Antifouling activity of isonitrosoacetanilides against microfouling and macrofouling.

Biofouling is responsible for structural and economic damage to man-made surfaces. Antifouling paints with biocides have been applied to structures to avoid organism adhesion; however, they have high toxicity and are not able to prevent all biofouling processes, necessitating the periodic mechanical removal of organisms and paint reapplication. Thus, there is an urgent demand for novel, effective, and environmentally friendly antifouling alternatives. As isonitrosoacetanilide is the precursor for many compounds with antibacterial activity, we believe that it could have antifouling activity against microfouling and, consequently, against macrofouling. The aim of this work was to investigate the antifouling potential of six isonitrosoacetanilide compounds and their toxicity. The compounds were employed at different concentrations (0.625-1.25-2.5-5-10 µg mL-1) in this study. The biofilm and planktonic bacteria inhibition and biofilm eradication potential were evaluated by crystal violet assay, while Amphibalus amphitrite barnacle settlement was evaluated by cyprid settlement assay. Toxicity evaluation (LC50 and EC50) was performed with A. amphitrite nauplii II and cyprid larvae. At least one of the tested concentrations of 4-Br-INA, 4-CH3-INA, and 2-Br-INA compounds showed nontoxic antifouling activity against microfouling (antibiofilm) and macrofouling (antisettlement). However, only 4-CH3-INA and 2-Br-INA also showed biofilm eradication potential. These compounds with antibiofilm activity and nontoxic effects could be combined with acrylic base paint resin or added directly into commercial paints in place of toxicant biocides to cover artificial structures as friendly antifouling agents.

Acartia tonsa Dana 1849 as a Model Organism: Considerations on Acclimation in Ecotoxicological Assays.

The copepod Acartia tonsa was standardized as model organism in acute toxicity bioassays due to its key position in coastal food chains and high sensitivity. Once bioassays are performed according to a protocol their results may become tools for the protection of aquatic ecosystems. However, there are divergences in bioassays methods using A. tonsa. This study aimed to investigate: (i) the need for acclimation of A. tonsa collected from the environment for use in acute toxicological bioassays; and (ii) differences in sensitivity between copepods collected from the environment and laboratory-grown copepods. Laboratory-grown copepods are more sensitive to SDS than A. tonsa from the environment. The acclimation time of 30 h helped organisms to recover from stress of collection/handling and changing environment/conditions. Therefore, laboratory-grown copepods showed to be more sensitive than organisms from environment; and for ecotoxicological bioassays acclimating A. tonsa collected from the environment for 30 h can be adopted.

Bacteria-invertebrate interactions as an asset in developing new antifouling coatings for man-made aquatic surfaces.

Economic losses can result from biofouling establishment on man-made structures. Macrofouling causes damage to artificial substrates, which justifies the need for its control. However, the antifouling coatings employed nowadays are typically not safe for the environment. Microfouling can affect macrofouling colonization, and thus represents a potential target for alternative antifouling control. From both ecological and economical points of view, information on the ecology and interactions between micro- and macrofouling are crucial to develop successful and safe control strategies, which will prevent biofouling development on man-made structures while preserving water quality and the safety of non-target organisms. This study presents a metabarcoding analysis of biofilm-associated marine bacteria (16S-rRNA-gene) and fungi (ITS-region), with the aim to understand invertebrate settlement over time on hard substrates exposed to natural condition (Control) and two treatments (Antimicrobials and Antifouling Painted). Biofouling composition changed with exposure time (up to 12 days) and showed differences among Control and Antimicrobials and Painted treatments. Antimicrobial treatment influenced more the biofouling composition than traditional antifouling paint (Cu2O-based). Both treatments caused microbial resistance. Macrofouling establishment was strongly influenced by Gram-negative heterotrophic bacteria (mostly Proteobacteria and Bacteroidetes). Nevertheless, each macrofouling taxon settled in response to a specific biofilm bacterial composition, although other factors can also affect the biofouling community as the condition of the substrate. We suggest that proper friendly antifouling technologies should be focused on inhibiting bacterial biofilm adhesion.

Surface coatings select their micro and macrofouling communities differently on steel.

Previous studies have shown the effect of surface coatings on biofouling; however, they did not take into account the interaction of the micro and macrofouling communities, the effect of substrate orientation and the zooplankton-zoobenthic coupling together. Therefore, the aim of this study was to evaluate the effect of Zn- and Cu2O-based coatings on micro and macrofouling on steel surfaces, while also observing the role of substrate orientation and zooplankton supply. An experiment was carried out in the Patos Lagoon Estuary in southern Brazil for three months between spring and summer, where ASTM-36 steel plates represented different coatings (Zn- and/or Cu2O-based) and orientations (vertical and horizontal). To assess the zooplankton supply, sampling was carried out weekly using a 200 µm plankton net. Zn-based coating positively affected microfouling density compared to uncoated surfaces. The same pattern was observed with macrofouling, associated with vagile fauna preference, which represented 70% of the settled macrofoulers. Cu2O-based antifouling painted surfaces showed the highest microfouling density inhibition, while Zn + Cu2O-based coating did not affect the bacteria adhesion but showed lower density compared to Zn-based coating alone. The coatings combination showed the highest invertebrate inhibition. In this way, the macrofouling community was more sensitive than microfouling was to the antifouling coatings tested. The substrate orientation only affected macrofouling, horizontal surfaces being more attractive than vertical. Meroplankton, tychoplankton and holoplankton were recorded on the surfaces, although their representation in plankton was not proportional to the recruits recorded on the substrates. This was probably due to fast dispersion, the interactions of other factors and/or ecological succession stage. Surface coating, substrate orientation, and zooplankton supply interacted with the biofouling process on steel in different ways depending on the organism evaluated. Therefore, copper oxide- and zinc-based coatings were not suitable as coatings to avoid the total biofouling establishment.

Natural and non-toxic products from Fabaceae Brazilian plants as a replacement for traditional antifouling biocides: an inhibition potential against initial biofouling.

In this study, we screened for the antifouling activity of 15 species plant extracts from Brazilian the Brazilian Caatinga Fabaceae against the initial colonization of natural marine bacterial biofilm. We also investigated the potential toxicity of extracts against planktonic and benthic non-target organisms. Aqueous extracts of plants collected in the Caatinga biome (PE, Brazil) were prepared and tested at different concentration levels (0, 0.5, 1, 2, 4, and 8 mg mL-1). Natural marine bacterial consortium was inoculated in multi-well plates and incubated with the different treatments for 48 h. The biofilm and planktonic bacterial density and biomass inhibition were evaluated along with biofilm biomass eradication. The extracts that showed the highest bacterial biofilm inhibition were evaluated for toxicity against microalgae and crustaceans. The biofilm and planktonic bacterial inhibition potential were evaluated through flow cytometry and spectrophotometry. The selected treatments were evaluated for their toxicity using the microalgae Chaetoceros calcitrans, the copepod Nitokra sp., and the brine shrimp Artemia salina as bioindicators. Our work demonstrates the biotechnological potential of Fabaceae plant compounds as a safe antifouling alternative. Anadenanthera colubrina var. cebil fruits and Apuleia leiocarpa leaf extracts showed antibiofilm activity (≥ 80%), while Myroxylon peruiferum and Dioclea grandiflora leaf extracts showed antibiotic activity. These extracts were safe to planktonic and benthic non-target organisms. The results of this study point to potential substitutes to highly toxic antifouling paints and shed light on the prospect of a yet to be explored biome for more sustainable alternatives in biofouling research.

Could some procedures commonly used in bioassays with the copepod Acartia tonsa Dana 1849 distort results?

Many organizations have suggested the use of the Calanoid copepod Acartia tonsa in protocols for acute toxicity tests. Nevertheless, these protocols present some problems, such as using 60-180µm meshes to separate specific stages of A. tonsa or carrying out the tests using small volumes that reflect high densities of A. tonsa that do not occur in nature, which could lead to distorted results. In addition, ecotoxicological studies may use statistical approaches that are inadequate for the type of data being analysed. For these reasons, some methodological approaches for bioassays using A. tonsa need to be clarified and revised. In this study, we present information about (i) the retention of copepodite stages of A. tonsa on 180, 330 and 500µm net meshes; (ii) tested storage volumes of 1 organism per 5, 10 or 20mL in each test container (TC); and (iii) considerations about the statistics employed. The results demonstrated that a net mesh of 180µm is capable of retaining all copepodite stages (CI to CVI), contrasting with the recommendation of using a 180µm mesh to separate out adults only. Coarser meshes (330 and 500µm) can also retain different proportions of all copepodite stages, but cannot separate out one developmental stage only. Twenty-five millilitres of medium in an open TC, commonly employed in bioassays simulating densities of 1 organism 5mL-1, completely evaporated, and the results showed that the TCs need to be covered (e.g., PVC film) and filled with a minimum of 100mL of culture medium (simulating densities of 1 organism 20mL-1) to avoid evaporation and increases in salinity. The current use of ANOVA in ecotoxicological studies with proportions of surviving organisms should also be reconsidered since the data are discrete and have a binomial distribution; general linear models (GLMs) are considered more adequate. The information presented here suggests some adjustments that hopefully will enable the improvement of the procedures and methods employed in studies of acute toxicity using the copepod A. tonsa.