Marco Faimali

Non-toxic antifouling activity of polymeric 3-alkylpyridinium salts from the Mediterranean sponge Reniera sarai (Pulitzer-Finali).

The antifouling activity and toxicity of polymeric 3-alkylpyridinium salts (poly-APS) isolated from the Mediterranean sponge Reniera sarai were studied. The activity of these natural products was compared to that of zinc and copper complexes of pyrithione, two non-persistent booster biocides successfully used in current antifouling coatings. Larvae of Balanus amphitrite (cyprids and nauplii) were used to monitor settlement inhibition and the extent to which inhibition was due to toxicity. The microalga Tetraselmis suecica and larvae of the mussel Mytilus galloprovincialis were used in toxicity bioassays. Compared to the booster biocides, poly-APS were less effective at inhibiting cyprid settlement, but their effects were non toxic and reversible, with very low toxicity against the organisms used in the toxicity bioassays. Although encouraging, these results are not enough to warrant the use of poly-APS as a potential commercial antifoulant. They however justify possible future efforts to chemically synthesize poly-APS analogues for further tests.

Effects of selected metal oxide nanoparticles on Artemia salina larvae: evaluation of mortality and behavioural and biochemical responses

The aim was to investigate the toxicity of selected metal oxide nanoparticles (MO-NPs) on the brine shrimp Artemia salina, by evaluating mortality and behavioural and biochemical responses. Larvae were exposed to tin(IV) oxide (stannic oxide (SnO2)), cerium(IV) oxide (CeO2) and iron(II, III) oxide (Fe3O4) NPs for 48xa0h in seawater, with MO-NP suspensions from 0.01 to 1.0xa0mg/mL. Mortality and behavioural responses (swimming speed alteration) and enzymatic activities of cholinesterase, glutathione-S-transferase and catalase were evaluated. Although the MO-NPs did not induce any mortality of the larvae, they caused changes in behavioural and biochemical responses. Swimming speed significantly decreased in larvae exposed to CeO2 NPs. Cholinesterase and glutathione-S-transferase activities were significantly inhibited in larvae exposed to SnO2 NPs, whereas cholinesterase activity significantly increased after CeO2 NP and Fe3O4 NP exposure. Catalase activity significantly increased in larvae exposed to Fe3O4 NPs. In conclusion, swimming alteration and cholinesterase activity represent valid endpoints for MO-NP exposure, while glutathione-S-transferase and catalase activities appear to be NP-specific.

High surface adsorption properties of carbon-based nanomaterials are responsible for mortality, swimming inhibition, and biochemical responses in Artemia salina larvae

We investigated the effects of three different carbon-based nanomaterials on brine shrimp (Artemia salina) larvae. The larvae were exposed to different concentrations of carbon black, graphene oxide, and multiwall carbon nanotubes for 48 h, and observed using phase contrast and scanning electron microscopy. Acute (mortality) and behavioural (swimming speed alteration) responses and cholinesterase, glutathione-S-transferase and catalase enzyme activities were evaluated. These nanomaterials were ingested and concentrated in the gut, and attached onto the body surface of the A. salina larvae. This attachment was responsible for concentration-dependent inhibition of larval swimming, and partly for alterations in the enzyme activities, that differed according to the type of tested nanomaterials. No lethal effects were observed up to 0.5mg/mL carbon black and 0.1mg/mL multiwall carbon nanotubes, while graphene oxide showed a threshold whereby it had no effects at 0.6 mg/mL, and more than 90% mortality at 0.7 mg/mL. Risk quotients calculated on the basis of predicted environmental concentrations indicate that carbon black and multiwall carbon nanotubes currently do not pose a serious risk to the marine environment, however if uncontrolled release of nanomaterials continues, this scenario can rapidly change.

Toxicity and transfer of metal oxide nanoparticles from microalgae to sea urchin larvae

Nanoparticles (NPs) contained in commercial products are released and enter into the aquatic ecosystem, posing serious possible risks to the environment and affecting the food chain. Therefore, investigating the potential toxicity of NPs on aquatic organisms has become an important issue. This study assessed the toxicity and trophic transfer of metal oxide NPs from marine microalgae (Cricosphaera elongata) to the larvae of the sea urchin Paracentrotus lividus. Larvae (24 h old) were fed on 2000 cell mL−1 48 h of microalgae contaminated with 5 mg L−1 of several metal oxide NPs (SiO2, SnO2, CeO2, Fe3O4) for 15 days. Larval viability and development were monitored from the 4-arm stage to the 8-arm pluteus stage. A significant decrease in survival was observed in larvae fed with microalgae exposed to SiO2 and CeO2 NPs. Abnormal development, characterised by skeletal degeneration and altered rudiment growth, was observed in all larvae fed with contaminated NP algae. Our findings revealed that SiO2 and CeO2 NPs exerted a toxic effect in the trophic interaction analysed, by reducing sea urchin larval viability, and all metal oxide NPs induced toxicological effects. In conclusion, metal oxide NPs may enter the food chain and become bioavailable for marine organisms, affecting their development.

Evaluation of the antifouling properties of 3-alyklpyridine compounds

One of the most promising alternative technologies to antifouling (AF) biocides based on toxic heavy metals lies in the development of natural eco-friendly biocides. The present study evaluates the AF potential of structurally different compounds containing a 3-alkylpyridine moiety. The products, namely poly 3-alkylpyridinium salts, saraine, and haminols, were either extracted or derived from natural sources (the sponges Haliclona sp. and Reniera sarai and the mollusc Haminoea fusari), or obtained by chemical synthesis. All the molecules tested showed generally good anti-settlement activity against larvae of the barnacle Amphibalanus (=Balanus) amphitrite (EC50 values between 0.19 and 3.61u2009μgu2009ml−1) and low toxicity (LC50 values ranging from 2.04 to over 100u2009μgu2009ml−1) with non-target organisms. For the first time, the AF potential of a synthetic monomeric 3-alkylpyridine was demonstrated, suggesting that chemical synthesis is as a realistic way to produce large amounts of these compounds for future research and development of environmentally-friendly AF biocides.

Imposex in Hexaplex trunculus at some sites on the North Mediterranean Coast as a base-line for future evaluation of the effectiveness of the total ban on organotin based antifouling paints

Imposex — the superimposition of male sexual organs (penis and vas deferens) onto female Neogastropods such as Hexaplex trunculus (Linne , 1758) — is used world-wide as a biomarker of ecological impact of organotin based antifouling biocides (TBT and TPhT). To limit the impact of organotin pollution, since January 1, 2003, the International Maritime Organization (IMO) has enacted a global ban on the use of organotin compounds in antifouling systems. It is important to record imposex levels and organotin contamination before the implementation of the ban, in order to assess the current situation and be able, in the future, to verify the effects of the International Protocol. In this paper, recent imposex data measured in populations of Hexaplex trunculus from three different Mediterranean regions are compared: the Ligurian Sea (Italy), the Lagoon of Venice (Italy) and the western coast of Istria (Croatia). In the two former locations, a partial ban on TBT has been in force for vessels less than 25 m since 1982, while in the latter region no restrictions on organotin antifouling paints have been applied yet. Gastropod samples collected from the Venice lagoon were analysed with an acid extraction followed by Grignard derivatisation, clean up and GC-MS determination, in order to relate the levels of TBT, TPhT and their metabolites with the imposex degree detected. Biological data show that the levels of imposex were very high (VDS from 4.3 to 5) in all the sampling sites considered, particularly in the Croatian coast stations. The concentrations of organotin compounds — butyltins and phenyltins — measured in the samples from the lagoon of Venice were found to partition differently in the visceral coil and in the rest of the soft body of the analysed organisms.

Chemical, molecular, and eco-toxicological investigation of Ostreopsis sp. from Cyprus Island: structural insights into four new ovatoxins by LC-HRMS/MS

Blooms of benthic dinoflagellates of the genus Ostreopsis (mainly O. cf. ovata and occasionally O. cf. siamensis) represent a serious concern for humans in the Mediterranean area, due to production of palytoxin-like compounds listed among the most potent marine toxins known. In this work, six strains of Ostreopsis sp. from Cyprus Island were analyzed through an integrated approach based on molecular, chemical, and eco-toxicological methods. Cypriot Ostreopsis sp. was found to be a species distinct from O. cf. ovata and O. cf. siamensis, belonging to the Atlantic/Mediterranean Ostreopsis spp. clade. Some variability in toxin profiles emerged: three strains produced ovatoxin-a (OVTX-a), OVTX-d, OVTX-e, and isobaric palytoxin, so far found only in O. cf. ovata; the other three strains produced only new palytoxin-like compounds, which we named ovatoxin-i, ovatoxin-j1, ovatoxin-j2, and ovatoxin-k. The new ovatoxins present the same carbon skeleton as ovatoxin-a, differing primarily in an additional C2H2O2 moiety and an unsaturation in the region C49–C52. Other minor structural differences were found, including the presence of a hydroxyl group at C44 (in OVTX-j1 and OVTX-k) and the lack of a hydroxyl group in the region C53–C78 (in OVTX-i and OVTX-j1). The toxin content of the analyzed Ostreopsis sp. strains was in the range 0.06–2.8xa0pgxa0cell−1, definitely lower than that of a Ligurian O. cf. ovata strain cultured under the same conditions. Accordingly, an eco-toxicological test on Artemia salina nauplii demonstrated that Ostreopsis sp. presents a very low toxicity compared to O. cf. ovata. The whole of these data suggest that Ostreopsis sp. from Cyprus Island poses a relatively low risk to humans.

Antisettlement activity of synthetic analogues of polymeric 3-alkylpyridinium salts isolated from the sponge Reniera sarai

Based on the high, non-toxic and reversible antifouling activity of the polymeric 3-alkylpyridinium salts isolated from the sponge Reniera sarai, the anti-settlement activity and toxicity of a series of synthetic analogues has been studied. All the test compounds were less efficient than the natural polymers, suggesting that the high and reversible anti-macrofouling activity of the natural polymers could derive from their detergent-like properties. The values obtained for EC50sett. of inhibition of cyprid settlement and EC50imm. as naupliar toxicity for the synthetic compounds indicate that the presence of single or multiple charges in the structure is not relevant for the antifouling activity which, conversely, is favoured by increasing the length of the alkyl chain, or by the presence of uncharged pyridine units. The compound 1,8-di(3-pyridyl)octane was the most efficient (EC50sett.= 0.44u2009μgu2009ml–1), although with a higher toxicity on naupliar stage of B. amphitrite than the natural polymers.

Multidisciplinary screening of toxicity induced by silica nanoparticles during sea urchin development

The aim of this study was to investigate the potential toxicity of Silica nanoparticles (SiO2 NPs) in seawater by using the sea urchin Paracentrotus lividus as biological model. SiO2 NPs exposure effects were identified on the sperm of the sea urchin through a multidisciplinary approach, combining developmental biology, ecotoxicology, biochemistry, and microscopy analyses. The following responses were measured: (i) percentage of eggs fertilized by exposed sperm; (ii) percentage of anomalies and undeveloped embryos and larvae; (iii) enzyme activity alterations (acetylcholinesterase, AChE) in the early developmental stages, namely gastrula and pluteus. Sperms were exposed to seawater containing SiO2 NPs suspensions ranging from 0.0001mg/L to 50mg/L. Fertilization ability was not affected at any concentration, whereas a significant percentage of anomalies in the offspring were observed and quantified by means of EC50 at gastrula stage, including undeveloped and anomalous embryos (EC50=0.06mg/L), and at pluteus stage, including skeletal anomalies and delayed larvae (EC50=0.27mg/L). Moreover, morphological anomalies were observed in larvae at pluteus stage, by immunolocalizing molecules involved in larval development and neurotoxicity effects - such as acetylated tubulin and choline acetyltransferase (ChAT) - and measuring AChE activity. Exposure of sea urchins to SiO2 NPs caused neurotoxic damage and a decrease of AChE expression in a non-dose-dependent manner. In conclusion, through the multidisciplinary approach used in this study SiO2 NPs toxicity in sea urchin offspring could be assessed. Therefore, the measured responses are suitable for detecting embryo- and larval- toxicity induced by these NPs.

New implications in the use of imposex as a suitable tool for tributyltin contamination: experimental induction in Hexaplex trunculus (Gastropoda, Muricidae) with different stressors

Imposex, i.e. the development of additional male sex organs (penis and/or vas deferens), in females of gonochorist marine and freshwater gastropods, is known to be caused by tributyltin (TBT), and it has been widely used as a biomonitoring tool in environmental surveys for TBT pollution assessment. In this study, we experimentally tested the potential to induce imposex by another endocrine disruptor (polychlorinated biphenyls [PCBs] mixture—Aroclor 1260). Adults of Hexaplex trunculus with low imposex level, coming from an Italian Marine Protected Area, were injected separately with different doses of tributyltin chloride (TBTCl) and Aroclor 1260. The compounds were dissolved in ethanol and the organisms were narcotised by immersion in MgCl2 solution before injection. Before and after the experiment, butyltin compounds (BuTs) and PCB tissue concentrations were determined. A significant increase in imposex with respect to non-treated organisms was observed in all treatments, including artefact controls. No clear correlation was observed between BuTs and PCB tissue concentrations and indices of imposex incidence. Based on these results, no assumption can be formulated about PCB effect on imposex development. Nevertheless, they suggest that the imposex level increase, at least in H. trunculus, in laboratory conditions might not be caused by TBT only, but it would rather be a non-specific response to different stress stimuli.