Integrated use of biomarkers (superoxide dismutase, catalase and lipid peroxidation) in mussels Mytilus galloprovincialis for assessing heavy metals' pollution in coastal areas from the Saronikos Gulf of Greece

Mussels are used as sentinel organisms and bioindicators to evaluate the toxic effects of chemical pollutants in marine organisms, especially heavy metals, representing an important tool for biomonitoring environmental pollution in coastal areas. Antioxidant defence enzymes play an important role in cellular antioxidant defence systems and protect from oxidative damage by reactive oxygen species (ROS). Indigenous mussels Mytilus galloprovincialis of the Saronikos Gulf of Greece were used for monitoring heavy metal pollution in three polluted sites in the area and in one unpolluted site. Seasonal variations of the activity of antioxidant defence enzymes, superoxide dismutase (SOD) and catalase (CAT), as well as lipid peroxidation (LP) were measured as biomarkers in a period of three years in relation to concentrations of trace metals in their gills and mantle and compared to mussels from an unpolluted sampling site. SOD activity increased at least 2 fold at the polluted sites when compared to the control site (the high activity was recorded in the spring time). CAT activity was increased 2-3 times at the polluted sites, with high activity in the winter and spring time, compared to the control site. LP concentration was twice higher at the polluted sites, following the same seasonal pattern. Trace metals contents in mussels collected at polluted sites were 3-4 fold higher compared to the control site and showed moderate variations along the months, with a winter maximum followed by a summer pre-spawning minimum matching the seasonal trends of temperature and salinity. Our results showed that metal pollution in the Elefsis Bay (the most polluted coastal area) causes relatively medium levels of oxidative stress in tissues of mussels due to cellular oxy-radical generation. This study, which is the first in the area, showed that seasonal variations of the activity of antioxidant defence enzymes and LP concentrations in mussels can be used as potential biomarkers of toxicity for long-term monitoring in marine coastal ecosystems.     

Imposex levels and concentrations of organotin compounds (TBT and its metabolites) in Nassarius nitidus from the Lagoon of Venice

Specimens of Nassarius nitidus were collected in seven stations of the Venice Lagoon to assess the levels of tributyltin (TBT) and its metabolites monobutyltin and dibutyltin in the tissues and monitor their effect on organisms, in particular the phenomenon of imposex (superimposition of male sexual characteristics on females). The following values of population indices were found: vas deferens sequence: 1.2+/-0.7-4.0+/-0.5; relative penis length: 6-47%. The least impacted station was situated in the northern part of the Lagoon, where females without imposex were found and Butyltin (BuTs) concentrations in the organisms (average sum of BuTs=43+/-14 ngSng(-1)w.) were significantly lower than in the other stations (range of average sum of BuTs: 101+/-22-217+/-27 ngSng(-1)d.w.). Population indices were found to be related to the TBT content in the tissues. In particular VDSI had a significant logarithmic correlation: r=0.95, n=8, p<0.05.     

Chloropigments a in the Gulf of Gdańsk (Baltic Sea) as markers of the state of this environment

The Gulf of Gdańsk receives the waters of the Wis?a (Vistula), the largest Polish river and the second largest river flowing into the Baltic Sea. Chloropigments a (chlorophyll a and its derivatives) were determined in water and sediments from the Gulf. Samples were collected during cruises of R.V. 'Oceania' at 19 stations located at different water depths and distances from the shore, in May (2003) and October (2004), i.e., at the beginning and at the end of the growing season, respectively. Seawater was also sampled on the beach at Sopot in June, July and August 2004, i.e., during the height of the growing season. All water samples were collected with a bathometer and passed through Whatman GF/F filters. Recent sediments (0-10 cm) were collected with a core sampler and divided into sub-samples (0-1, 1-5 and 5-10 cm). The pigments were determined using HPLC-DAD. The results were first correlated with a set of parameters measured in situ during sample collection (salinity, temperature, oxygen content of seawater) and determined in the same material (organic carbon content, granulometry), then worked up statistically. Although this investigation shows that particular chloropigments a in recent sediments are indicative of the prevailing environmental conditions in the study area and the trends in their variations, they should be treated as a relative and not an absolute measure, since this is the first such study based on a large and coherent set containing both chloropigment a and environmental data. In addition, a chlorophyll a budget is estimated for the Gulf of Gdańsk on the basis of the literature and the authors' own data. Only c. 10% of the chlorophyll a produced annually in the Gulf of Gdańsk is transferred to sediments in the form of chloropigments a; 2.8% is in the form of undecomposed chlorophyll a and 7.2% in the form of chlorophyll a derivatives. About 90% of chlorophyll a is decomposed to colourless products.     

Recolonization and recovery dynamics of the macrozoobenthos after sand extraction in relict sand bottoms of the Northern Adriatic Sea

The long-term effects of sand extraction on macrozoobenthic communities were investigated in an offshore area in the Northern Adriatic Sea characterised by relict sands formed during the last Adriatic post-glacial transgression. Surveys were carried out before, during and 1, 6, 12, 18, 24 and 30 months after extraction at three impacted and seven reference stations. The operations did not influence the physical characteristics of the sediment, but they caused almost complete defaunation at dredged sites. Univariate and multivariate analyses highlighted that the macrozoobenthic community responses to the dredging operations were (1) a rapid initial recolonisation phase by the dominant taxa present before dredging, which took place 6-12 months after sand extraction; (2) a slower recovery phase, that ended 30 months after the operations, when the composition and structure of the communities were similar in the dredged and reference areas. This pattern of recolonisation-recovery fits well with the commonly encountered scenario where the substratum merely remains unchanged after marine aggregate extraction.     

Filter-feeder macroinvertebrates as key players in culturable bacteria biodiversity control: a case of study with Sabella spallanzanii (Polychaeta: Sabellidae)

The present study investigates the effect of the filtering activity of Sabella spallanzanii on the culturable heterotrophic bacterial community through the comparison of the bacterial diversity in transplanted polychaetes and the surrounding seawater. For isolation of culturable heterotrophic bacteria, seawater samples as well as polychaete homogenates were plated in triplicates onto Bacto Marine Agar 2216 (Difco). All the colonies grown were isolated, subcultured and identified by several morphological, biochemical and cultural methods. Some bacterial genera showed higher average abundances in polychaetes than in seawater (i.e. Lucibacterium and Photobacterium). Aeromonas represented a conspicuous component of the bacterial community both in S. spallanzanii and seawater. The presence of Cytophaga and Pseudomonas was also relevant in the examined seawater samples. The selective concentrations of some bacterial genera inside S. spallanzanii either by grazing on bacteria or their capability as bacterial reservoirs, provides evidence for the role of macrobenthic invertebrates as key determinants for microbial diversity.     

Hepatic biomarkers of sediment-associated pollution in juvenile turbot, Scophthalmus maximus L

Hatchery-reared turbot (Scophthalmus maximus L.) were exposed for 3 weeks, under laboratory conditions, to sediment collected from polluted sites in Cork Harbour and a reference site at Ballymacoda, Co. Cork, Ireland. The potential of surficial sediment for inducing hepatic biomarkers was assessed at two levels of biological organisation: expression of cytochrome P450 [Western blotting analysis and 7-ethoxy-resorufin O-dealkylase (EROD), 7-benzoxy resorufin O-dealkylase (BROD), 7-methoxy resorufin O-dealkylase (MROD), 7-pentoxy-resorufin O-dealkylase (PROD) activities] and DNA integrity (Comet assay). Positive controls were generated, either by exposing turbot to cadmium chloride-spiked seawater (Comet assay) or to beta-naphthaflavone by intraperitoneal injection (cytochrome P450 induction). The induction of cytochrome P450 activity (EROD, MROD and PROD) in animals following a 7-day exposure to contaminated sediments was significantly higher than those exposed to reference site sediment and remained elevated thereafter; BROD was not induced. DNA single-strand breaks were also significantly higher following exposure to contaminated sediments throughout the experiment. Although no direct correlation between induction of alkoxyresorufin O-dealkylase activities and a particular chemical class was established, the induction of MROD and PROD activities in fish exposed to sediments containing complex contaminant mixtures, appeared to be more sensitive than conventional EROD activity assays. We conclude from the present laboratory study that S. maximus is a suitable sentinel species for the assessment of moderately contaminated sediments and therefore allows for the further development of this model for future, ecologically relevant, field studies.     

Short-term response of the slow growing seagrass Posidonia oceanica to simulated anchor impact

Experimental evaluations about the impact of anchors of small vessels have previously shown that each anchoring can on average damage up to six shoots of Posidonia oceanica, removing small amount of biomass and, at the same time, interrupting continuity among shoots. The aim of the paper was to investigate the response of P. oceanica to different damage intensity at two levels of substrata compactness. Three treatments were considered: control (no damage); low damage (simulated anchor damage by three strokes of a hoe); and high damage (six strokes). Disturbance was higher where the substratum was highly penetrable and after one year significant variation was observed among treatments for both the number of leaves per shoot and shoot density. Conversely, the number of leaves per shoot by the end of the study was similar among all treatment combinations, suggesting that this was the only phenological feature that recovered, and probably the only result that through clonal integration could be achieved. These data strongly highlighted the role that anchoring might have on the slow growing seagrass P. oceanica meadow.     

Effect of exposure to benzo[a]pyrene on SODs, CYP1A1/1A2- and CYP2E1 immunopositive proteins in the blood clam Scapharca inaequivalvis

The effects of water-borne exposure to benzo[a]pyrene (36 h; celite-bound 0.44 mg L(-1) B[a]P) on cytochrome P450 (CYP) and superoxide dismutases (SODs) were examined in digestive gland of the blood clam, Scapharca inaequivalvis. B[a]P accumulation and elimination were rapid, with maximum whole-body concentrations of 1.78 ng g(-1) wet wt after 12 h of treatment, followed by a progressive decline to 0.89 ng g(-1) at 36 h. The presence of B[a]P resulted in an increase in total CYP of digestive gland microsomes from 54+/-14 to 108+/-21 pmol/mg protein (mean+/-SD; p<0.05, 24 h). Increases were also seen in microsomal CYP1A1/1A2-immunopositive protein (50.5 kDa app. mol. wt; p<0.05), but not CYP2E1-immunopositive protein (49 kDa app. mol. wt.), indicating a specific response of the former isoform. Exposure to B[a]P produced a steady increase in Mn-SOD digestive gland activity (p<0.01; p<0.05) but no significant change in Cu/Zn-SOD activity. The respective proteins, measured by western blotting, were not significant induced after B[a]P exposure. Cu/Zn-SOD and Mn-SOD activities were correlated with total CYP levels (r=0.96 and 0.63, respectively), indicating a role for CYP in reactive oxygen species (ROS) production during exposure. Both 'NADPH-independent' and NADPH-dependent metabolism of B[a]P by digestive gland microsomes was seen, producing mainly 1,6-, 3,6- and 6,12-diones, with some phenols and 7,8-dihydrodiol; putative protein adducts were also formed. Redox cycling of the diones may also have contributed to ROS production, leading to the increased SOD activities.     

The effect of long-term depuration on levels of oxidative stress biomarkers in mullets (Mugil cephalus) chronically exposed to contaminants

The present study aimed to obtain additional data on the effect of long-term depuration on the levels of oxidative stress biomarkers, and to clarify the role of mullets for monitoring pollution in River Douro estuary. Mullets chronically exposed to a mixture of contaminants in Douro estuary were captured in Spring of 2001, 2002 and 2003. The activities of antioxidant enzymes catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPX); and oxidative damages in lipids (lipid peroxidation) and in proteins (protein carbonyl content) were assessed at capture day and after transfer to unpolluted seawater for 1, 4 and 8 months. An overall decrease in the activities of the antioxidant enzymes was detected, except for the GPX after 4 months depuration. CAT activity exhibited the more significant decrease at the end of the long-term depuration. The decrease in SOD activity after 1 month of depuration was then maintained during the remaining depuration period. Regarding oxidative damages, a decrease in lipid peroxidation as well as the content of oxidised proteins was observed during depuration. Indeed, at capture the activities of antioxidant defences were higher as a result of the formation of reactive oxygen species (ROS) from the metabolism of pollutants. The oxidative damaged molecules were repaired or degraded during the depuration period, supporting the use of such damages as indicators of exposure to pro-oxidant pollutants.     

Evaluation of the applicability of a fuzzy index of ecosystem integrity (FINE) to characterize the status of Tyrrhenian lagoons

The new index FINE, a multimetric, fuzzy-based index for the evaluation of environmental quality for Mediterranean transitional waters, was calculated using biotic data gathered between 2000 and 2006 at 15 stations in 4 Tyrrhenian transitional water ecosystems (Orbetello Lagoon, Padrongiano Delta, Stagno di S. Teodoro, and Stagno di Tortolì), i.e. in a different biogeographic sub-province respect to the one in which the index was developed and validated. The rationale of FINE is that certain attributes, selected on the basis of established principles of benthic ecology, are fundamental for lagoon ecosystem function. A set of other indices (Simpson's 1-lambda', the W-statistics, AMBI, and BOPA) was also calculated and compared to FINE outputs. FINE, 1-lambda', and the W-statistics were significantly correlated with sedimentary organic matter content. Some stations were unequivocally assigned to the same ecological status, independently from the index used; for others, some discrepancies were evident. Mediterranean transitional waters probably share ecosystem complexities which are not yet fully captured by indices developed and validated elsewhere.