V. Moschino

Assessing the significance of Ruditapes philippinarum as a sentinel for sediment pollution: bioaccumulation and biomarker responses.

The present study assessed whether the bivalve Ruditapes philippinarum may be appropriately deployed as a bioindicator in monitoring transitional environments, in terms of bioaccumulation potential and biomarker responses. The concentrations of trace metals, PAHs and PCBs were determined in sediments and clam tissue, and biomarkers were estimated at various levels of biological complexity (i.e. metallothioneins, lipofuscins, survival-in-air and reburrowing behaviour). Sediments and clams were collected biannually in 2004 and 2005 at eight sites within Venice lagoon, which were influenced differently by natural and anthropogenic impacts. Results highlighted that the broad variations of pollutant concentrations in sediments were not consistent either with the body residuals or with the biomarker responses. Consequently, on the basis of the observed weak responsiveness and sensitivity to anthropogenic stressors we suggest a more cautious use for R. philippinarum as sentinel organism, at least in estuarine sediments in the north Adriatic transitional areas.

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.

Sediment elutriate toxicity testing with embryos of sea urchin (Paracentrotus lividus)

Several laboratory conditions for preparing and testing of elutriates from sediments of industrial and urban contaminated areas of the Lagoon of Venice (Italy) were assessed in experiments using embryos of the sea urchin Paracentrotus lividus . Elutriates were obtained by mixing of aerated offshore sea water and fresh aliquots of sediments (5, 20, 200 g l -1 ) for four hours. Sediments were then allowed to settle for 12 hours, the supernatant was filtered (at 1 µm) and subsequently tested, undiluted and diluted, at ratios of 1:4 and 1:10. Each diluted sample was again tested after further filtering at 0.45 µm. Bioassays were carried out on embryos from post-fertilisation to the pluteus stage. The following parameters were observed: survival, frequency of developmental stages, and embryonic growth. The latter was most sensitive to varying experimental conditions, as the length of the skeletal somatic rods of plutei decreased significantly with increasing sediment concentration. Percent survival was slightly reduced in non-diluted and non-filtered treatments only.The frequency of developmental stages was always significantly different in 200 g l -1 elutriates 24 hours after fertilisation. In bioassays, no direct relationship was observed between sediment concentrations in elutriates and toxic effects, fitting the contamination levels revealed by chemical analysis of elutriates. When short mixing times were used, the most convenient sediment:water ratio was 1:4 (without further dilution or filtering) which more clearly showed the different degrees of toxicity among sediments. The elutriate bioassay with lividus embryos is proposed as a rapid and highly sensitive approach for monitoring sediment toxicity in water-column organisms.

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.

An ecotoxicological approach for the Boka Kotorska Bay (south-eastern Adriatic Sea): First evaluation of lysosomal responses and metallothionein induction in mussels

Mytilus galloprovincialis was used as a biomonitoring organism in Boka Kotorska Bay, a coastal transitional ecosystem in south-western Montenegro. Native mussels were collected in June 2008 at four sites thought to be differently impacted. Biological effects were investigated analysing both generic and specific biomarkers at cellular level (metallothionein content, lysosomal membrane stability, lipofuscin and neutral lipid accumulation, lysosomal structural changes). Trace element levels in mussels were quite low, only Cu and Zn exhibiting slight increases in the two sampling sites of Tivat Bay, the first one in front of a small working dockyard and the other in the water area of a former Naval dockyard. Mussels collected from these sites exhibited the highest values in neutral lipid and lysosomal volume density and the lowest neutral red retention times. Metallothionein content was always low, reflecting the minor body burden of the trace elements and suggesting a non-relevant environmental induction.

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.

An ecotoxicological approach to evaluate the effects of tourism impacts in the Marine Protected Area of La Maddalena (Sardinia, Italy)

In the Marine Protected Area of La Maddalena Archipelago, environmental protection rules and safeguard measures for nautical activities have helped in reducing anthropogenic pressure; however, tourism related activities remain particularly significant in summer. With the aim of evaluating their impacts, the biomarker approach using transplanted Mytilus galloprovincialis as sentinel organisms coupled with POCIS deployment was applied. Mussels, translocated to four marine areas differently impacted by tourism activities, were sampled before, during and after the tourist season. Moreover, endocrine disruptors in passive samplers POCIS and the cellular toxicity of whole POCIS extracts on mussel haemocytes were evaluated to integrate ecotoxicological information. Lysosomal biomarkers, condition index and mortality rate, as well as metals in tissues suggested an alteration of the health status of mussels transplanted to the most impacted sites. The cellular toxicity of POCIS extracts was pointed out, notwithstanding the concentrations of the examined compounds were always below the detection limits.

Effects of hydraulic dredging on target species Chamelea gallina from the northern Adriatic Sea: physiological responses and shell damage

The impact of experimental dredging was assessed on the venus clam Chamelea gallina from seabeds off the Lagoon of Venice. Commercial-sized clams were collected using high water pressure and mechanical sorting (HP), as in professional fishing, and low water pressure without sorting (LP). To highlight the effects of acute stress due to the different approaches in harvesting clams, various biological responses were evaluated at both organism and cell level. Filtration and respiration rates showed opposite behaviour, the former significantly decreasing, the latter increasing, when mechanical stress was higher. Consequently, lower values of scope for growth in HP clams were observed. The survival in air test exhibited significantly lower median survival time values (LT 50 ) in more severely stressed animals. The haematocrit value and phagocytic index significantly decreased in HP samples, revealing a detrimental effect on mechanisms of immunological defence. Acid phosphatase and β-glucuronidase activity was also affected by increasing mechanical stress, even though an opposite trend was shown in the two investigated enzymes. Lastly, higher damage levels were detected in shells of HP clams.