, which results in an impact over a limited distance from the cages.

Marine biofouling is the accumulation of organisms on underwater surfaces, causing increased ship hydrodynamic drag, which results in higher fuel consumption and decreased speed and range. Biofilms constitute a major component of the overall biofouling and may lead to a 14 % increase in ship fuel costs. Past solutions to antifouling (AF) have used toxic coatings which have subsequently been shown to severely affect marine life. The prohibited use of these antifoulants has led to the search for bio-inspired AF strategies. Current approaches towards the production of alternative coatings include the incorporation of natural AF compounds into paints. Screening assays for novel AF compounds are often separated into two categories; toxicity and AF assays. Increasingly there is evidence that active compounds affect organisms at non-toxic concentrations, hence, the necessity for more insightful AF testing, such as bacterial and diatom attachment. This study assessed natural product (NP) antifouling performance of two marine seaweeds (Chondrus crispus and Bifurcaria bifurcata) and two isolated pure compounds from terrestrial sources (usnic acid and juglone) against two marine biofilm bacteria, Cobetia marina and Marinobacter hydrocarbonoclasticus. Overall it was found that all NPs affected bacterial attachment, however, juglone demonstrated the best AF performance against both bacterial species at a concentration range between 5 - 20 ppm. Biofilm colonisation is a surface related phenomenon, thus novel bioassays have been developed to directly test biofilm attachment and growth on NP-containing coatings for both static and hydrodynamic conditions. This study has incorporated NPs into a model coating system, using two formulations in order to assess their effect on biofilm growth. Laboratory screening of NP-containing coatings is often largely unexplored mainly due to difficulties in assessing their activity over short experimental time scales (typically only a maximum of a few days). To date there are only a limited number of reports on laboratory assessment for antifouling paints and their effect on biofilm growth and/or attachment. In this study, NP-containing model paints were applied on to coupons, placed in 24-well plates and then inoculated with the marine biofilm forming bacteria. This has been achieved by the development of a novel bioassay protocol that has allowed the in situ observation of biofilm formation and growth, by corroborating different techniques such as a multidetection microplate reader and confocal laser scanning microscopy (through nucleic acid staining). There was good correlation between the two techniques which showed that the NP containing coatings significantly inhibited biofilm growth and also revealed marked differences in biofilm structure (e.g. bio-volume, morphology and thickness). The goal of this study was to develop a new protocol to allow assessment of biofilm formation on coatings in a high throughput non-invasive manner. New protocols and methods using microfluidic devices were developed for the assessment of bacterial attachments and initial biofilm formation in the presence and absence of a NP under hydrodynamic conditions. This led to the development and fabrication of a novel lab-on-a-chip device for the investigation of the biofilm response to different hydrodynamic conditions. The microfluidic flow channels were designed using computational fluid dynamic simulations so as to have a pre-defined, homogeneous wall shear stress in the channels, ranging from 0.03 to 4.30 Pa, which are relevant to in-service conditions on a ship hull.

O presente trabalho apresenta o estudo do desempenho de revestimentos antifouling obtidos a partir de tintas contendo polianilina ou derivados de polianilina aplicados sobre um substrato metálico e a comparação com tintas comerciais. As tintas foram processadas utilizando equipamento adequado visando a obtenção de produtos com qualidade equivalente aos disponíveis no mercado. Tanto a polianilina e seus derivados como as tintas contendo estes polímeros em sua formulação foram caracterizadas através de diferentes técnicas como espectroscopia de infravermelho, análise termogravimétrica, condutividade elétrica e microscopia eletrônica de varredura. Os revestimentos obtidos foram também avaliados através de ensaios de névoa salina, lixiviação e erosão. O desempenho antifouling dos revestimentos foi avaliado através de ensaios de imersão in situ tanto em ambiente marinho como em água doce. Algumas das tintas elaboradas contendo polianilina na sua formulação apresentaram resultados antiincrustantes similares ao da tinta comercial INTERMARINE (International).
Antifouling coatings were prepared with paints containing polyaniline (PAni) or its derivatives and evaluated by antifouling performance on metallic or polyvinyl chloride substrates. The paints were prepared in particular equipment in order to produce a paint with similar characteristics to those commercially available. The paints, the PAni and its derivatives were characterized with FT-IR spectrophotometry, thermogravimetric analysis, electrical conductivity and scanning electron microscopy. Beside that, the coatings were also characterized by salt spray test, leaching and erosion. The antifouling coatings performance was evaluated with immersion tests in marine environment or in fresh water. Some of the paints containing PAni showed antifouling performance similar to commercial antifouling paint.

Os processos de separação com membranas estão presentes nas mais diversas aplicações industriais. Em especial, a microfiltração e a ultrafiltração vêm sendo extensivamente utilizadas no tratamento de água e de efluentes. Com o aumento da demanda, muitos estudos têm sido feitos para melhorar o desempenho dos processos com membranas, porém a escolha apropriada da membrana é um fator crucial para atingir esse objetivo. As membranas à base de polietersulfona (PES) estão entre as mais utilizadas industrialmente para esse tipo de aplicação devido às suas excelentes propriedades mecânicas, estabilidade térmica e resistência química. No entanto, por serem pouco hidrofílicas, essas membranas apresentam baixos fluxos de água e elevada tendência ao fouling e ao biofouling. A fim de melhorar essas propriedades, muitos autores têm proposto modificações nas membranas para torná-las mais hidrofílicas e, portanto, aumentar o fluxo de água e diminuir o fouling. Dentro desse contexto, o presente trabalho teve como objetivo preparar membranas de ultrafiltração de PES, utilizando a ftaloil-quitosana (FQ) como aditivo, a fim de se obter membranas com propriedade antifouling. Membranas de PES e PES/FQ foram preparadas pelo processo de inversão de fases e caracterizadas quanto à morfologia, à estrutura química, à estabilidade térmica, ao caráter hidrofílico, à permeância hidráulica, à massa molar de corte (MMC) e ao desempenho em ultrafiltração de solução proteica. As membranas modificadas com o aditivo apresentaram estrutura mais heterogênea e com macrovazios maiores, maior caráter hidrofílico e maior permeância hidráulica. Os resultados de MMC não foram conclusivos. No teste de ultrafiltração, essas membranas apresentaram maiores fluxos e menor tendência ao fouling, indicando que o aditivo utilizado foi adequado na melhoria das propriedades desejadas. A retenção proteica, a perda de fluxo e a recuperação de fluxo após limpezas não apresentaram diferenças significativas.
In recent years, membrane separation processes have been widely used in all kinds of industries and applications. Particularly microfiltration and ultrafiltration have been extensively used for drinking water and wastewater treatments. With the increasing demand, many efforts have been done in order to enhance the process performance, but the choice of the appropriate membrane is a crucial factor to achieve this goal. Polyethersulfone (PES) based membranes are among the most commonly used for such applications due to their excellent chemical resistance, thermal stability and mechanical properties. However, because of its low hydrophilicity, these membranes have low water flux and high fouling and biofouling tendency. In order to improve these properties, many authors have proposed membrane modifications to make them more hydrophilic and thus increase the water flux and reduce fouling. Within this context, this work aimed to prepare PES ultrafiltration membranes using phthaloyl-chitosan (FQ) as an additive in order to obtain membranes with antifouling property. PES and PES/FQ membranes were prepared by phase inversion process and their morphology, chemical structure, thermal stability, hydrophilicity, hydraulic permeance, molecular weight cutoff (MWCO) and performance in ultrafiltration of protein solution were characterized. The membranes modified with additives showed more heterogeneous structure with larger macrovoids, higher hydrophilicity and higher hydraulic permeance. The MWCO results were inconclusive. In the ultrafiltration test, these membranes exhibited higher fluxes and lower fouling tendency, indicating that the additive used was adequate in improving the desired properties. The protein retention, flux reduction and flux recovery after cleaning showed no significant differences.

L'interdiction de produits antifouling à base de TBT a contraint les entreprises productrices de peintures à trouver rapidement un substituant approprié afin de limiter le phénomène de biofouling sur les coques des bateaux. Les autres biocides utilisés dans les peintures antifouling ont également soulevé de nombreuses questions quant à leurs effets sur l'environnement marin. Une alternative peut être apportée par le développement de revêtements dans lesquels les ingrédients actifs seraient des composés issus d'organismes marins. Des études récentes confirment le potentiel d'ingrédients actifs à base d'extraits d'algues marines dans les préparations antifouling. Nous avons démontré au cours de notre étude que les extraits de Sargassum muticum (Heterokonta, Fucales), de Polysiphonia lanosa (Rhodobionta, Céramiales) et de Ceramium botryocarpum (Rhodobionta, Céramiales) présentent une activité antifouling intéressante comparés aux biocides utilisés actuellement dans les peintures antifouling. Des tests d'incorporation d'extraits d'algues bruts dans des revêtements antifouling ont été effectués durant les étés 2004 et 2005 dans le port de Lorient, et les premiers résultats sont prometteurs. La purification de l'extrait éthanol/dichlorométhane de S. Muticum a permis d'isoler une fraction active dont la composition a été analysée. Les techniques de Spectrométrie de Masse, de Résonance Magnétique Nucléaire et de Chromatographie en Phase Gazeuse ont mis en évidence la présence d'acide palmitique et de dioctyl phthalate dans cette fraction. Les tests effectués sur les bactéries, microalgues et spores d'algues ont démontré l'efficacité antifouling de l'acide palmitique commercial
The ban of TBT-based antifouling products has forced the coating companies to find a suitable substitute to limit the development of fouling on ship hulls. The other organic biocides used in antifouling paints are suspected to have negative effects on the marine environment. An alternative exists with the development of antifouling coatings in which active ingredients would be isolated from marine organisms. Recent studies have demonstrated the potential of active compounds extracted from marine algae in antifouling paints. We have shown in our study that the extracts from Sargassum muticum (Heterokonta, Fucales), Polysiphonia lanosa (Rhodobionta, Ceramiales) and Ceramium botryocarpum (Rhodobionta, Ceramiales) present an interesting antifouling activity, which can be compared to the activity of the organic biocides used in antifouling paints. In situ tests have been conducted during the summer 2004 and 2005 in the harbour of Lorient, and the first results of crude extracts incorporated in coatings are promising. An active fraction has been isolated after the purification of the ethanol/dichloromethane extract from S. Muticum. Mass Spectrometry, Nuclear Magnetic Resonance and Gas Chromatography technics have shown the presence of palmitic acid and dioctyl phthalate in the active fraction. The tests conducted on bacteria, microalgae and algae spores have demonstrated the antifouling efficiency of the palmitic acid

Tributyltin is arguably the most toxic compound ever to be deliberately introduced into the marine environment as an ingredient of antifouling paints. It has had widespread toxic effects on a range of marine organisms, with some gastropod species being particularly sensitive. Effects of TBT on non-target species have resulted in partial bans on its use in many countries, so that new inputs to the water column have decreased in most areas. One of the physicochemical features of TBT is that it is readily sequestered by suspended particulates due to its low solubility and its hydrophobicity, therefore becoming incorporated into estuarine sediments. The availability of this sediment-bound TBT has been investigated through its potential for re-release back to the water column, and directly from the sediment using the sediment dwelling gastropod Hinia reticulata. The sorption process itself has been investigated using natural components to determine the sediment-water partition coefficient (Kd) together with factors affecting its magnitude. Sorption by sediments has been shown to be rapid (minutes), although the achievement of equilibrium may take longer (hours), and exhibits a Freundlich-like dependence on the TBT concentration due to the variable energies of TBT sorption sites on sediment particles. The major determinant of Kd is sediment type, greater adsorption occurring in fine-grained organic rich sediments compared to low organic sands; although both salinity and pH modify the degree of adsorption. The sorption process has been shown to be reversible, so that previously contaminated sediments may act as reservoirs of TBT, releasing the compound back to the overlying water for many years. Hinia reticulate has been shown to be an effective and quantitative accumulator of both dissolved and sediment-bound TBT, principally acquiring TBT from water across the respiratory surfaces. When additionally exposed to sediments, significantly higher body burdens were accumulated, with up to 80% of the total attributable to the sediment. Uptake of TBT across the surface of the headtfoot appears to be an important pathway for sediment-exposed Hinia reticulata, while the ingestion of contaminated sediment does not appear to occur. Hinia reticulata is capable of metabolising TBT to lesser butylated and presumably less toxic products which are excreted, making its accumulated body burdens responsive to changing environmental TBT levels, and increasing its value as a biomonitor When exposed to a range of TBT contaminated sediments, Hinia reticulata showed there to be greater TBT availability from sediments with a low sorptive capacity (sands), principally through desorption of TBT to the overlying water. Fine-grained organic-rich muds, which have a greater capacity for TBT, produced lower accumulated burdens in Hinia reticulata, but may represent more important long-term sources of TBT to benthic organisms in estuaries.