Alexandra Cravo

Genotoxicity of copper oxide and silver nanoparticles in the mussel Mytilus galloprovincialis

Though there is some information on cytotoxicity of copper nanoparticles and silver nanoparticles on human cell lines, there is no information on their genotoxic and cytotoxic behaviour in bivalve molluscs. The aim of this study was to investigate the genotoxic impact of copper oxide and silver nanoparticles using mussels Mytilus galloprovincialis. Mussels were exposed to 10 μg L⁻¹ of CuO nanoparticles and Cu²⁺ and Ag nanoparticles and Ag⁺ for 15 days to assess genotoxic effects in hemocytes using the comet assay. The results obtained indicated that copper and silver forms (nanoparticles and ionic) induced DNA damage in hemolymph cells and a time-response effect was evident when compared to unexposed mussels. Ionic forms presented higher genotoxicity than nanoparticles, suggesting different mechanisms of action that may be mediated through oxidative stress. DNA strand breaks proved to be a useful biomarker of exposure to genotoxic effects of CuO and Ag nanoparticles in marine molluscs.

A multibiomarker approach in Mytilus galloprovincialis to assess environmental quality

A multibiomarker approach was carried out for the first time in the South Portuguese Coast using Mytilus galloprovincialis, to assess environmental quality, establish if there are adverse biological responses associated to different sources of anthropogenic contamination and to determine spatial and seasonal trends. For this purpose the battery of biomarkers selected was: superoxide dismutase (SOD), catalase (CAT), glutathione peroxidases (GPx total and Se dependent), Cytochrome P450 component system, Glutathione-S-transferase (GST) and acetylcholinesterase (AChE), metallothionein (MT) and lead-delta-aminolevulinic acid dehydratase (ALAD), lipid peroxidation (LPO) and Condition Index (CI) along with the determination of PAHs and metals (Cd, Cu, Ni, Pb and Zn). Results show that despite the levels of both organic and metallic contaminants in these eight spots in the South Coast of Portugal not being particularly high compared with other contaminated/polluted sites worldwide, the selected battery of biomarkers responded efficiently to the environmental changes and allowed an environmental assessment between seasons and sites. Different spatial and seasonal responses were evident along the South Coast of Portugal, meaning that the contamination is not homogeneous. This does not only reflect different competition, origin and intensity of contamination, but also different environmental conditions (e.g. temperature, salinity). Along the South Portuguese Coast site 8 was the most contaminated, while site 2 was considered the least contaminated. Despite environmental factors possibly causing difficulties in the general interpretation of biomarker data, those that better responded to environmental contamination were CYP450, SOD-mit and T-GPx for the summation SigmaPAHs, MT (digestive gland) for metals (especially Cu), ALAD for Pb and LPO for both organic and metallic contamination. These biomarkers were also positively correlated with temperature in summer, revealing this as a more stressful/critical season. In future environmental contamination assessments there is no need to analyse the components b5, P418, NADH and NADPH of phase I MFO system, and MT in the gills, since their responses are not evident.

A multibiomarker approach in the clam Ruditapes decussatus to assess the impact of pollution in the Ria Formosa lagoon, South Coast of Portugal

The Ria Formosa lagoon is an ecosystem whose water quality reflects the anthropogenic influence upon the surrounding areas. In this lagoon, the clam Ruditapes decussatus has a great economical importance and has been widely used as a biomonitor. A multibiomarker approach (δ-aminolevulinic acid dehydratase, metallothionein, lipid peroxidation, acetylcholinesterase, alkali-labile phosphates, DNA damage) was applied to assess the environmental quality of this ecosystem and the accumulation of contaminants and their potential adverse effects on clams. Clams were sampled in different shellfish beds in the period between July 2007 and December 2008 and abiotic parameters (temperature, salinity, pH and dissolved oxygen of seawater and organic matter in the sediment), condition index, metals (Cd, Cu, Zn, Ni, Pb), TBTs and PAHs concentrations were measured in clam tissues. Data was integrated using Principal Component Analyses and biomarker indices: IBR (Integrated Biomarker Response) and HSI (Health Status Index). This multibiomarker approach enabled discrimination of a time and space trend between sites with different degrees of anthropogenic contamination, identifying one of them (site 2) as the most stressful and summer months as the most critical period for clams due to an increase of environmental stress (anthropogenic pressure along with extreme environmental conditions, e.g. temperature, dissolved oxygen, organic matter in the sediments, etc). The selected biomarkers provided an integrated response to assess the environmental quality of the system, proving to be a useful approach when complex mixtures of contaminants occur.

Partitioning of trace metals between soft tissues and shells of Patella aspera.

This paper describes the partitioning of iron, manganese, zinc, copper, cadmium, cobalt, nickel and lead between the soft tissues and the shells of the gastropod mollusc Patella aspera. Specimens of the limpet P. aspera were collected from a clean coastal marine site (CMS) and from an estuarine contaminated site (ECS) on the south coast of Portugal. Fe and Zn concentrations were significantly lower on both populations and less variable in the shells than in the soft tissues. In contrast Mn concentrations were higher in the shells than in the soft tissues at ECS. The partitioning factor (PF), defined as the ratio between the mean metal concentrations in soft tissues and in the shells, was maximum for iron and minimum for manganese. Between the soft tissues and the shells, only manganese at ECS recorded a significant relationship between metal concentrations, suggesting that the mechanism that controls the accumulation of this essential metal in both tissues was different from the others. From the obtained data, in biomonitoring sewage contamination studies, shells of P. aspera can be considered as a good indicator for Mn while the soft tissues for Zn. As copper, cadmium, nickel and cobalt concentrations were much higher in the soft tissues than in the shells (< 0.1 microg g(-1)), this indicates that the shells were not a good indicator for these metals because they do not reflect the environmental bioavailability as do the soft tissues.

Using biochemical and isotope geochemistry to understand the environmental and public health implications of lead pollution in the lower Guadiana River, Iberia: A freshwater bivalve study

Lead is a natural component of aquatic ecosystems with no known biological role and is highly toxic. Its toxicity stems from its ability to mimic biologically important metals and to produce membrane damage through lipid peroxidation (LPO). Most lead poisoning symptoms are thought to occur by interfering with an essential enzyme, delta-aminolevulinic acid dehydratase (ALAD), the activity of which is markedly inhibited by lead. The purpose of this work was to study the levels and effects of lead pollution (responses of ALAD and oxidative stress biomarker LPO) in the freshwater bivalve Corbicula fluminea along the lower Guadiana River (Portugal and Spain); a major river system impacted by historic mining pollution and more recent anthropogenic inputs. The results show that the enzymatic activity of ALAD is negatively correlated with the total Pb concentration of the whole tissue suggesting that ALAD has considerable potential as a biomarker of lead exposure in C. fluminea. To identify the sources of lead to which bivalves have been exposed, high precision (206)Pb/(204)Pb, (207)Pb/(204)Pb, (208)Pb/(204)/Pb ratios for C. fluminea confirm that historical mining activities in the Iberian Pyrite Belt are the dominant source of lead pollution in the lower Guadiana River. The isotope patterns however exhibit marked seasonal and geographic variation in response to rainfall and river water management. Locally, other anthropogenic sources of lead have been detected in C. fluminea close to population centres, thus adding to its versatility as a freshwater bio-indicator. Overall, the study highlights the value of natural ecosystems as monitors of water quality and their importance for public health assessment and surveillance.

Integrated approach to assess ecosystem health in harbor areas.

UNLABELLED Harbors are critical environments with strategic economic importance but with potential environmental impact: health assessment criteria are a key issue. An ecosystem health status approach was carried out in Portimão harbor as a case-study. Priority and specific chemical levels in sediments along with their bioavailability in mussels, bioassays and a wide array of biomarkers were integrated in a biomarker index (IBR index) and the overall data in a weight of evidence (WOE) model. Metals, PAHs, PCBs and HCB were not particularly high compared with sediment guidelines and standards for dredging. Bioavailability was evident for Cd, Cu and Zn. Biomarkers proved more sensitive namely changes of antioxidant responses, metallothioneins and vittellogenin-like proteins. IBR index indicated that site 4 was the most impacted area. Assessment of the health status by WOE approach highlighted the importance of integrating sediment chemistry, bioaccumulation, biomarkers and bioassays and revealed that despite some disturbance in the harbor area, there was also an impact of urban effluents from upstream. CAPSULE ABSTRACT Environmental quality assessment in harbors.

Minor and trace elements in the shell of Patella aspera (Röding 1798)

Specimens of the limpet Patella aspera were collected from a clean, coastal marine site and a contaminated estuary on the south coast of Portugal. The shells were analysed individually for their minor (Mg, Sr) and trace element (Fe, Mn, Zn) content. Mean concentrations of these elements in the shell of P. aspera were 4651, 1318, 35.8, 29.9 and 5.5 microg g(-1), respectively. The elemental concentrations exhibited both a marked intra- and inter-population variability. Despite the variability within individual populations, significant differences in the trace element composition were apparent between the shells taken from the two sites. Small shells (< 2 g) provided the best resolution between sites for both manganese and iron. Differences in zinc were best resolved for larger shells. The shell of P. aspera has an extraordinarily high magnesium concentration, which is insensitive to gross salinity differences, and a trace metal assemblage that can be interpreted in terms of environmental exposure. On these grounds, it is recommended that the shell of P. aspera is a tissue for potential use in environmental trace metal monitoring.

Metallothionein concentrations in a population of Patella aspera: variation with size.

The determination of metallothionein (MT) concentrations in species with different feeding habits is important from the ecotoxicological point of view because it provides a better understanding of the role of these proteins in metal uptake pathways. The main objective was study the variation of MT and metal (Cd, Cu and Zn) concentrations with size and weight in the limpets Patella aspera. In addition investigate the relationship between MT and metal concentrations in limpets from different metal load environments of the south coast of Portugal with the aim to use MT in P. aspera as a biomarker of metal exposure. MT concentrations in the whole soft tissues of P. aspera increased with size and weight while metals decrease with size and weight. MT concentrations showed no significant relationship with Cd or Cu concentrations in the limpets from the South Portuguese Coast. However, a negative exponential relationship detected between MT and Zn concentrations suggest that Zn bound to MT might be displaced by Cd or Cu ions. The ability of limpets to store both Cd and Cu bound to MT may be responsible for the tolerance of this species to contaminated environments. The relationship between MT concentrations and Zn indicates that MT seems to play a minor role in binding Zn in Patella species.

DNA damage as a biomarker of genotoxic contamination in Mytilus galloprovincialis from the south coast of Portugal

DNA damage was evaluated in the haemolymph of Mytilus galloprovincialis from nine sites along the south coast of Portugal using the comet assay. DNA damage was low, in the same range of sites considered to suffer low impact from genotoxic contaminants. Even so, differences between sites, seasons and genders were found. Highest values were in mussels from the main estuaries and the fishery harbour, reflecting higher genotoxin levels, whereas the lowest values can be used as a baseline for future work. Non-contaminant related factors (e.g. temperature and oxygen) were also shown to influence DNA damage. Between seasons, highest values were in summer related not only to the increase of tourism in this region (∼10-fold), but also to temperature. Between genders, males were found to be more sensitive. The condition index was also generally higher in summer. Lipid peroxidation, another damage biomarker, was measured in gills to assess if there is any association between the responses of both biomarkers and if they are similarly affected by the same environmental conditions. LPO like DNA damage was higher in summer. This work confirms that DNA damage is a sensitive biomarker to discriminate genotoxic contamination, even in areas considered to suffer low impact from genotoxins.

Mercury concentrations in invertebrates from Mid-Atlantic Ridge hydrothermal vent fields

Mercury determinations were carried out in mussels ( Bathymodiolus azoricus ) from three Mid-Atlantic Ridge hydrothermal vents (Lucky Strike, Menez Gwen and Rainbow) and shrimps ( Rimicaris exoculata and Mirocaris fortunata ) from Rainbow. Among the three hydrothermal vents, mussels of Menez Gwen show the highest levels of total Hg and comparing mussels and shrimps from Rainbow the former show more Hg than shrimps. Mussels from different hydrothermal vents are exposed to different kinds of environment which may result in distinct bioaccumulation processes. Detoxification processes in shrimps are related to the low concentrations found. When compared with coastal species from unpolluted sites, mussels show higher concentrations of total Hg and shrimps lower levels. The methyl-mercury concentrations found were very low, not exceeding the detection limit of the technique.