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- Genotoxicity of copper oxide and silver nanoparticles in the mussel Mytilus galloprovincialisPublication . Gomes, Tânia; Araújo, Olinda; Pereira, Rita; Catarina Almeida, Ana; Cravo, Alexandra; Bebianno, Maria JoãoThough 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.
- Efficiency of 2-phenoxyethanol and Clove Oil for Reducing Handling Stress in Reared Meagre, Argyrosomus regius (Pisces: Sciaenidae)Publication . Barata, Marisa; Soares, Florbela; Almeida, Ana Catarina; Pousão-Ferreira, Pedro; Ribeiro, Laura; Aragão, CláudiaThe effect of two anesthetics, 2-phenoxyethanol (2-PE) and clove oil (COil) were studied in meagre, Argyrosomus regius. This study aimed to determine the adequate dose of anesthesia for different aquaculture procedures, to assess a sedative or stressor effect of low anesthetic concentrations, and to assess the effect of low anesthetic doses at high fish densities for longer periods of time on fish survival and behavior. Anesthetics were tested at different concentrations: 2-PE at 100, 250, 400, 550, and 700mg/L; COil at 25, 40, 55, 70, and 85mg/L. Meagre became anesthetized within 10min when using concentrations above 250mg/L for 2-PE and above 40mg/L for COil. Deep anesthesia was achieved at 700 and 85mg/L, for 2-PE and COil, respectively. The fish did not react when blood was collected at these concentrations. Plasma cortisol and glucose levels were similar between sedated meagre with both 2-PE-100mg/L and COil-10mg/L, and the control (not sedated; P>0.05). This suggests that low concentrations of these anesthetics induce similar stress response as handling without anesthesia during routine activities. No mortality was registered when meagre was maintained at high densities with low concentrations of each anesthetic for 2h.
- Genotoxicity in two bivalve species from a coastal lagoon in the south of PortugalPublication . Catarina Almeida, Ana; G. Pereira, Catarina; Gomes, Tânia; Cardoso, Cátia; Bebianno, Maria João; Cravo, AlexandraDNA damage was evaluated by comet assay in the haemolymph of two bivalve species Ruditapes decussatus and Mytilus galloprovincialis from the Ria Formosa lagoon (south Coast of Portugal). Clams and mussels were sampled from sites close to each other to determine interspecific responses to similar environmental conditions, considering also seasonal and gender differences. Coupled with genotoxic effect, another damage biomarker (lipid peroxidation) was analysed to verify if the conditions that instigate DNA damage can be related with injury to cell membranes. For both species, DNA damage was low, reflecting the low levels of genotoxic contaminants in the lagoon, and no interspecific differences were found. However, seasonal differences were established for both bivalve species, reflecting higher environmental stress in summer. Regarding gender susceptibility, only clams showed differences in percentage of Tail DNA, with females more sensitive to DNA damage than males. Additionally, results for clams point out that factors responsible for LPO may not be the same as those causing genotoxicity. This study demonstrated that DNA damage is a sensitive biomarker to discriminate spatial, temporal and gender differences, being an appropriate biomarker for genotoxicity evaluation even in places of low contamination, such as the Ria Formosa lagoon.
- Metal concentrations in the shell of Bathymodiolus azoricus from contrasting hydrothermal vent fields on the mid-Atlantic ridgePublication . Cravo, Alexandra; Foster, P.; Catarina Almeida, Ana; Bebianno, Maria João; Company, RuiSpecimens of Bathymodiolus azoricus were sampled along the Mid-Atlantic Ridge at the Menez Gwen, Lucky Strike and Rainbow hydrothermal fields. Individual shells (n = 51), through the weight range 0.62 to 15.70 g, were analyzed for their magnesium, strontium, iron, manganese, copper, zinc and cadmium concentrations. Amongst the marine molluscs the shell of B. azoricus is confirmed as being particularly impoverished in strontium (mean 943 microg g(-1)). Trace metal concentrations in the shells decreased in the order Fe> Mn> Zn> Cu> Cd. Despite originating from trace metal rich environments mean concentrations were low (37.9, 13.2, 10.7, 1.1 and 0.7 microg g(-1), respectively). Irrespective of geographical origin magnesium, strontium and copper concentrations were primarily dictated by shell weight. In contrast cadmium concentrations were elevated in shells from the Rainbow field and ambient seawater chemistry imparted site specific chemical fingerprints to the shells with respect to the iron to manganese ratio.
- Interspecific variability of endocrine disruption and oxidative stress in two bivalve species from the Ria Formosa Lagoon (south coast of Portugal)Publication . Pereira, Catarina; Gomes, Tânia; Cardoso, Catia; Almeida, Ana Catarina; Araújo, Olinda; Bebianno, Maria João; Cravo, AlexandraEstuaries are highly productive ecosystems that have been increasingly affected by anthropogenic activities. In this study, the presence of endocrine disrupting compounds (EDCs) and their relation to oxidative stress were assessed in two bivalve species (mussels Mytilus galloprovincialis and clams Ruditapes decussatus) from the Ria Formosa Lagoon (south Portugal). We applied the alkali-labile phosphate (ALP) assay to determine vitellogenin-like protein levels, and the lipid peroxidation (LPO) method to assess oxidative damage. Mussels and clams exhibited the same pattern of ALP concentrations, with no differences between genders and higher levels in winter. R. decussatus showed higher ALP values than M. galloprovincialis, possibly associated with a stronger responsiveness of clams to EDCs or due to the influence of the reproductive cycle. Mussels exhibited a more evident seasonal effect for LPO, indicating that these organisms are more influenced by seasonal fluctuations in the water column compared to the sediment-associated clams. Both species appear to be under the influence of EDC exposure (which could induce Vg-like protein synthesis) and oxidative stressors (leading to lipid peroxidation) in the Ria Formosa Lagoon, particularly in winter, reflecting the presence of different contaminants in Ria Formosa and environmental changes, as well as complex interactions between them.
- DNA damage as a biomarker of genotoxic contamination in Mytilus galloprovincialis from the south coast of PortugalPublication . Catarina Almeida, Ana; G. Pereira, Catarina; Gomes, Tânia; Bebianno, Maria João; Cravo, AlexandraDNA 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.
- Metals in the shell of Bathymodiolus azoricus from a hydrothermal vent site on the Mid-Atlantic RidgePublication . Cravo, Alexandra; Foster, P.; Catarina Almeida, Ana; Company, Rui; Cosson, R.P.; Bebianno, Maria JoãoSpecimens of the mussel Bathymodiolus azoricus were collected from Menez Gwen, a relatively shallow (850 m) hydrothermal vent field on the Mid-Atlantic Ridge. Each bivalve shell (n = 21) was individually cleaned by selective chemical. The residual crystal matrix of each shell was individually analysed for the concentrations of the minor elements magnesium and strontium and the trace elements iron, manganese, copper and zinc. The chemical composition of the crystal matrix is unusual. B. azoricus is identified as a species having one of the most strontium impoverished shells amongst the marine molluscs. For a bimineral species the magnesium concentration is also extraordinary low. Despite originating from a trace metal rich environment; the metal concentrations in the shells were exceptionally low. Mean concentrations of iron, manganese, copper and zinc were 20.6, 3.7, 0.6 and 9.4 microg g(-1) respectively. Minor and trace element concentrations exhibited a marked intra-population variability. Copper concentrations increased and iron and zinc concentrations decreased with increasing shell weight. Due to its insensitivity to the high environmental levels of trace elements and the variability in intra-population concentrations induced by shell weight the crystal matrix of the shell of B. azoricus has little potential for use in environmental trace metal monitoring in areas contiguous to deep-sea hydrothermal vents.
- DNA damage in two bivalve species: Mytilus galloprovincialis and Ruditapes decussatus from the South coast of PortugalPublication . Catarina Almeida, Ana; Cravo, AlexandraIn the last years, several studies have been carried out to assess the environmental “health” of the South Coast of Portugal. The aim of the present thesis was to evaluate DNA damage as a biomarker of genotoxic contamination in two bivalve species from the south coast of Portugal, mussels Mytilus galloprovincialis and clams Ruditapes decussatus. Mussels were sampled at nine sites along the coast, and clams were collected at four shellfish beds in the Ria Formosa Lagoon. The alkaline version of the Comet assay was used to estimate the DNA damage present in the haemolymph of bivalves. An image analysis system (Komet 5.5, Kinetic Imaging Ltd) was applied and 3 parameters were calculated, Tail DNA (%), Olive Tail Moment (OTM; arbitrary units) and Tail length (μm). Data obtained for both species were low, in the same order of magnitude for sites considered as reference. These values can be used as baseline/reference levels of the genotoxic contamination for future studies made in the South coast of Portugal. The nature and magnitude of contaminants in this coast were not particularly genotoxic. DNA damage present in mussel and clams haemolymph revealed to be a sensitive biomarker of genotoxic contamination. DNA damage in both specimens responded to the existing differences between sites and seasons, with highest values during summer. This damage biomarker was also sensitive to gender differences concerning both species and the overall data indicates that females have higher DNA damage than males. Moreover, this study demonstrated that information about the condition index (and Abstract - iii - reproductive cycle) and the local abiotic conditions are essential for the appropriate explanation of data. When comparing the DNA damage between the two analysed species, collected from close sites, DNA damage was relatively similar for both bivalve species. However, while mussels had highest DNA damage in August, clams showed maximum values in February/March, suggesting some interspecific differences possibly associated with dissimilar trophic strategies and/or habitats. Lipid peroxidation (LPO) was also analysed, to verify if the environmental conditions (contaminants and abiotic factors) can induce injuries in both cellular membranes and DNA. For mussels, the compounds/factors affecting LPO can also promote (low) genotoxicity. For clams, this relationship was only verified during July. Concluding, the present thesis demonstrates that the DNA damage present in M. galloprovincialis and R. decussatus is a sensitive biomarker to discriminate spatial, temporal and genders differences, revealing to be an appropriate tool for the evaluation of genotoxic contamination along the South coast of Portugal.