Sewage sludge ash-based mortar as construction material: Mechanical studies, macrofouling, and marine toxicity.

Incinerated sewage sludge ash is tested here as a cement and aggregate substitute in mortar blocks. It can be used at various percentages to reduce the overall cost of production and promote ash recycling. The compressive strength of the cast blocks was tested at 28 days to determine the optimal combination of ball milled ash (replacing cement) and sewage sludge ash (replacing sand). This was compared with a control block made of cement and sand only. The cast blocks with the optimal ash formulation were tested for their flexural strength and other properties such as surface functional groups, constituent phases and porosity. The control and ash mortars exhibited similar properties. A potential application of these blocks is to use them as part of seawalls. These blocks were thus suspended in the sea for 6 months. Marine organism attachment was observed over time in both control and ash mortar blocks. There was no significant difference between the mortars after 6 months. The mortar blocks were also subjected to leaching tests (NEN-7345). The leachates did not exhibit toxicity to microalgae. In contrast, mild toxicity was observed in the sea urchin embryo development assay. Overall, the study suggests that sewage sludge ash is a potential material to be used for seawall construction as it has the desirable mechanical properties. However, there remain some residual marine toxicity concerns that need to be further addressed.

A comparative investigation of toxicity of three polymer nanoparticles on acorn barnacle (Amphibalanus amphitrite).

Pollution from plastic waste is increasingly prevalent in the environment and beginning to generate significant adverse impact on the health of living organisms. In this study, we investigate the toxicity of polymer nanoparticles exposed to Acorn Barnacle (Amphibalanus amphitrite) nauplii, as an animal model. Highly stable aqueous dispersion of luminescent nanoparticles from three common polymers: polymethylmethacrylate (PMMA), polystyrene (PS), and polyvinylchloride (PVC), were prepared via nanoprecipitation and fully characterised. Exposure studies of these polymer particles to freshly spawned barnacle nauplii were performed within a concentration range from 1 to 25 mg/L under laboratory-controlled conditions. The exposure to PMMA and PS nanoparticles did not show detrimental toxicity and did not cause sufficient mortality to compute a LC50 value. However, PVC nanoparticles were significantly toxic with a mortality rate of up to 99% at 25 mg/L, and the calculated LC50 value for PVC nanoparticles was 7.66 ± 0.03 mg/L, 95% CI. Interestingly, PVC nanoparticle aggregates were observed to adhere to the naupliar carapace and appendages at higher concentrations and could not be easily removed by washings. To explore the possibility of chemical toxicity of polymer nanoparticles, analysis of the polymer powders which was used to prepare the nanoparticles was conducted. The presence of low molecular weight oligomers such as dimers, trimers and tetramers were observed in all polymer samples. The chemical nature and concentration of such compounds are likely responsible for the observed toxicity to the barnacle nauplii. Overall, our study shows that care should be exercised in generalising the findings of exposure studies performed using one type of plastic particles, as the use of different plastic particles may elicit different responses inside a living organism.

Ascidians collected during the 2013 Singapore Strait International Marine Biodiversity Workshop.

During the 2013 Singapore Strait International Marine Biodiversity Workshop, hundreds of ascidians were collected along with numerous other marine phyla. Collections were made intertidally as well as subtidally by snorkel, SCUBA, trawling and dredging. While many ascidian specimens remain unidentified, 14 aplousobranchs, nine phlebobranchs and 17 stolidobranchs have been identified; a surprising 50% of these comprise 20 new records for Singapore. An additional new record of the phlebobranch Perophora namei collected from the same area in 2014 and 2015 is included here. Of the 21 new records, Aplidium cf. grisiatum, Distaplia regina, Euherdmania cf. digitata, Cnemidocarpa irene, Monandrocarpa monotestis and Polyandrocarpa rollandi are also first records for the entire South China Sea region.

Incinerated Sewage Sludge Bottom Ash- Chemical processing, Leaching patterns and Toxicity testing.

Sewage sludge bottom ash, which is the major fraction obtained from the incineration of sewage sludge was treated with various organic and inorganic acids for heavy metal removal, along with a comparative phosphate treatment for heavy metal fixation. Malonic acid, an organic acid, was found to remove heavy metals better as compared to nitric acid, a strong inorganic acid. The acid treated samples were further examined for heavy metal leaching, followed by marine toxicity/abnormality testing of the leachates, where acid treated and phosphate treated ash leachate displayed higher (with malonic acid proving to be most toxic) and similar toxicity profiles as compared to raw ash leachate respectively. Raw ash was tested for its leaching patterns at different liquid/solid ratios(L/S = 5 and 10), salinities and time points (24, 48 and 72 h), where the leaching was found to saturate at L/S = 5 and at 24 h with varied salinity effecting the leaching insignificantly. When raw ash was benchmarked against concrete sand for marine toxicity, a material commonly used for land reclamation, acute toxicity patterns were found to be mostly similar except in case of the sea urchin embryonic assay, where toxicity was detected, indicating the sensitivity of the assay to residual levels of heavy metals. The raw ash was also tested against human cell lines where it displayed size and dose-dependent toxicity. To enable the use of ash for environment applications such as coastal reclamation, appropriate treatments are required to minimize leaching of potential harmful contaminants and this study demonstrates the importance of post-treatment of ash on its subsequent toxicity to organisms.

Measuring protein isoelectric points by AFM-based force spectroscopy using trace amounts of sample.

Protein charge at various pH and isoelectric point (pI) values is important in understanding protein function. However, often only trace amounts of unknown proteins are available and pI measurements cannot be obtained using conventional methods. Here, we show a method based on the atomic force microscope (AFM) to determine pI using minute quantities of proteins. The protein of interest is immobilized on AFM colloidal probes and the adhesion force of the protein is measured against a positively and a negatively charged substrate made by layer-by-layer deposition of polyelectrolytes. From the AFM force-distance curves, pI values with an estimated accuracy of ±0.25 were obtained for bovine serum albumin, myoglobin, fibrinogen and ribonuclease A over a range of 4.7-9.8. Using this method, we show that the pI of the 'footprint' of the temporary adhesive proteins secreted by the barnacle cyprid larvae of Amphibalanus amphitrite is in the range 9.6-9.7.

Polyion multilayers with precise surface charge control for antifouling.

We report on a molecular fabrication approach to precisely control surface ζ potentials of polymeric thin layers constructed by electrostatic layer-by-layer (LbL) assembly methods. The protocol established allows us to achieve surface isoelectric points (IEP) in the pH range of 6-10. Poly(acrylic acid) (PAA, a weak polyanion) and poly(diallyldimethylammonium chloride) (PDADMAC, a strong polycation) were chosen to build up the bulk films. The weak polycation polyethylenimine (PEI) was applied as a top layer. A unique feature of this approach is that the chemical composition of the top layer is not affected by the manipulation of the ζ potential of the films. Surface charge tuning is achieved by controlling the degree of ionization of the weak polyelectrolytes at various pH values and subsequent manipulation of the amount of polyelectrolyte deposited in the penultimate and last layers, respectively. Following assembly and characterization, the films were used as candidates for antifouling surfaces. The fouling behavior of barnacle cyprids and bacteria on the LbL films with similar hydrophilicity and roughness but different surface charge densities were studied. We found that more cyprids of Amphibalanus amphitrite settled on the negatively charged LbL film compared to the neutral or positively charged LbL film. In bacterial adhesion tests employing Pseudomonas, Escherichia coli, and Staphylococcus aureus, more bacteria were observed on the positively charged LbL film compared with the neutral and negatively charged LbL films, possibly as a result of the negative potential of the bacterial cell wall. The procedures proposed allow one to adjust surface isoelectric points of LbL architectures to achieve optimal antifouling performance of a given material taking into account specific pH values of the environment and the character of the fouler.

Barnacle larvae exploring surfaces with variable hydrophilicity: influence of morphology and adhesion of "footprint" proteins by AFM.

Interaction forces of adhesive proteins employed by cyprid larvae of Amphibalanus amphitrite for temporary attachment during surface exploration in marine fouling were studied by AFM force spectroscopy using chemically modified, reactive colloidal probes. The proteins were covalently attached to the surfaces of the probes by incubation in the protein deposits (footprints) left behind at the surface by the cyprids. This covalent coupling enabled robust and reproducible probing of adhesion of the attachment proteins to model surfaces with variable hydrophilicity. Three model monolayer surfaces were designed and prepared that exhibited different wettabilities derived from variations in the monolayer chemical composition. The morphology and size of cyprid protein deposits was imaged by AFM. The deposits showed larger area of spreading on more hydrophobic surfaces, whereas the overall volume of the secreted proteins exhibited no significant variation. Notable difference in adhesion forces was found among the surfaces by force spectroscopy, with substantially higher values measured on the hydrophobic surface (21 ± 2 nN) than that measured on the more hydrophilic surface (7.2 ± 1 nN). The same surfaces were also tested in laboratory essays. Rather surprisingly, no significant differences were found in values of fractional cyprid settlement among the surfaces studied, indicating that variations of surface wettability and adhesion strength of settlement proteins may be insufficient to explain settlement trends.

Sulfobetaine-based polymer brushes in marine environment: is there an effect of the polymerizable group on the antifouling performance?

Three different zwitterionic polymer brush coatings for marine biofouling control were prepared by surface-initiated atom transfer radical polymerization (ATRP) of sulfobetaine-based monomers including methacrylamide (SBMAm), vinylbenzene (SBVB) and vinylimidazolium (SBVI). None of these brush systems have been assessed regarding marine antifouling performance. Antifouling tests performed indicate that surfaces featuring these three brush systems substantially reduce the adhesion of the marine microalgae, Amphora coffeaeformis, and the settlement of cyprid larvae of the barnacle, Amphibalanus amphitrite, in a similar way, displaying comparable performance. Thus, it appears that the chemical structure of the polymerizable group has no substantial influence on marine antifouling performance.

Cross-linked polyelectrolyte multilayers for marine antifouling applications.

A polyionic multilayer film was fabricated by layer-by-layer (LbL) sequential deposition followed by cross-linking under mild conditions on a substrate surface to inhibit marine fouling. A novel polyanion, featuring methyl ester groups for an easy cross-linking was used as a generic solution for stabilization of LbL films in a harsh environment. Covalent cross-linking was confirmed by FTIR and XPS spectroscopy. AFM was used to observe film morphology and its variation because of cross-linking, as well as to measure the thickness of the LbL films. Cross-linking improved the stability of the LbL film when it was immersed in artificial seawater, natural seawater, and in a polar organic solvent (DMSO). No changes in the thickness and topography of the film were observed in these media. The LbL films prevented settlement of Amphibalanus amphitrite barnacle cyprids and reduced adhesion of the benthic diatom Amphora coffeaeformis. Assay results indicated that the cross-linking process did not weaken the antifouling effect of LbL films. The high stability and low degree of fouling make these coatings potentially promising candidates in marine applications.

A novel geometry for a laboratory-based larval settlement assay.

A novel configuration, consisting of two apposing surfaces bounding a vertical water column, is presented and evaluated for settlement assays using cyprids of Balanus amphitrite. Assays were conducted on planar surfaces, ranging from hydrophobic polystyrene to hydrophilic glass and including CH(3)- and NH(3) (+)-terminated self-assembled monolayers (SAMs). Identical apposing surfaces generated settlement rates comparable to those obtained in prior studies, while a choice assay yielded consistent results, with individual replicates each indicating the preferred surface for settlement. As gravity favours contact with the lower apposing surface, cyprids trapped at the air/water interface settled on or around the perimeter where the water column meets the lower substratum. These cyprids are capable of selecting a settlement location and are thus not lost to the assay. The assay geometry lends itself to assessing cyprid exploration and settlement on planar surfaces with chemical patterning, including relief microstructures, without using a confining material or requiring the coating of a three-dimensional well.

Functional polymer brushes via surface-initiated atom transfer radical graft polymerization for combating marine biofouling.

Dense and uniform polymer brush coatings were developed to combat marine biofouling. Nonionic hydrophilic, nonionic hydrophobic, cationic, anionic and zwitterionic polymer brush coatings were synthesized via surface-initiated atom transfer radical polymerization (SI-ATRP) of 2-hydroxyethyl methacrylate, 2,3,4,5,6-pentafluorostyrene, 2-(methacryloyloxy)ethyl trimethylammonium chloride, 4-styrenesulfonic acid sodium and N,N'-dimethyl-(methylmethacryloyl ethyl) ammonium propanesulfonate, respectively. The functionalized surfaces had different efficacies in preventing adsorption of bovine serum albumin (BSA), adhesion of the Gram-negative bacterium Pseudomonas sp. NCIMB 2021 and the Gram-positive Staphylococcus aureus, and settlement of cyprids of the barnacle Amphibalanus amphitrite (=Balanus amphitrite). The nonionic hydrophilic, anionic and zwitterionic polymer brushes resisted BSA adsorption during a 2 h exposure period. The nonionic hydrophilic, cationic and zwitterionic brushes exhibited resistance to bacterial fouling (24 h exposure) and cyprid settlement (24 and 48 h incubation). The hydrophobic brushes moderately reduced protein adsorption, and bacteria and cyprid settlement. The anionic brushes were least effective in preventing attachment of bacteria and barnacle cyprids. Thus, the best approach to combat biofouling involves a combination of nonionic hydrophilic and zwitterionic polymer brush coatings on material surfaces.

Natural antifoulants from the marine cyanobacterium Lyngbya majuscula.

Filamentous benthic marine cyanobacteria are a prolific source of structurally unique bioactive secondary metabolites. A total of 12 secondary metabolites, belonging to the mixed polyketide-polypeptide structural class, were isolated from the marine cyanobacterium, Lyngbya majuscula, and were tested to determine if they showed activity against barnacle larval settlement. The assays revealed four compounds, dolastatin 16, hantupeptin C, majusculamide A, and isomalyngamide A, that showed moderate to potent anti-larval settlement activities, with EC(50) values ranging from 0.003 to 10.6 microg ml(-1). In addition, field testing conducted over a period of 28 days (using the modified Phytagel method) based on the cyanobacterial compound, dolastatin 16, showed significantly reduced barnacle settlement as compared to controls at all the concentrations tested. The results of this study highlight the importance of marine cyanobacteria as an underexplored source of potential environmentally friendly antifoulants.

Marine biofouling field tests, settlement assay and footprint micromorphology of cyprid larvae of Balanus amphitrite on model surfaces.

Atomic force microscopy (AFM), laboratory settlement assays and field tests were used to correlate cyprid footprint (FP) morphology with the behaviour of cyprids on different substrata. AFM imaging under laboratory conditions revealed more porous and larger FPs on glass exposing a CH3-surface than on aminosilane functionalised (NH2-) surfaces. The secreted FP volume was found to be similar on both substrata (2.1-2.6 microm(3)). Laboratory settlement assays and marine field tests were performed on three substrata, viz. untreated clean glass, NH2-glass, and CH3-glass. The results distinguished settlement preferences for NH2-glass and untreated glass over CH3-terminated surfaces, suggesting that cyprids favour settling on hydrophilic over hydrophobic surfaces. On combining observations from different length scales, it is speculated that the confined FP size on NH2-glass may induce a higher concentration of the settlement inducing protein complex. Settlement may be further facilitated by a stronger adherence of FP adhesives to the NH2-surface via Coulombic interactions.