Assessing chemical contamination in the coastal waters of the Adriatic Sea using active mussel biomonitoring with Mytilus galloprovincialis.

The MYTIAD project focused on the assessment of chemical contamination (metals, PAHs, PCBs and organochlorine pesticides) in the Adriatic coastal waters by active mussel watching. Its purpose was harmonising and standardising strategies and methodologies used to assess the contamination status of the Adriatic Sea, in the framework of the WFD, the MSFD and UNEP/MAP Integrated Monitoring and Assessment Programme. Data on metals pointed out some hotspots along the eastern and western Adriatic Sea coasts, with the highest values of total PAH concentration detected in Split, Trieste, and Taranto (Ionian Sea). PCB and endrin contamination was higher in the Adriatic Sea than in the western Mediterranean Sea. This is the first comprehensive overview of contamination in the Adriatic Sea with critical comparisons of related studies over the Mediterranean Sea. It provides a useful harmonised dataset to support a coordinated definition of baselines, targets and thresholds, and further management of chemical contamination.

Comparative assessment of cardiac activity and DNA damage in haemocytes of the Mediterranean mussel Mytilus galloprovincialis in exposure to tributyltin chloride.

This study gives an insight in sensitivity of heart rate (Hr) of Mytilus galloprovincialis as a physiological biomarker. Impact of tributyltin chloride (TBT-Cl) on Hr was studied in parallel with evaluation of mutagenic, genotoxic and cytotoxic potential of TBT-Cl (10, 100 and 1000µg/L) within 96h treatment in static conditions. Mutagenic potential was assessed by SOS/umuC assay while genotoxicity was assessed in haemocytes of M. galloprovincialis by using the comet assay and the micronucleus test. Benzo(a)pyrene (B(a)P) was used as a positive control. Hr variations detected in TBT-Cl treatments can be linked to data obtained in the genotoxicological assays indicating that Hr can be considered and used as a reliable physiological biomarker for detecting the presence of organotin compounds. However despite the observed genotoxic potential of B(a)P, a noteworthy Hr response was not observed which further questions the potential of Hr in the detection of different types of pollutants.

Synthesis and biological activity of amino acid derivatives of avarone and its model compound.

A series of eighteen derivatives of marine sesquiterpene quinone avarone and its model system tert-butylquinone with amino acids has been synthesized by nucleophilic addition of amino acids to the quinones. In vitro cytotoxic activity toward human cancer cell lines (HeLa, A549, Fem-X, K562, MDA-MB-453) and normal MRC-5 cell line was determined. Several compounds showed very strong inhibitory activity with IC50 values less than 10 µM. Avarone derivatives were more active than the corresponding tert-butylquinone derivatives. The results of the cytofluorimetric analysis of cell cycle of HeLa cells showed that apoptosis might be one of possible mechanism of action of these compounds in cancer cells. In order to examine the influence of caspases on cell death, the apoptotic mechanisms induced by the tested compounds were determined using specific caspases 3, 8 and 9 inhibitors. For all compounds antibacterial activities against six strains of Gram-positive and four strains of Gram-negative bacteria were determined, as well as antifungal activity against three fungal species.

Genotoxic potential and heart rate disorders in the Mediterranean mussel Mytilus galloprovincialis exposed to Superdispersant-25 and dispersed diesel oil.

The effects of ex situ exposure of Mytilus galloprovincialis to Superdispersant-25 (S-25), diesel oil and dispersed diesel oil mixtures were studied by the impact on level of DNA damage in haemocytes (comet assay) and the cardiac activity patterns of mussels. Specimens were exposed for 72 h in a static system to diesel oil (100 µL/L and 1 mL/L), S-25 (5 and 50 µL/L), and dispersed diesel oil mixtures M1 (diesel oil 100 µL/L + S-25 5 µL/L) and M2 (diesel oil 1 mL/L + S-25 50 µL/L). For positive control 40 µM CdCl2 was used. The comet assay results indicated genotoxic potential of S-25 while the effects of diesel oil alone were not observed. The highest response was detected for M1 while the effects of M2 were not detected. The heart rate disorders were recorded for the diesel oil (1 mL/L), S-25 (50 µL/L) and both dispersed diesel oil mixtures.

Evaluation of single-cell gel electrophoresis data: combination of variance analysis with sum of ranking differences.

Specimens of the mussel Mytilus galloprovincialis were collected from five sites in the Boka Kotorska Bay (Adriatic Sea, Montenegro) during the period summer 2011-autumn 2012. Three types of tissue, haemolymph, digestive gland were used for assessment of DNA damage. Images of randomly selected cells were analyzed with a fluorescence microscope and image analysis by the Comet Assay IV Image-analysis system. Three parameters, viz. tail length, tail intensity and Olive tail moment were analyzed on 4200 nuclei per cell type. We observed variations in the level of DNA damage in mussels collected at different sites, as well as seasonal variations in response. Sum of ranking differences (SRD) was implemented to compare use of different types of cell and different measure of comet tail per nucleus. Numerical scales were transferred into ranks, range scaling between 0 and 1; standardization and normalization were carried out. SRD selected the best (and worst) combinations: tail moment is the best for all data treatment and for all organs; second best is tail length, and intensity ranks third (except for digestive gland). The differences were significant at the 5% level. Whereas gills and haemolymph cells do not differ significantly, cells of the digestive gland are much more suitable to estimate genotoxicity. Variance analysis decomposed the effect of different factors on the SRD values. This unique combination has provided not only the relative importance of factors, but also an overall evaluation: the best evaluation method, the best data pre-treatment, etc., were chosen even for partially contradictory data. The rank transformation is superior to any other way of scaling, which is proven by ordering the SRD values by SRD again, and by cross validation.

Chitin-based renewable materials from marine sponges for uranium adsorption.

Marine sponges of the order Verongida form three-dimensional networks of fibrous chitin, which can easily be extracted. In the hydrated state, these networks are flexible, mechanically stable and can be cut or pressed into any desired form. Here, we show for the first time that chitin-based networks of sponge origin are useful for effective uranium adsorption. They adsorb uranium from solution with a higher adsorption capacity than many other chitinous sorbents. Up to 288 mg/g could be achieved. Solid-state NMR, infrared, and Raman spectroscopy indicated that the uranyl is bound to the chitin by weak interactions. 90% of the uranyl could be desorbed using diluted hydrochloric acid. Uranium adsorption and desorption did not result in any destruction of the chitin-based material.

Synthesis and biological activity of derivatives of the marine quinone avarone.

Nine alkyl(aryl)thio derivatives of the marine sesquiterpene quinone avarone were synthesized by nucleophilic addition of thiols or thiophenol to avarone. In most cases only one regioisomer was obtained. Their cytotoxic activities, brine shrimp lethality and antibacterial activity were evaluated, as well as those of some previously synthesized avarone derivatives. Anti-HIV activity of two derivatives was tested. Electrochemical properties were determined for all the derivatives in order to obtain more accurate information on structure-activity relationships. Most derivatives showed cytotoxic activity against tumor cell lines, with IC(50) values less than 10 microM for some of them, in particular those with electron-donating substituents. The most active compound was 4'-(methylamino)avarone, with IC(50) value of 2.4 microM to melanoma Fem-X cells, and no cytotoxicity to normal lymphocytes.

A novel lectin from the sponge Haliclona cratera: isolation, characterization and biological activity.

A lectin from the Adriatic sponge Haliclona cratera was purified by ion-exchange and gel chromatography. The molecular mass of the lectin is approximately 29 kDa. Purified lectin is rich in hydrophobic and basic amino acids and has an isoelectric point at pH 8.6. H. cratera lectin is relatively heat- and pH-stable. It agglutinates native and trypsinized, papainized and neuraminidase-treated human A, B, O, AB and sheep erythrocytes, and the hemagglutinating activity is independent of Ca(2+), Mn(2+) and Mg(2+) ions; D-galactose and N-acetyl-D-galactosamine are found to be moderate inhibitors of the activity. H. cratera lectin displays cytotoxic effect on HeLa and FemX cells and weak mitogenic effect on human T-lymphocytes pretreated with phytohemagglutinin (PHA).