Synthesis and Antifouling Activity Evaluation of Analogs of Bromosphaerol, a Brominated Diterpene Isolated from the Red Alga Sphaerococcus coronopifolius.

Marine biofouling is an epibiotic biological process that affects almost any kind of submerged surface, causing globally significant economic problems mainly for the shipping industry and aquaculture companies, and its prevention so far has been associated with adverse environmental effects for non-target organisms. Previously, we have identified bromosphaerol (1), a brominated diterpene isolated from the red alga Sphaerococcus coronopifolius, as a promising agent with significant antifouling activity, exerting strong anti-settlement activity against larvae of Amphibalanus (Balanus) amphitrite and very low toxicity. The significant antifouling activity and low toxicity of bromosphaerol (1) motivated us to explore its chemistry, aiming to optimize its antifouling potential through the preparation of a number of analogs. Following different synthetic routes, we successfully synthesized 15 structural analogs (2-16) of bromosphaerol (1), decorated with different functional groups. The anti-settlement activity (EC50) and the degree of toxicity (LC50) of the bromosphaerol derivatives were evaluated using cyprids and nauplii of the cirriped crustacean A. amphitrite as a model organism. Derivatives 2, 4, and 6-16 showed diverse levels of antifouling activity. Among them, compounds 9 and 13 can be considered as well-performing antifoulants, exerting their activity through a non-toxic mechanism.

Antifouling Activity of Meroterpenes Isolated from the Ascidian Aplidium aff. densum.

The settlement and growth of fouling organisms on man-made surfaces can be prevented by the application of antifouling paints containing active compounds (biocides, heavy metals), most of which are toxic to non-target organisms. As part of our research program in chemical ecology and blue biotechnology, we are conducting studies to investigate the natural defence mechanisms of marine organisms that are free from epibionts, with the aim of isolating molecules involved in surface defence that could be good candidates as antifouling agents. Ascidians were selected for our investigation because previous studies have shown that they contain abundant and diverse secondary metabolites, which play a defensive role and have been applied to drug discovery. It is therefore relevant to study the role of such secondary metabolites in surface protection. In this study, 5 meroterpenoids (cordiachromene A, didehydroconicol, epiconicol, methoxyconidiol, conidione) from Aplidium aff. densum (ascidian) were investigated as potential antifoulants towards the inhibition of bacterial growth and settlement inhibition of barnacles. Cardiochromene A (IC50 barnacle settlement = 6.04 µg/mL; MIC Gram positive = 125 µg/mL; MIC Gram negative = 32 µg/mL) and epiconicol (IC50 barnacle settlement = 8.05 µg/mL; MIC Bacillus = 63 µg/mL; MIC other strains = 32 µg/mL) were the most promising compounds among those tested in this study.

Effects of polystyrene microplastics on larval development, settlement, and metamorphosis of the intertidal barnacle Amphibalanus amphitrite.

The effects of microplastic on mortality and sublethal responses on larval development of meroplankton are still largely unknown. Present study investigated the effects of four sizes of virgin spherical polystyrene microplastics (diameter 1.7, 6.8, 10.4, 19.0 µm) on naupliar (stage II-VI) and cypris larvae of barnacle Amphibalanus amphitrite at environmentally relevant concentrations (1, 10, 100, 1000 beads mL-1). Essential life-history traits, including mortality, development time and rates of growth, settling, and metamorphosis were measured throughout the entire larval development. Feeding experiments were conducted to evaluate if microplastics decreased naupliar feeding due to physical impacts or selective feeding of nauplii. The results showed that A. amphitrite stage II nauplii were able to ingest and efficiently egest all sizes of microplastics. All the life-history endpoints measured were not significantly affected by all sizes of microplastics at any concentration tested. Presence of all sizes of microplastics did not cause physical interference on naupliar feeding and all stages of nauplius larvae (stage III-VI) did not selectively feed on microplastics. However, the feeding ability of stage III nauplius appeared to be affected by 1.7 µm at 1000 beads mL-1 which was possibly due to individual variations rather than microplastics' impacts. Overall, the full larval development of barnacle A. amphitrite was not affected by microplastics at environmentally relevant concentrations under laboratory condition.

(-)- and (+)-Asperginulin A, a Pair of Indole Diketopiperazine Alkaloid Dimers with a 6/5/4/5/6 Pentacyclic Skeleton from the Mangrove Endophytic Fungus Aspergillus sp. SK-28.

A pair of enantiomeric indole diketopiperazine alkaloid dimers [(-)- and (+)-asperginulin A (1a and 1b)] with an unprecedented 6/5/4/5/6 pentacyclic skeleton were isolated from the mangrove endophytic fungus Aspergillus sp. SK-28. The enantiomeric dimers were separated by chiral-phase HPLC. Their structures including absolute configurations were elucidated by spectroscopic analysis, X-ray diffraction, and quantum chemical calculation. (+)-Asperginulin A (1b) exhibited antifouling activity against the barnacle Balanus reticulatus.

Antifouling Properties of Dendritic Polyglycerols against Marine Macrofouling Organisms.

Dendritic polyglycerols (PGs) were synthesized and postmodified by grafting of poly(ethylene glycol) (PEG) and polypropylene glycol (PPG) diglycidyl ether groups, and their antifouling and fouling-release properties were tested. Coating characterization by spectroscopic ellipsometry, contact angle goniometry, attenuated total internal reflection-Fourier transform infrared spectroscopy (ATR-FTIR), and atomic force microscopy showed brushlike morphologies with a high degree of microscale roughness and the ability to absorb large amounts of water within seconds. PGs with three different thicknesses were tested in laboratory assays against settlement of larvae of the barnacle Balanus improvisus and against the settlement and removal of zoospores of the alga Ulva linza. Very low coating thicknesses, e.g., 11 nm, reduced the settlement of barnacles, under static conditions, to 2% compared with 55% for an octadecyltrichlorosilane reference surface. In contrast, zoospores of U. linza settled readily but the vast majority were removed by exposure to a shear force of 52 Pa. Both PEG and PPG modification increased the antifouling properties of the PG films, providing a direct comparison of the ultralow fouling properties of all three polymers. Both, the modified and the nonmodified PGs are promising components for incorporation into amphiphilic fouling-resistant coatings.

Phyco-linked vs chemogenic magnetite nanoparticles: Route selectivity in nano-synthesis, antibacterial and acute zooplanktonic responses.

Despite the fact that magnetic iron oxide nanoparticles (Fe3O4-MNPs) considered as the most promising nanoparticles (NPs) in biomedicine and environmental biotechnology, their safety and ecotoxicological impacts of biogenic and chemogenic routes of Fe3O4-MNPs in the marine aquatic system is scarcely studied. In this work, we report the optimized and suitable phyco-synthesis route for nano-Fe3O4 based on the six selected species of the Persian Gulf seaweeds: Ulva prolifera, U. flexuosa, U. linza, U. intestinalis, U. clathrata, and Sargassum boveanum. Moreover, antibacterial activities and acute zooplanktonic responses in Artemia salina and acorn barnacle Amphibalanus amphitrite to chemogenic and biogenic Fe3O4-MNPs, were evaluated. Although all the seaweeds extract showed reducing potential for Fe3O4-MNPs green synthesis - mainly on the basis of characterization results- the algal route selectivity has been demonstrated to be important for the biosynthesis of magnetite NPs. Herein, the cubo-spherical and polydisperse U. prolifera-derived Fe3O4-MNPs with particles sizes of 9.59 nm were the best ones. The comparative zooplanktonic cytotoxicity of chemo- and bio-route of Fe3O4-MNPs exhibited no acute toxicity in nauplii and adults of A. salina (96-h EC50 ≥ 1000 mg/L) and the potential of toxicity in A. amphitrite nauplii (48-h EC50 = 466.5 and 842.3 mg/L for chemo- and bio-route of Fe3O4-MNPs, respectively). The in vitro antimicrobial activity of both chemo- and bio-route of magnetite NPs to selective human pathogenic bacteria and fungi (i.e. n = 11) showed strong antagonistic activity against Staphylococcus epidermidis, Bacillus subtilis, B. pumulis, and Saccharomyces cerevisiae. In conclusion, these findings demonstrate the optimized phyco-fabrication of Fe3O4-MNPs as promising nontoxic approach in ecobiotechnology, the new insight about the potential adverse effects of chemosynthesized Fe3O4-MNPs to crustacean zoo-organisms after their possible entrance into the marine environments, and bio/chemo-route Fe3O4-MNPs as pivotal agent for nanoantimicrobials.

Correlative assays of barnacle cyprid behaviour for the laboratory evaluation of antifouling coatings: a study of surface energy components.

Laboratory evaluation of antifouling coatings is underpinned by settlement studies with specific fouling organisms. Established methods provide insight into the likelihood of failure of a particular coating system, but can neglect the process of surface selection that often precedes attachment. The present approach for quantifying the exploratory behaviour of barnacle cypris larvae suggested that inspection behaviour can be a rapid and predictive proxy for settlement. Two series of xerogels with comparable total surface energy, but different dispersive and polar components, were evaluated. Settlement assays with three-day-old cyprids of Balanus improvisus demonstrated that while attachment was not linked directly to dispersive free energy, the composition of the xerogel was nevertheless significant. Behavioural analysis provided insight into the mechanism of surface rejection. In the case of a 50:50 PH/TEOS (phenyltriethoxysilane-based) xerogel vs a 50:50 TFP/TEOS (3,3,3-trifluoropropyltrimethoxysilane-based) xerogel, wide-searching behaviour was absent on the former.

Isolation and Structure-Activity Relationship of Subergorgic Acid and Synthesis of Its Derivatives as Antifouling Agent.

In this study, as part of our continuous search for environmentally-friendly antifoulants from natural resources, subergorgic acid (SA) was identified from the gorgonian coral Subergorgia suberosa, demonstrating non-toxic, significant inhibitory effects (EC50 1.25 µg/mL, LC50 > 25 µg/mL) against the settlement of Balanus amphitrite. To further explore the bioactive functional groups of SA and synthesize more potent antifouling compounds based on the lead SA, the structure-activity relationships of SA were studied, followed by rational design and synthesis of two series of SA derivatives (one being benzyl esters of SA and another being SA derivatives containing methylene chains of various lengths). Our results indicated that (1) both the double bond and ketone carbonyl are essential elements responsible for the antifouling effect of SA, while the acid group is not absolutely necessary for maintaining the antifouling effect; (2) all benzyl esters of SA displayed good antifouling effects (EC50 ranged from 0.30 to 2.50 µg/mL) with the most potent compound being 5 (EC50 0.30 µg/mL, LC50 > 25 µg/mL), which was over four-fold more potent than SA; and (3) the introduction of a methylene chain into SA reduces the antifouling potency while the length of the methylene chain may differently influence the antifouling effect, depending on the functional group at the opposite site of the methylene chain. Not only has this study successfully revealed the bioactive functional groups of SA, contributing to the mechanism of SA against the settlement of B. amphitrite, but it has also resulted in the identification of a more potent compound 5, which might represent a non-toxic, high-efficiency antifoulant.

Harmful or harmless: Biological effects of marennine on marine organisms.

Marennine is a water-soluble blue-green pigment produced by the marine diatom Haslea ostrearia. The diatom and its pigment are well known from oyster farming areas as the source of the greening of oyster gills, a natural process increasing their market value in Western France. Blooms of blue Haslea are also present outside oyster ponds and hence marine organisms can be exposed, periodically and locally, to significant amounts of marennine in natural environments. Due to its demonstrated antibacterial activities against marine pathogenic bacteria (e.g. Vibrio) and possible prophylactic effects toward bivalve larvae, marennine is of special interest for the aquaculture industry, especially bivalve hatcheries. The present study aimed to provide new insights into the effects of marennine on a large spectrum of marine organisms belonging to different phyla, including species of aquaculture interest and organisms frequently employed in standardised ecotoxicological assays. Different active solutions containing marennine were tested: partially purified Extracellular Marennine (EMn), and concentrated solutions of marennine present in H. ostrearia culture supernatant; the Blue Water (BW) and a new process called Concentrated Supernatant (CS). Biological effects were meanwhile demonstrated in invertebrate species for the three marennine-based solutions at the highest concentrations tested (e.g., decrease of fertilization success, delay of embryonic developmental stages or larval mortality). Exposure to low concentrations did not impact larval survival or development and even tended to enhance larval physiological state. Furthermore, no effects of marennine were observed on the fish gill cell line tested. Marennine could be viewed as a Jekyll and Hyde molecule, which possibly affects the earliest stages of development of some organisms but with no direct impacts on adults. Our results emphasize the need to determine dosages that optimize beneficial effects and critical concentrations not to be exceeded before considering the use of marennine in bivalve or fish hatcheries.

Synthesis and Structure-Activity Relationship of Omaezallene Derivatives.

Omaezallene derivatives (nor-bromoallene, nor-bromodiene, and bromoenynes) were successfully synthesized. Their antifouling activity and toxicity to the cypris larvae of the barnacle Amphibalanus amphitrite and ecotoxicity to the marine crustacean Tigriopus japonicus were studied. It was revealed that the two side chains of omaezallene were essential to its antifouling activity because the activities of nor-bromoallene and nor-bromodiene were significantly diminished. The bromoenyne was found to exhibit potent antifouling activities comparable to omaezallene with low toxicity and ecotoxicity. Preparation of bromoenyne framework is much easier than that of bromodiene moiety in omaezallene. Based on the antifouling activities of the bromoenynes, the synthesis of fluorescent probes and evaluation of their biological activities were also carried out.

Four New C9 Metabolites from the Sponge-Associated Fungus Gliomastix sp. ZSDS1-F7-2.

Four new structurally related metabolites, one γ-lactone named gliomasolide F (1), one δ-lactone named gliomasolide G (2), and two medium-chain fatty acids named gliomacids A⁻B (3⁻4), each containing nine carbons in total, were identified from the sponge-associated fungus Gliomastix sp. ZSDS1-F7-2. The planar chemical structures of these novel C9 metabolites were elucidated by nuclear magnetic resonance (NMR) spectroscopic methods, in connection with the analysis of high-resolution mass spectrometry (HRMS) and infrared (IR) data. The absolute configuration of 1, was determined by comparisons of experimental circular dichroism (CD) and optical rotation (OR) value with corresponding ones computed by quantum chemistry. The relative configuration of 2 was determined by the Nuclear Overhauser effect spectroscopy (NOESY) spectrum, while its absolute configuration was tentatively determined in view of the biogenetic and biosynthetic relationships between 1 and 2. Compounds 3⁻4, originally as an inseparable mixture, were successfully isolated after chemical modifications. The stereo-chemistries of compounds 3⁻4 were assumed by comparison of 13C NMR with those of the similar moiety reported in literature, in addition to the biogenetic and biosynthetic relationships with 1. The plausible biosynthetic relationships among these four C9 metabolites were supposed. Biologically, compounds 1⁻4 showed no cytotoxic effect against HeLa cell line at concentrations up to 25 μg/mL, while 1 exhibited moderate antifouling activity against the settlement of Balanus amphitrite larvae with IC50 being 12.8 μg/mL and LC50 > 25 μg/mL. The co-occurrence of macrolides gliomasolides A—E and four C9 metabolites in the same fermentation culture made us assume that these C9 metabolites might be biosynthetic building blocks toward the construction of more complex macrolides such as gliomasolides A—E or other unidentified polyketides.

Evaluation of sewage sludge incineration ash as a potential land reclamation material.

This study evaluated the potential of utilising sewage sludge incineration ash as a land reclamation material. Toxicity assessment of the leachate of the ash was carried out for both terrestrial and marine organisms. Both the fruit fly Drosophila melanogaster and barnacle Amphibalanus amphitrite showed that both bottom and fly ash leached at liquid-to-solid (L/S) ratio 5 did not substantially affect viabilities. The leachate carried out at L/S 10 was compared to the European Waste Acceptance Criteria and the sewage sludge ashes could be classified as non-hazardous waste. The geotechnical properties of the sewage sludge ash were studied and compared to sand, a conventional land reclamation material, for further evaluation of its potential as a land reclamation material. It was found from direct shear test that both bottom and fly ashes displayed similar and comparable shear strength to that of typical compacted sandy soil based on the range of internal friction angle obtained. However, the consolidation profile of bottom ash was significantly different from sand, while that of fly ash was more similar to sand. Our study showed that the sewage sludge ash has the potential to be used as a land reclamation material.

Assessing the chronic toxicity of nickel to a tropical marine gastropod and two crustaceans.

The mining and processing of nickel ores from tropical regions contributes 40% of the global supply. The potential impact of these activities on tropical marine ecosystems is poorly understood. Due to the lack of ecotoxicity data for tropical marine species, there is currently no available water quality guideline value for nickel that is specific to tropical species. In this study, we investigated the toxicity of nickel to three tropical marine invertebrates, the gastropod Nassarius dorsatus, the barnacle Amphibalanus amphitrite, and the copepod Acartia sinjiensis. All toxicity tests used chronic endpoints, namely larval growth, metamorphosis (transition from nauplii to cyprid larvae) and larval development for the snail, barnacle and copepod respectively. Toxicity tests were carried out under environmentally relevant conditions (i.e. 27-30ᵒC, salinity 34-36‰, pH 8.1-8.4). Copper was also tested for quality assurance purposes and to allow for comparisons with previous studies. The copepod was the most sensitive species to nickel, with development inhibited by 10% (EC10) at 5.5 (5.0-6.0) µg Ni/L (95% confidence limits (CL)). Based on EC10 values, the gastropod and barnacle showed similar sensitivities to nickel with growth and metamorphosis inhibited by 10% at 64 (37-91) µg Ni/L and 67 (53-80) µg Ni/L, respectively. Based on existing data available in the literature, the copepod A. sinjiensis is so far the most sensitive tropical marine species to nickel. This study has provided high quality data which will contribute to the development of a water quality guideline value for nickel in tropical marine waters. A species sensitivity distribution of chronic nickel toxicity used the data generated in this paper supplemented by available literature data, comprising 12 species representing 6 taxonomic groups. A 5% hazard concentration (HC5) was determined as 8.2 µg/L Ni.

Design and Biological Evaluation of Antifouling Dihydrostilbene Oxime Hybrids.

By combining the recently reported repelling natural dihydrostilbene scaffold with an oxime moiety found in many marine antifoulants, a library of nine antifouling hybrid compounds was developed and biologically evaluated. The prepared compounds were shown to display a low antifouling effect against marine bacteria but a high potency against the attachment and growth of microalgae down to MIC values of 0.01 µg/mL for the most potent hybrid. The mode of action can be characterized as repelling via a reversible non-toxic biostatic mechanism. Barnacle cyprid larval settlement was also inhibited at low µg/mL concentrations with low levels or no toxicity observed. Several of the prepared compounds performed better than many reported antifouling marine natural products. While several of the prepared compounds are highly active as antifoulants, no apparent synergy is observed by incorporating the oxime functionality into the dihydrostilbene scaffold. This observation is discussed in light of recently reported literature data on related marine natural antifoulants and antifouling hybrids as a potentially general strategy for generation of improved antifoulants.

Biogenic corrosion inhibitor on mild steel protection in concentrated HCl medium.

Turbinaria ornata (TO) extract was tested as green corrosion inhibitor on mild steel (MS) coupons in conc. HCl medium with an efficiency of 100% at 25 g l-1 during 5 min exposure. Antibacterial efficacy performed against 16 S rDNA identified marine biofilming bacteria (MBB) and human pathogenic bacteria (HPB). Maximum inhibition growth was 16 mm on MBB observed in Bacillus megaterium (MBF14 - AB894827) and 20 mm on HPB in Escherichia coli (B5 - NCIM 2931). Similarly, minimum of 10 mm on MBB witnessed in Pseudomonas sp., (MBF9 - AB894829). Toxicity studies proved 50.0% LC50 at 500 µg ml-1 in 24 hrs, whereas Balanus amphitrite resulted in 100% mortality within 12 hrs. Results including weight loss, potentiodynamic polarization and electrochemical impedance spectroscopy, FT-IR and GC-MS confirm 10-Octadecaonic acid as a major corrosion inhibitor from T. ornata and is discovered as a novel antifoulant. Anticorrosion formulation will become available soon.

Linking stable isotopes and biochemical responses in Balanus glandula under sewage influence.

In the present study, we analyzed the influence of untreated sewage exposure on carbon (δ13C) and nitrogen (δ15N) isotopic composition and several biochemical responses in the barnacle Balanus glandula. The main objective was to evaluate whether changes in stable isotopes signature do reflect biochemical sub-lethal effects in a sewage influence gradient. Stable isotopes analysis showed differences in isotope signatures between close sewage influence and distant sites, being δ13C signatures stronger than that of δ15N. Regarding biochemical effects, although organisms close to the effluent would be clearly exposed to contaminants (increased GST activity) the oxidative stress would not be too evident (peroxidases and ACAP not affected). The most affected physiological aspect was the digestive one, reflected in increased alkaline proteases and lipases activities. A clear relation between δ15N and GST activity was found, showing to δ15N as an indicator of potential exposure to chemical contaminants.

Isocyanides Derived from α,α-Disubstituted Amino Acids: Synthesis and Antifouling Activity Assessment.

Herein, we contribute to the development of environmentally friendly antifoulants by synthesizing eighteen isocyanides derived from α,α-disubstituted amino acids and evaluating their antifouling activity/toxicity against the cypris larvae of the Balanus amphitrite barnacle. Almost all isocyanides showed good antifouling activity without significant toxicity and exhibited EC50 values of 0.07 - 7.30 µg/mL after 120-h exposure. The lowest EC50 values were observed for valine-, methionine-, and phenylalanine-derived isocyanides, which achieved > 95% cypris larvae settlement inhibition at concentrations of less than 30 µg/mL without exhibiting significant toxicity. Thus, the prepared isocyanides should be useful for further research focused on the development of environmentally friendly antifouling agents.

Scopuquinolone B, a new monoterpenoid dihydroquinolin-2(1H)-one isolated from the coral-derived Scopulariopsis sp. fungus.

Further chemical investigation of the secondary metabolites of the fungus Scopulariopsis sp. led to the discovery of a new alkaloid, scopuquinolone B (1). The structure of compound 1 was elucidated by extensive NMR spectroscopic data, CD spectrum and analysis of its Dess-Martin oxidation derivative. Compound 1 was evaluated for antilarval settlement activity of barnacle Balanus amphitrite and showed promising antifouling activity with an EC50 value of 0.103 µM and a high therapeutic ratio of 222.

Environmentally benign antifouling activity and toxic properties of bioactive metabolites from mangrove Excoecaria agallocha L.

This study was aimed to investigate the antifouling (AF) potentials and toxic properties of methanol extract from leaves of mangrove Excoecaria agallocha. Antimicrofouling activity results inferred that this extract strongly inhibited fouling bacterial and microalgal growth. This extract had also inhibited the settlement of brown mussel Perna indica and larvae of barnacle Balanus amphitrite. Further, EC50 < LC50 and therapeutic ratio > 1 together propagated non-toxic nature of the extract. Mollusk foot adherence assay result showed complete inhibition of foot spreading and loss of attachment of common rocky fouler Patella vulgata to the substrata. Field assay results affirmed that this extract effectively deterred settlement of biofoulers. Purification and GC-MS analysis of bioassay-guided active spot evidenced presence of three major compounds (> 85%) responsible for the promising AF activity. The identified lead compounds subjected to an estimation (BIOWIN™) program developed by United States Environmental Protection Agency (USEPA) predicts that they are biodegradable in nature. Graphical abstract.

Hymerhabdrin A, a novel diterpenoid with antifouling activity from the intertidal sponge Hymerhabdia sp.

Hymerhabdrin A (1), a diterpenoid possessing a novel 6/6/5 fused-ring skeleton, together with four known sterols were isolated from an intertidal marine sponge Hymerhabdia sp. Their structures were elucidated by extensive spectroscopic methods, and the relative and absolute configurations of 1 were determined by NOESY analysis and electronic circular dichrosim calculations, respectively. Hymerhabdrin A (1) exhibited significant antifouling activity against Balanus amphitrite larval with LC50 (lethal concentration 50) value of 3.6 µg ml-1.