Luisa Da Ros

Apparent induction of a cytochrome P450 with immunochemical similarities to CYP1A in digestive gland of the common mussel (Mytilus galloprovincialis L.) with exposure to 2,2′,3,4,4′,5′-hexachlorobiphenyl and Arochlor 1254

Abstract The induction of a cytochrome P450 with immunochemical similarities to CYP1A, and accompanying changes in microsomal NADPH-dependent benzo[a]pyrene (BaP) metabolism, were examined in digestive gland of the common mussel (Mytilus galloprovincialis L.) with exposure to 20 ppb water-borne polychlorobiphenyl (PCB) mixture (Arochlor 1254) for 4 or 10 days, or 4 days after a single injection into the mantle cavity of the mixed-type inducer PCB congener 2,2′,3,4,4′,5′-hexachlorobiphenyl (CB-138; 2.5 μg g−1 wet weight). Whole animal tissue levels of PCB following water-column exposure or injection were similar to those for mussel species from polluted field sites, viz. 0.8 to 1.9 μg g−1 wet weight. Levels of microsomal CYP1A-immunopositive protein increased 59% (CB-138) and 72% (Arochlor 1254; 10 days exposure) as determined by Western blot analysis using polyclonal antibodies to hepatic CYP1A of perch (Perca fluviatilis). No changes were seen in levels of digestive gland CYP1A-like mRNA 4 days after injection of CB-138 as determined by Northern analysis using cDNA to hepatic CYP1A1 of rainbow trout (Oncorhynchus mykiss). The increases in levels of CYP1A-immunopositive protein were accompanied by a shift in microsomal NADPH-dependent BaP metabolism towards phenol and diol and away from dione production, the former increasing from 32 to 85% of total free metabolites. The marked decrease in dione production (which is the major BaP metabolite formed in control microsomes) resulted in no increase in total microsomal BaP metabolism with exposure to PCBs. The Type I ligand α-naphthoflavone markedly inhibited microsomal phenol but had no affect on dione production, whereas the Type II ligand clotrimazole markedly inhibited dione, but had much less effect on phenol production. The overall results are interpreted in terms of the existence of an inducible CYP1A-like enzyme catalysing predominantly 2-electron monooxygenation leading to epoxide (and hence phenol and diol) formation, and a constitutive non-inducible cytochrome P450 catalysing predominantly 1-electron oxidation leading to dione formation. Both Arochlor 1254 or CB-138 produced cellular damage in the digestive gland in the form of decreased epithelial digestive cell height and decreased lysosomal membrane stability.

Biomonitoring approach with mussel Mytilus galloprovincialis (Lmk) and clam Ruditapes philippinarum (Adams and Reeve, 1850) in the Lagoon of Venice

Transplanted Mytilus galloprovincialis and native Ruditapes philippinarum were deployed in 10 sampling stations with different pollution impact within the Lagoon of Venice to evaluate the temporal variations and the suitability of the following cytochemical and histochemical biomarkers just as indicators of environmental stress: lysosomal membrane stability, lipofuscins, neutral lipids and lysosome to cytoplasm volume ratio. The physiological status of the organisms was also investigated by determining the survival in air capability and the reburrowing rate (clams). The biological parameters were assessed in June and October. Furthermore, for a better definition of the environmental aspects of the study sites, heavy metal, PAH and PCB concentrations were also evaluated in the sediments. As a whole, the biological responses examined in both species from all the sampling sites showed significant differences between the two seasonal campaigns, only lysosomal membrane stability exhibited less variability. Pollutants in sediments generally showed low-intermediate contamination levels, few hotspots persisting mostly in the inner areas of the lagoon, the most influenced by the industrial zone. Transplanted mussels were more responsive than native clams and the biological responses of both species varied temporally. The range of the spatial variability was always narrow and reflected only partially the broader variability shown by the chemical content in the sediments. In this sense, biological responses seemed to be particularly influenced by the high temporal and spatial heterogeneity that characterise the Lagoon of Venice, as well as most of the transitional environments.

Assessment of the impact of chemical pollutants on mussel, Mytilus galloprovincialis, from the Venice Lagoon, Italy

Abstract The purpose of this study was to identify possible relationships between biochemical—Catalase, Superoxide dismutase (SOD) and Propionaldehyde dehydrogenase (PDH)—and histochemical—Neutral Red Retention time (NRR)—parameters of mussels ( Mytilus galloprovincialis ) and chemical (Hg, Pb, Cd, Zn, Cr, DDTs and PCBs) contaminants from the most polluted area of the Venice Lagoon which lies around Porto Marghera (industrial pollution) and the city of Venice (domestic pollution). Sampling of mussels was undertaken at the beginning of March 1995 and repeated in March 1996, just before spawning, when tissue concentrations of chemical pollutants reach their annual maxima. The results of the organic contaminant analysis showed generally good agreement between sites for the two sampling periods, with values broadly 10% lower during 1996 as compared to 1995. Metal contaminant concentrations in tissues revealed greater variability than organic compounds. Distribution pattern of biological parameters were more complex and variable. The apparent lack of significant positive correlations between antioxidant enzyme activities and pollutant body burdens as well as the low values of NRR are consistent with the present debate on the use of these biological indices as markers of contaminant-mediated stress for molluscs.

Integrated use of biomarkers in the mussel Mytilus galloprovincialis for assessing off-shore gas platforms in the Adriatic Sea: Results of a two-year biomonitoring program

Despite a large number of gas platforms existing in the Adriatic Sea, which is a semi-enclosed basin characterized by a slow turnover rate and increasing industrial as well as other anthropogenic activities, the effects of these structures on the aquatic ecosystem require further investigation. Since 1998, multidisciplinary studies have been performed by CNR-ISMAR to comply with legislation and to support the development of protocols for the monitoring of offshore activities in the Adriatic Sea. The present study was developed to implement a biomonitoring plan to assess the ecotoxicological effects of the extraction activities of an off-shore gas platform. Biomarkers were evaluated in mussels collected from the platform in relation to physiological stress, DNA damage, cellular damage, oxidative stress and exposure effects. Organic contaminants and trace element bioaccumulation were also assessed in the soft body of the mussels to correlate bioaccumulation of pollutants with biomarker responses. The results indicate an absence of platform-related environmental stress.

Reproductive cycle of the mussel (Mytilus galloprovincialis Lmk) in Venice Lagoon (North Adriatic)

Abstract The reproductive cycle of Mytilus galloprovincialis Lmk cultured in Venice Lagoon has been studied from 1979 to 1982. Histology of the gonads shows that the gametes are ripe from September to May, when several spawnings occur with a peak at the end of winter (January -February). Reproductive activity is quiescent between July and August but release of gametes is possible even in summer, if only to a limited extent.

Biological effects of environmental pollutants in American Oyster, Crassostrea virginica: a field study in Laguna de Terminos, Mexico

The response of Crassostrea virginica to a complex mixture of toxic contaminants was studied at four sites in Laguna de Terminos, Mexico. Contaminants assessed were heavy metals, organochlorine compounds, and hydrocarbons. Biomarkers (cholinesterase activity, neutral red retention, and metallothionein), histopathology and prevalence of Perkinsus marinus were used to evaluate the effects of contaminants. The resultant contaminant body burden was moderately as high as a whole, and biomarker levels were also moderate. Oedema in the gills and systemic haemocytosis were the only histopathological lesions observed. P. marinus was never evidenced in the oyster tissues examined. Statistical results from principal components analysis show that metallothioneins are positively correlated with copper and chromium, neutral red and oedema with chlordanes, pentachloroanisol, drins, polychlorobiphenyls and total pesticides, total hydrocarbons and polycyclic aromatic hydrocarbons, whereas cholinesterase activity is negatively correlated to Cu and Cr. The biomarkers used were sensitive indicators responding to moderate levels of pollutants.

Biomonitoring of a polluted coastal area (Bay of Muggia, Northern Adriatic Sea): A five-year study using transplanted mussels.

The subcellular effects of pollution were evaluated using two lysosomal biomarkers in mussels, Mytilus galloprovincialis, deployed periodically over a period of 5 years in a harbour area in the Bay of Muggia (Gulf of Trieste, North Adriatic Sea) that is strongly influenced by anthropogenic activities. Mussels were collected from a clean marine farm and analysed (sample T0). A sub-sample was transplanted to the harbour site (sample M) and analysed after about 12 weeks. An additional sub-sample was relocated within the farm as a control and was also tested at the end of the 12-week period (sample T1). The transplantation procedures were repeated twice yearly for 5 consecutive years, starting in 2009. Two well-established lysosomal biomarkers, i.e. lysosomal membrane stability and lipofuscin accumulation, were evaluated in hepatopancreas cells. The body condition index and mortality rate were also assessed. Moreover, various pollutants were determined in both mussel flesh, for a better comprehension of the biological response, and sediments, for a general characterization of the study area. As a whole, the applied biomarkers were found to be appropriate for determining the responses of mussels to environmental pollutant loads over time. Variations in lysosomal membrane stability and lipofuscin content were mostly related to total PAHs and metals respectively. Our results confirm the usefulness of active biomonitoring in evaluating pollution trends in marine coastal areas and in particular the value of lysosomal biomarkers as a rapid screening tool for highlighting pollutant effects at least at organism level.

Use of biomarkers to assess the welfare of the edible clam, Ruditapes philippinarum: may it be a tool for proving areas of origin?

The use of biomarkers as early-warning signals to assess the impact of pollutants and other environmental stressors in coastal habitats has been internationally recognized as a useful tool in marine coastal monitoring. We propose a similar approach to verify the welfare of edible shellfish, Ruditapes philippinarum, thus exploiting its possible application as an effective tool to verify the origin of fishing/aquaculture products. A selected battery of biomarkers was chosen and applied to organisms from two different habitats in the Lagoon of Venice (a farming site and a natural area where fishing is currently banned, because of pollution problems) and from a local market. Biological responses were evaluated physiologically (survival in air test and condition index), behaviourally (reburrowing rate), and histochemically (quantification of neutral lipids and lipofuscin). Micropollutants (heavy metals, PCBs, and PAHs) were also determined in the soft tissues. Results indicated clams from the farming site were in the best condition with regard to both chemical and biological measurements. In contrast, the market sample contained the highest concentrations of PAHs. PCBs and heavy metals varied slightly and quite irregularly among the three samples. The worst values of the biomarkers were observed for clams collected at the polluted site, where, in particular, the lowest physiological and behavioural indexes and the highest accumulation of lipofuscin in the digestive tissue were recorded.

Biochemical and lysosomal biomarkers in the mussel Mytilus galloprovincialis from the Mar Piccolo of Taranto (Ionian Sea, Southern Italy)

Biomarkers are internationally recognized as useful tools in marine coastal biomonitoring, in particular, as early-warning signals at the level of individual organisms to assess biological effects of pollutants and other stressors. In the present study, Mytilus galloprovincialis has been employed as a sentinel organism to assess biological pollution effects in the Mar Piccolo of Taranto (Southern Italy), a coastal lagoon divided into two small inlets, connected to the open sea through one natural and one artificial narrow openings. Mussels were collected in June 2013 at three sites located within each of the two inlets of the Mar Piccolo. Biological effects were investigated through a suite of biomarkers suitable to reflect effects and/or exposure to contaminants at biochemical and cellular levels. Biochemical biomarkers included glutathione-S-transferase (GST) and acetylcholinesterase (AChE) enzyme activities; as histochemical biomarkers, lysosomal membrane stability, lipofuscin and neutral lipid accumulation, and lysosomal structural changes were considered. As a whole, results highlighted differences among the three study sites, particularly for GST, AChE, and lipofuscins, which are consistent with the variations of the chemical pollutants in sediments. The applied biomarkers showed that a stress syndrome likely to be ascribed to environmental pollutants is occurring in mussels living in the Mar Piccolo of Taranto, in particular, the ones inhabiting the first inlet.