Bioassays and field immersion tests: a comparison of the antifouling activity of copper-free poly(methacrylic)-based coatings containing tertiary amines and ammonium salt groups

This paper focuses on the activity spectrum of three dimethylalkyl tertiary amines as potential active molecules and the corresponding ammonium salt-based antifouling (AF) paints. Bioassays (using marine bacteria, microalgae and barnacles) and field tests were combined to assess the AF activity of coatings. Bioassay results demonstrated that the ammonium salt-based paints did not inhibit the growth of microorganisms (except the dimethyldodecylammonium-based coatings) and that the tertiary amines were potent towards bacteria, diatoms, and barnacle larvae at non-toxic concentrations (therapeutic ratio, LC₅₀/EC₅₀, < 1). The results from field tests indicated that the ammonium salt-based coatings inhibited the settlement of macrofouling and the dimethylhexadecylammonium-based coatings provided protection against slime in comparison with PVC blank panels. Thus, results from laboratory assays did not fully concur with the AF activity of the paints in the field trial.     

A flow-through fluorescence polarization detection system for measuring GPCR-mediated modulation of cAMP production

A flow-through fluorescence polarization (FP) detection system that makes use of a novel high-performance liquid chromatography (HPLC) fluorescence detector modified with polarization filters was developed. This flow-through FP detection system was evaluated by using a novel and very cost-effective bioassay for cyclic adenosine monophosphate (cAMP). The bioassay was first evaluated and optimized in an FP plate reader format and subsequently in a flow-through bioassay setup. The principle of the bioassay is based on the competition of cAMP and a fluorescent cAMP derivative for the cAMP binding domain of protein kinase A. cAMP could accurately be determined over a range of 0.8 to 30 pmol/well in the plate reader FP assay and over a range of 0.3 to 50 pmol/well in the flow-through FP assay setup. High Z' factors (i.e., 0.89 for the plate reader and 0.93 for the flow-through FP cAMP assay, respectively) indicated robust assays. Finally, functional cAMP signaling of the human histamine H(3) G-protein-coupled receptor (GPCR) in cell cultures was measured with both assay formats with good sensitivities and assay windows. The pEC(50) values obtained in both assay formats were in accordance with those obtained with standard methods. The flow-through FP detection system could thus be used as a cost-effective alternative to FP plate reader assays. Moreover, the novel flow-through FP detection system for cAMP constitutes a good analytical tool to be used in the GPCR research field as an alternative to the use of FP plate readers or radioactive laboratories nowadays used for cAMP measurements.     

Combinatorial materials research applied to the development of new surface coatings IX: an investigation of novel antifouling/fouling-release coatings containing quaternary ammonium salt groups

Polysiloxane coatings containing chemically-bound ("tethered") quaternary ammonium salt (QAS) moieties were investigated for potential application as environmental-friendly coatings to control marine biofouling. A combinatorial/high-throughput approach was applied to the investigation to enable multiple variables to be probed simultaneously and efficiently. The variables investigated for the moisture-curable coatings included QAS composition, ie alkyl chain length, and concentration as well as silanol-terminated polysiloxane molecular weight. A total of 75 compositionally unique coatings were prepared and characterized using surface characterization techniques and biological assays. Biological assays were based on two different marine microorganisms, a bacterium, Cellulophaga lytica and a diatom, Navicula incerta, as well as a macrofouling alga, Ulva. The results of the study showed that all three variables influenced coating surface properties as well as antifouling (AF) and fouling-release (FR) characteristics. The incorporation of QAS moieties into a polysiloxane matrix generally resulted in an increase in coating surface hydrophobicity. Characterization of coating surface morphology revealed a heterogeneous, two-phase morphology for many of the coatings investigated. A correlation was found between water contact angle and coating surface roughness, with the contact angle increasing with increasing surface roughness. Coatings based on the QAS moiety containing the longest alkyl chain (18 carbons) displayed the highest micro-roughness and, thus, the most hydrophobic surfaces. With regard to AF and FR properties, coatings based on the 18 carbon QAS moieties were very effective at inhibiting C. lytica biofilm formation and enabling easy removal of Ulva sporelings (young plants) while coatings based on the 14 carbon QAS moities were very effective at inhibiting biofilm growth of N. incerta.     

Siloxane-based biocatalytic films and paints for use as reactive coatings

We have developed a new methodology for preparing films and paints suitable for use as biocatalytic coatings. The hydrolytic enzymes pronase and alpha-chymotrypsin were immobilized by either sol-gel entrapment or by covalent attachment into a polydimethylsiloxane (PDMS) matrix and cast into thin films or incorporated into an oil-based paint formulation. All of the coatings retained enzymatic activity and adhered to several different materials. The enzymatic films and paints also exhibited higher thermostability than enzyme free in solution or covalently attached to the outer surface of PDMS. A porous membrane based on a PDMS-immobilized enzyme was also prepared by an immersion precipitation process. Protein adsorption measurements showed that the enzyme-containing films and paints adsorbed less protein than enzyme-free controls, and that protein adsorption decreased with increasing proteolytic activity of the coating. These coatings thus provide the means to apply a stable enzymatic surface to a wide range of materials, and may be generally useful as biocatalytic paints and films.     

A methodology for evaluating biocide release rate, surface roughness and leach layer formation in a TBT-free, self-polishing antifouling coating

Due to the forthcoming IMO ban on the use of tributyltin (TBT) antifouling paints, a new generation of TBT-free coatings has been developed that typically contain cuprous oxide and an organic co-biocide. Accurate and reproducible test methods are needed to evaluate the performance and environmental impact of these new coatings. This study investigated a methodology for evaluating TBT-free, AF coatings containing cuprous oxide. A commercially available AF coating underwent rotary immersion testing at 0, 0.51 and 2.05 m s-1. Scanning electron microscopy (SEM) and energy dispersive x-ray (EDX) analysis were used to assess leach layer formation, percentage cuprous oxide by weight and particle size distribution (PSD). Biocide release rates and surface roughness were also measured. An increase in rotary speed caused a spike in Cu2+ release rate after which the release rate stabilised to previous levels. An increase in leach layer thickness was also observed after the rotary speed increase. A model is suggested to account for the observations.     

Combinatorial materials research applied to the development of new surface coatings IX: An investigation of novel antifouling/fouling-release coatings containing quaternary ammonium salt groups

Polysiloxane coatings containing chemically-bound (“tethered”) quaternary ammonium salt (QAS) moieties were investigated for potential application as environmental-friendly coatings to control marine biofouling. A combinatorial/high-throughput approach was applied to the investigation to enable multiple variables to be probed simultaneously and efficiently. The variables investigated for the moisture-curable coatings included QAS composition, ie alkyl chain length, and concentration as well as silanol-terminated polysiloxane molecular weight. A total of 75 compositionally unique coatings were prepared and characterized using surface characterization techniques and biological assays. Biological assays were based on two different marine microorganisms, a bacterium, Cellulophaga lytica and a diatom, Navicula incerta, as well as a macrofouling alga, Ulva. The results of the study showed that all three variables influenced coating surface properties as well as antifouling (AF) and fouling-release (FR) characteristics. The incorporation of QAS moieties into a polysiloxane matrix generally resulted in an increase in coating surface hydrophobicity. Characterization of coating surface morphology revealed a heterogeneous, two-phase morphology for many of the coatings investigated. A correlation was found between water contact angle and coating surface roughness, with the contact angle increasing with increasing surface roughness. Coatings based on the QAS moiety containing the longest alkyl chain (18 carbons) displayed the highest micro-roughness and, thus, the most hydrophobic surfaces. With regard to AF and FR properties, coatings based on the 18 carbon QAS moieties were very effective at inhibiting C. lytica biofilm formation and enabling easy removal of Ulva sporelings (young plants) while coatings based on the 14 carbon QAS moities were very effective at inhibiting biofilm growth of N. incerta.     

A methodology for evaluating biocide release rate,surface roughness and leach layer formation in a TBT-free,self-polishing antifouling coating

Abstract

Due to the forthcoming IMO ban on the use of tributyltin (TBT) antifouling paints, a new generation of TBT-free coatings has been developed that typically contain cuprous oxide and an organic co-biocide. Accurate and reproducible test methods are needed to evaluate the performance and environmental impact of these new coatings. This study investigated a methodology for evaluating TBT-free, AF coatings containing cuprous oxide. A commercially available AF coating underwent rotary immersion testing at 0, 0.51 and 2.05 m s^?1. Scanning electron microscopy (SEM) and energy dispersive x-ray (EDX) analysis were used to assess leach layer formation, percentage cuprous oxide by weight and particle size distribution (PSD). Biocide release rates and surface roughness were also measured. An increase in rotary speed caused a spike in Cu²⁺ release rate after which the release rate stabilised to previous levels. An increase in leach layer thickness was also observed after the rotary speed increase. A model is suggested to account for the observations.     

Pioneer marine biofilms on artificial surfaces including antifouling coatings immersed in two contrasting French Mediterranean coast sites

Marine biofilm communities that developed on artificial substrata were investigated using molecular and microscopic approaches. Polystyrene, Teflon® and four antifouling (AF) paints were immersed for 2 weeks at two contrasting sites near Toulon on the French Mediterranean coast (Toulon military harbour and the natural protected area of Porquerolles Island). Biofilms comprising bacteria and diatoms were detected on all the coatings. The population structure as well as the densities of the microorganisms differed in terms of both sites and coatings. Lower fouling densities were observed at Porquerolles Island compared to Toulon harbour. All bacterial communities (analysed by PCR-DGGE) showed related structure, controlled both by the sites and the type of substrata. Pioneer microalgal communities were dominated by the same two diatom species, viz. Licmophora gracilis and Cylindrotheca closterium, at both sites, irrespective of the substrata involved. However, the density of diatoms followed the same trend at both sites with a significant effect of all the AF coatings compared to Teflon and polystyrene.     

Pioneer marine biofilms on artificial surfaces including antifouling coatings immersed in two contrasting French Mediterranean coast sites

Marine biofilm communities that developed on artificial substrata were investigated using molecular and microscopic approaches. Polystyrene, Teflon? and four antifouling (AF) paints were immersed for 2 weeks at two contrasting sites near Toulon on the French Mediterranean coast (Toulon military harbour and the natural protected area of Porquerolles Island). Biofilms comprising bacteria and diatoms were detected on all the coatings. The population structure as well as the densities of the microorganisms differed in terms of both sites and coatings. Lower fouling densities were observed at Porquerolles Island compared to Toulon harbour. All bacterial communities (analysed by PCR-DGGE) showed related structure, controlled both by the sites and the type of substrata. Pioneer microalgal communities were dominated by the same two diatom species, viz. Licmophora gracilis and Cylindrotheca closterium, at both sites, irrespective of the substrata involved. However, the density of diatoms followed the same trend at both sites with a significant effect of all the AF coatings compared to Teflon and polystyrene.     

Screening of Marine Algal Extracts for Anti-settlement Activities against Microalgae and Macroalgae

The ban on the use of TBT-based antifouling paints for boats under 25 m in length has lead to a search for new non-toxic antifoulants. One of the most promising alternative technologies to heavy metal based antifouling paint is the development of antifouling coatings whose active ingredients are naturally occurring compounds from marine organisms. This is based on the principle that marine organisms also face the problem of the presence of epibionts on their own surfaces. In this study, the antifouling activity of a series of aqueous, ethanolic and dichloromethane extracts from thirty algae from the North East Atlantic coast was investigated. The extracts were tested in laboratory assays against species representative of two major groups of fouling organisms, viz . macroalgae and microalgae. The activity of several extracts was comparable to that of heavy metals and biocides (such as TBTO and CuSO 4 ) currently used in antifouling paints and their lack of toxicity with respect to the larvae of oysters and sea urchins suggests a potential for novel active ingredients.     

The potential for translocation of marine species via small-scale disruptions to antifouling surfaces

Vessel hull fouling is a major vector for the translocation of nonindigenous species (NIS). Antifouling (AF) paints are the primary method for preventing the establishment and translocation of fouling species. However, factors such as paint age, condition and method of application can all reduce the effectiveness of these coatings. Areas of hull that escape AF treatment (through limited application or damage) constitute key areas that may be expected to receive high levels of fouling. The investigation focused on whether small-scale (mm² to cm²) areas of unprotected surface or experimental ‘scrapes’ provided sufficient area for the formation of fouling assemblages within otherwise undamaged AF surfaces. Recruitment of fouling taxa such as algae, spirorbids and hydroids was recorded on scrapes as narrow as 0.5 cm wide. The abundance and species richness of fouling assemblages developing on scrapes ≥1 cm often equalled or surpassed levels observed in reference assemblages totally unprotected by AF coatings. Experiments were conducted at three sites within the highly protected and isolated marine park surrounding Lady Elliott Island at the southernmost tip of the Great Barrier Reef, Australia. Several NIS were recorded on scrapes of AF coated surfaces at this location, with 1-cm scrapes showing the greatest species richness and abundance of NIS relative to all other treatments (including controls) at two of the three sites investigated. Slight disruptions to newly antifouled surfaces may be all that is necessary for the establishment of fouling organisms and the translocation of a wide range of invasive taxa to otherwise highly protected marine areas.     

The potential for translocation of marine species via small-scale disruptions to antifouling surfaces

Vessel hull fouling is a major vector for the translocation of nonindigenous species (NIS). Antifouling (AF) paints are the primary method for preventing the establishment and translocation of fouling species. However, factors such as paint age, condition and method of application can all reduce the effectiveness of these coatings. Areas of hull that escape AF treatment (through limited application or damage) constitute key areas that may be expected to receive high levels of fouling. The investigation focused on whether small-scale (mm(2) to cm(2)) areas of unprotected surface or experimental 'scrapes' provided sufficient area for the formation of fouling assemblages within otherwise undamaged AF surfaces. Recruitment of fouling taxa such as algae, spirorbids and hydroids was recorded on scrapes as narrow as 0.5 cm wide. The abundance and species richness of fouling assemblages developing on scrapes > or =1 cm often equalled or surpassed levels observed in reference assemblages totally unprotected by AF coatings. Experiments were conducted at three sites within the highly protected and isolated marine park surrounding Lady Elliott Island at the southernmost tip of the Great Barrier Reef, Australia. Several NIS were recorded on scrapes of AF coated surfaces at this location, with 1-cm scrapes showing the greatest species richness and abundance of NIS relative to all other treatments (including controls) at two of the three sites investigated. Slight disruptions to newly antifouled surfaces may be all that is necessary for the establishment of fouling organisms and the translocation of a wide range of invasive taxa to otherwise highly protected marine areas.     

海洋真菌抗污损活性天然产物研究

黏附于海洋船舶或人工设施表面的污损生物给人类海洋生产活动与生态环境带来诸多不利影响.将具有抗污损活性的化合物开发成防污涂料是目前防治海洋生物污损的最常用手段之一.而大量传统有机金属防污剂因其严重毒副作用被禁用,亟须开发高效、环境友好型抗污损涂料.海洋真菌能够产生大量结构新颖、作用机制独特的高效、低毒/无毒抗污损活性次级...     

Short-term testing of antifouling surfaces: the importance of colour

Data from short-term biofouling assays are frequently used to evaluate the performance of antifouling (AF) coatings. There are a large number of factors, however, that may influence community development. One such factor is colour. The hypothesis was that differences in colour may impact the short-term development of a biofouling community and therefore bias the results. An experiment was designed to investigate the effect of black and white substrata on settlement of fouling organisms in the field. Both Ulva sp. and Spirorbis sp. had significantly higher settlement on black surfaces. This result emphasises the importance of considering colour and other factors when undertaking short-term testing of AF coatings.     

Effects of crosslink density in zwitterionic hydrogel coatings on their antifouling performance and susceptibility to silt uptake

Abstract

Hydrogel coatings effectively reduce the attachment of proteins and organisms in laboratory assays, in particular when made from zwitterionic monomers. In field experiments with multiple species and non-living material, such coatings suffer from adsorption of particulate matter. In this study, the zwitterionic monomer 3-[N-(2-methacryloyloxyethyl)-N,N-dimethylammonio] propanesulfonate (SPE) was copolymerized with increasing amounts of the photo-crosslinker benzophenon-4-yloxyethyl methacrylate (BPEMA) to systematically alter the density of crosslinks between the polymer chains. The effect of increasing crosslink density on the antifouling (AF) performance of the coatings was investigated in laboratory assays and fields tests. In both cases, the AF performance was improved by increasing the crosslinker content. The coatings reduced protein, diatom, and barnacle accumulation, and showed better resistance to biomass accumulation. The findings underline that the marine AF performance of hydrogel coatings does not only depend on the specific chemical structure of the polymers, but also on their physico-chemical properties such as rigidity and swelling.     

Laboratory assessment of the antifouling potential of a soluble-matrix paint laced with the natural compound polygodial

Polygodial is a potent and selective inhibitor of ascidian metamorphosis that shows promise for controlling fouling by ascidians in bivalve aquaculture. The current study examined the potency of, and associated effects of seawater exposure on, a rosin-based soluble-matrix paint laced with 0.08–160?ng?polygodial?g^?1 wet paint matrix. Paint-coated surfaces were soaked in seawater for 0, 2, 4 or 12?weeks prior to screening for antifouling activity using a bioassay based on the nuisance ascidian Ciona savignyi Herdman. Mortality was greater (mean 50% lethal concentration: 5?±?2?ng?g^?1; mean 75% lethal concentration: 17?±?4?ng?g^?1) and metamorphosis was inhibited (mean 50% anti-metamorphic concentration: 2?±?0.4?ng?g^?1; mean 75% anti-metamorphic concentration: 15?±?10?ng?g^?1) in C. savignyi larvae exposed to polygodial-laced soluble-matrix paints, relative to control paints without polygodial. Soaking in seawater prior to testing reduced the efficacy of the formulation up to nearly 12-fold, but even after soaking for 12?weeks paints laced with polygodial at 160?ng?g^?1 wet paint matrix prevented ?90% of the larvae of C. savignyi from completing metamorphosis. The outcome of this experiment provides a positive first step in evaluating the suitability of polygodial-laced soluble-matrix paints for use in aquaculture.     

Development and characteristics of an adhesion bioassay for ectocarpoid algae

Species of filamentous brown algae in the family Ectocarpaceae are significant members of fouling communities. However, there are few systematic studies on the influence of surface physico-chemical properties on their adhesion. In the present paper the development of a novel, laboratory-based adhesion bioassay for ectocarpoid algae, at an appropriate scale for the screening of sets of experimental samples in well-replicated and controlled experiments is described. The assays are based on the colonization of surfaces from a starting inoculum consisting of multicellular filaments obtained by blending the cultured alga Ectocarpus crouaniorum. The adhesion strength of the biomass after 14 days growth was assessed by applying a hydrodynamic shear stress. Results from adhesion tests on a set of standard surfaces showed that E. crouaniorum adhered more weakly to the amphiphilic Intersleek? 900 than to the more hydrophobic Intersleek? 700 and Silastic? T2 coatings. Adhesion to hydrophilic glass was also weak. Similar results were obtained for other cultivated species of Ectocarpus but differed from those obtained with the related ectocarpoid species Hincksia secunda. The response of the ectocarpoid algae to the surfaces was also compared to that for the green alga, Ulva.     

On-line monitoring of antifouling and fouling-release surfaces using bioluminescence and fluorescence measurements during laminar flow

A laminar flow biofilm-monitoring system was used to determine the efficacies of three antifouling (AF) coatings and five fouling-release (FR) coatings againstVibrio harveyi attachment. On-line measurements of tryptophan fluorescence and bioluminescence from each coating, normalized to an upstream stainless steel coupon, were used to determine the effects of AF and FR surfaces on biofilm formation. The AF coatings consisted of 5, 10, and 35 wt% Sea Nine 211 (C9211) incorporated into a vinyl copolymer. Both the 10 and 35 wt% coatings significantly inhibited biofilm biomass development measured by tryptophan fluorescence compared to the stainless steel control.V. harveyi bioluminescence was significantly greater than tryptophan fluorescence in cells attached to these coatings, suggesting that bioluminescence expression may be a marker for cellular stress or toxicity in biofilms. Five different polydimethylsiloxane (PDMS) FR coatings did not inhibit biofilm formation under low flow conditions. However, four PDMS coatings demonstrated decreased biomass levels compared to stainless steel after exposure to a shear stress of 330 dynes cm^–2. There was no toxic additive in these coatings; bioluminescence and tryptophan fluorescence were proportional.     

Short-term testing of antifouling surfaces: the importance of colour

Abstract

Data from short-term biofouling assays are frequently used to evaluate the performance of antifouling (AF) coatings. There are a large number of factors, however, that may influence community development. One such factor is colour. The hypothesis was that differences in colour may impact the short-term development of a biofouling community and therefore bias the results. An experiment was designed to investigate the effect of black and white substrata on settlement of fouling organisms in the field. Both Ulva sp. and Spirorbis sp. had significantly higher settlement on black surfaces. This result emphasises the importance of considering colour and other factors when undertaking short-term testing of AF coatings.     

Natural products as antifouling compounds: recent progress and future perspectives

Since early 2008, an increasing number of countries have ratified an international treaty to ban the application of antifouling (AF) coatings based on organotin compounds (eg tributyltin (TBT) and triphenyltin). As a result, the demand for environmentally friendly, non-toxic or low-toxicity AF compounds and technologies (green AF agents) has become an urgent reality. Marine coatings based on Cu₂O and various other biocides have a negative impact on the environment and they must eventually be replaced by new, effective, and environmentally friendly AF compounds. This mini-review describes important AF compounds discovered from a variety of organisms from 2004 until mid 2009, and discusses recent and general trends in the discovery of AF compounds. Finally, a perspective on the future of AF compound development is presented. The discussion is aimed at updating scientists and engineers on the current challenges facing AF research.