PDMS networks meet barnacles: a complex and often toxic relationship.

The biological impact of chemical formulations used in various coating applications is essential in guiding the development of new materials that directly contact living organisms. To illustrate this point, an investigation addressing the impact of chemical compositions of polydimethylsiloxane networks on a common platform for foul-release biofouling management coatings was conducted. The acute toxicity of network components to barnacle larvae, the impacts of aqueous extracts of crosslinker, silicones and organometallic catalyst on trypsin enzymatic activity, and the impact of assembled networks on barnacle adhesion was evaluated. The outcomes of the study indicate that all components used in the formulation of the silicone network alter trypsin enzymatic activity and have a range of acute toxicity to barnacle larvae. Also, the adhesion strength of barnacles attached to PDMS networks correlates to the network formulation protocol. This information can be used to assess action mechanisms and risk-benefit analysis of PDMS networks.

Resistance of Zwitterionic Peptide Monolayers to Biofouling.

Self-assembled monolayers (SAMs) are widely used in science and engineering, and recent progress has demonstrated the utility of zwitterionic peptides with alternating lysine (K) and glutamic acid (E) residues for antifouling purposes. Aiming at developing a peptide-based fouling-resistant SAM suitable for presentation of surface-attached pheromones for barnacle larvae, we have investigated five different peptide SAMs, where four are based on the EK motif, and the fifth was designed based on general principles for fouling resistance. The SAMs were formed by self-assembly onto gold substrates via cysteine residues on the peptides, and formation of SAMs was verified via ellipsometry, wettability, infrared reflection-absorption spectroscopy and cyclic voltammetry. Settlement of cypris larvae of the barnacle Balanus (=Amphibalanus) amphitrite, the target of pheromone studies, was tested. SAMs were also subjected to fouling assays using protein solutions, blood serum, and the bacterium Mycobacterium marinum. The results confirm the favorable antifouling properties of EK-containing peptides in most of the assays, although this did not apply to the barnacle larvae settlement test, where settlement was low on only one of the peptide SAMs. The one peptide that had antifouling properties for barnacles did not contain a pheromone motif, and would not be susceptible to degredation by common serine proteases. We conclude that the otherwise broadly effective antifouling properties of EK-containing peptide SAMs is not directly applicable to barnacles, and that great care must be exercised in the design of peptide-based SAMs for presentation of barnacle-specific ligands.

Effects of Toxic Leachate from Commercial Plastics on Larval Survival and Settlement of the Barnacle Amphibalanus amphitrite.

Plastic pollution represents a major and growing global problem. It is well-known that plastics are a source of chemical contaminants to the aquatic environment and provide novel habitats for marine organisms. The present study quantified the impacts of plastic leachates from the seven categories of recyclable plastics on larval survival and settlement of barnacle Amphibalanus (=Balanus) amphitrite. Leachates from plastics significantly increased barnacle nauplii mortality at the highest tested concentrations (0.10 and 0.50 m(2)/L). Hydrophobicity (measured as surface energy) was positively correlated with mortality indicating that plastic surface chemistry may be an important factor in the effects of plastics on sessile organisms. Plastic leachates significantly inhibited barnacle cyprids settlement on glass at all tested concentrations. Settlement on plastic surfaces was significantly inhibited after 24 and 48 h, but settlement was not significantly inhibited compared to the controls for some plastics after 72-96 h. In 24 h exposure to seawater, we found larval toxicity and inhibition of settlement with all seven categories of recyclable commercial plastics. Chemical analysis revealed a complex mixture of substances released in plastic leachates. Leaching of toxic compounds from all plastics should be considered when assessing the risks of plastic pollution.

Incorporation of silicone oil into elastomers enhances barnacle detachment by active surface strain.

Silicone-oil additives are often used in fouling-release silicone coatings to reduce the adhesion strength of barnacles and other biofouling organisms. This study follows on from a recently reported active approach to detach barnacles, which was based on the surface strain of elastomeric materials, by investigating a new, dual-action approach to barnacle detachment using Ecoflex®-based elastomers incorporated with poly(dimethylsiloxane)-based oil additives. The experimental results support the hypothesis that silicone-oil additives reduce the amount of substratum strain required to detach barnacles. The study also de-coupled the two effects of silicone oils (ie surface-activity and alteration of the bulk modulus) and examined their contributions in reducing barnacle adhesion strength. Further, a finite element model based on fracture mechanics was employed to qualitatively understand the effects of surface strain and substratum modulus on barnacle adhesion strength. The study demonstrates that dynamic substratum deformation of elastomers with silicone-oil additives provides a bifunctional approach towards management of biofouling by barnacles.

Dynamic surface deformation of silicone elastomers for management of marine biofouling: laboratory and field studies using pneumatic actuation.

Many strategies have been developed to improve the fouling release (FR) performance of silicone coatings. However, biofilms inevitably build on these surfaces over time. Previous studies have shown that intentional deformation of silicone elastomers can be employed to detach biofouling species. In this study, inspired by the methods used in soft-robotic systems, controlled deformation of silicone elastomers via pneumatic actuation was employed to detach adherent biofilms. Using programmed surface deformation, it was possible to release > 90% of biofilm from surfaces in both laboratory and field environments. A higher substratum strain was required to remove biofilms accumulated in the field environment as compared with laboratory-grown biofilms. Further, the study indicated that substratum modulus influences the strain needed to de-bond biofilms. Surface deformation-based approaches have potential for use in the management of biofouling in a number of technological areas, including in niche applications where pneumatic actuation of surface deformation is feasible.

Barnacle cement: a polymerization model based on evolutionary concepts.

Enzymes and biochemical mechanisms essential to survival are under extreme selective pressure and are highly conserved through evolutionary time. We applied this evolutionary concept to barnacle cement polymerization, a process critical to barnacle fitness that involves aggregation and cross-linking of proteins. The biochemical mechanisms of cement polymerization remain largely unknown. We hypothesized that this process is biochemically similar to blood clotting, a critical physiological response that is also based on aggregation and cross-linking of proteins. Like key elements of vertebrate and invertebrate blood clotting, barnacle cement polymerization was shown to involve proteolytic activation of enzymes and structural precursors, transglutaminase cross-linking and assembly of fibrous proteins. Proteolytic activation of structural proteins maximizes the potential for bonding interactions with other proteins and with the surface. Transglutaminase cross-linking reinforces cement integrity. Remarkably, epitopes and sequences homologous to bovine trypsin and human transglutaminase were identified in barnacle cement with tandem mass spectrometry and/or western blotting. Akin to blood clotting, the peptides generated during proteolytic activation functioned as signal molecules, linking a molecular level event (protein aggregation) to a behavioral response (barnacle larval settlement). Our results draw attention to a highly conserved protein polymerization mechanism and shed light on a long-standing biochemical puzzle. We suggest that barnacle cement polymerization is a specialized form of wound healing. The polymerization mechanism common between barnacle cement and blood may be a theme for many marine animal glues.

Recyclable plastics as substrata for settlement and growth of bryozoans Bugula neritina and barnacles Amphibalanus amphitrite.

Plastics are common and pervasive anthropogenic debris in marine environments. Floating plastics provide opportunities to alter the abundance, distribution and invasion potential of sessile organisms that colonize them. We selected plastics from seven recycle categories and quantified settlement of (i) bryozoans Bugula neritina (Linnaeus, 1758) in the lab and in the field, and of (ii) barnacles Amphibalanus (= Balanus) amphitrite (Darwin, 1854) in the field. In the laboratory we cultured barnacles on the plastics for 8 weeks and quantified growth, mortality, and breaking strength of the side plates. In the field all recyclable plastics were settlement substrata for bryozoans and barnacles. Settlement depended on the type of plastic. Fewer barnacles settled on plastic surfaces compared to glass. In the lab and in the field, bryozoan settlement was higher on plastics than on glass. In static laboratory rearing, barnacles growing on plastics were initially significantly smaller than on glass. This suggested juvenile barnacles were adversely impacted by materials leaching from the plastics. Barnacle mortality was not significantly different between plastic and glass surfaces, but breaking strength of side plates of barnacles on polyvinyl chloride (PVC) and polycarbonate (PC) were significantly lower than breakage strength on glass. Plastics impact marine ecosystems directly by providing new surfaces for colonization with fouling organisms and by contaminants shown previously to leach out of plastics and impact biological processes.

Modification of Silicone Elastomer Surfaces with Zwitterionic Polymers: Short-Term Fouling Resistance and Triggered Biofouling Release.

We present a method for dual-mode-management of biofouling by modifying surface of silicone elastomers with zwitterionic polymeric grafts. Poly(sulfobetaine methacrylate) was grafted from poly(vinylmethylsiloxane) elastomer substrates using thiol-ene click chemistry and surface-initiated, controlled radical polymerization. These surfaces exhibited both fouling resistance and triggered fouling-release functionality. The zwitterionic polymers exhibited fouling resistance over short-term (∼hours) exposure to bacteria and barnacle cyprids. The biofilms that eventually accumulated over prolonged-exposure (∼days) were easily detached by applying mechanical strain to the elastomer substrate. Such dual-functional surfaces may be useful in developing environmentally and biologically friendly coatings for biofouling management on marine, industrial, and biomedical equipment because they can obviate the use of toxic compounds.

Bioinspired surfaces with dynamic topography for active control of biofouling.

Dynamic change of the surface area and topology of elastomers is used as a general, environmentally friendly approach for effectively detaching micro- and macro-fouling organisms adhered on the elastomer surfaces. Deformation of elastomer surfaces under electrical or pneumatic actuation can debond various biofilms and barnacles. The bio-inspired dynamic surfaces can be fabricated over large areas through simple and practical processes. This new mechanism is complementary with existing materials and methods for biofouling control.

The effects of model polysiloxane and fouling-release coatings on embryonic development of a sea urchin (Arbacia punctulata) and a fish (Oryzias latipes).

In recent decades attention has focused on the development of non-toxic fouling-release coatings based on silicone polymers as an alternative to toxic antifouling coatings. As fouling-release coatings gain market share, they will contribute to environmental contamination by silicones. We report effects of eight model polysiloxane and three commercial foul-release coatings on embryonic development of sea urchins and fish, Japanese medaka. We used model coatings because they have known composition and commercially available components and molecules leaching from these coatings have been partially characterized. The commercial fouling-release coatings are purported to be non-toxic and components are proprietary. Our goal was to expose embryos of well studied model animals to the coatings to determine if the complex mixtures leaching from the coatings impact development. Urchins were chosen because development is rapid and embryos can enter the non-slip layer over surfaces. Medaka was chosen because the female deposits the sticky eggs onto the anal fin and then scrapes them off onto surfaces. Embryos were confined in water over coatings in 24 well plates. Fresh model coatings had no effect on urchin development while commercial fouling-release coatings inhibited development. Fish embryos had delayed hatching, increased mortality of hatchlings and dramatically decreased ability of hatchlings to inflate the swim bladder and reduced hatching success on all coatings. After one-month immersion of coatings in running seawater to simulate initial application in the marine environment, sea urchin embryos died when placed over model silicones. Effects of the commercial coatings were reduced but included retarded development. Effects on fish embryos over leached coating were reduced compared to those of fresh coating and included decreased hatching success, decreased hatchling survival and inability to inflate the swim bladder for commercial coatings. These findings suggest, similar to medical conclusions, compounds leaching from silicone coatings can impact development and the topic deserves study.

Noradrenaline-functionalized hyperbranched fluoropolymer-poly(ethylene glycol) cross-linked networks as dual-mode, anti-biofouling coatings.

The strategy of decorating antibiofouling hyperbranched fluoropolymer-poly(ethylene glycol) (HBFP-PEG) networks with a settlement sensory deterrent, noradrenaline (NA), and the results of biofouling assays are presented. This example of a dual-mode surface, which combines both passive and active modes of antibiofouling, works in synergy to improve the overall antibiofouling efficiency against barnacle cyprids. The HBFP-PEG polymer surface, prior to modification with NA, was analyzed by atomic force microscopy, and a significant distribution of topographical features was observed, with a nanoscopic roughness measurement of 110 ± 8 nm. NA attachment to the surface was probed by secondary ion mass spectrometry to quantify the extent of polymer chain-end substitution with NA, where a 3- to 4-fold increase in intensity for a fragment ion associated with NA was observed and 39% of the available sites for attachment were substituted. Cytoskeletal assays confirmed the activity of tethered NA on adhering oyster hemocytes. Settlement assays showed deterrence toward barnacle cyprid settlement, while not compromising the passive biofouling resistance of the surface. This robust strategy demonstrates a methodology for the incorporation of actively antibiofouling moieties onto a passively antibiofouling network.

Compounds from silicones alter enzyme activity in curing barnacle glue and model enzymes.

BACKGROUND: Attachment strength of fouling organisms on silicone coatings is low. We hypothesized that low attachment strength on silicones is, in part, due to the interaction of surface available components with natural glues. Components could alter curing of glues through bulk changes or specifically through altered enzyme activity. METHODOLOGY/PRINCIPAL FINDINGS: GC-MS analysis of silicone coatings showed surface-available siloxanes when the coatings were gently rubbed with a cotton swab for 15 seconds or given a 30 second rinse with methanol. Mixtures of compounds were found on 2 commercial and 8 model silicone coatings. The hypothesis that silicone components alter glue curing enzymes was tested with curing barnacle glue and with commercial enzymes. In our model, barnacle glue curing involves trypsin-like serine protease(s), which activate enzymes and structural proteins, and a transglutaminase which cross-links glue proteins. Transglutaminase activity was significantly altered upon exposure of curing glue from individual barnacles to silicone eluates. Activity of purified trypsin and, to a greater extent, transglutaminase was significantly altered by relevant concentrations of silicone polymer constituents. CONCLUSIONS/SIGNIFICANCE: Surface-associated silicone compounds can disrupt glue curing and alter enzyme properties. Altered curing of natural glues has potential in fouling management.