Biomonitoring of arsenic through mangrove oyster (Crassostrea corteziensis Hertlein, 1951) from coastal lagoons (SE Gulf of California): occurrence of arsenobetaine and other arseno-compounds.

In this study, we examined the bioavailability of arsenic through the mangrove oyster Crassostrea corteziensis sampled from seven coastal lagoons in SE Gulf of California during the rainy and dry seasons. As concentrations in soft tissue of oysters C. corteziensis fluctuated between 5.2 and 11.6 µg/g on dry weight; organisms from the control site presented the lowest As concentrations in the two sampling seasons. As speciation was evaluated in selected samples and indicated that arsenobetaine was the major arseno-compound accounting for 53.5-74.7 % of total As. Lower percentage contributions were obtained for nonextractable As (9.7-25.5 %) and other molecules such as arsenocholine and methyl-arsonate (<5 %). Inorganic As was detectable in only two samples, at concentrations lower than 0.1 µg/g. These As data are the first generated in NW Mexico and indicate that C. corteziensis is safe for human consumption in terms of arseno-compounds. It is evident that As bioavailability in these lagoons is low.

Arsenic and arsenic species in cultured oyster (Crassostrea gigas and C. corteziensis) from coastal lagoons of the SE Gulf of California, Mexico.

The aim of this study was to evaluate the bioavailability of arsenic (As) through cultured oyster Crassostrea gigas and Crassostrea corteziensis from four coastal lagoons (SE Gulf of California). Organisms were collected in two seasons (rainy and dry season), and they were analyzed for total arsenic and chemical speciation of this element. The concentrations of As in oyster soft tissue fluctuated between 5.44 and 9.56 µg/g for rainy season and 6.46 and 8.33 µg/g for dry season (dry weight) in C. gigas. In C. corteziensis, the As concentrations were <5 µg/g for both seasons (dry weight). Arsenic speciation indicated arsenobetaine as the major arseno-compound accounting for 43.2-76.3 % of total content of As. Lower contributions were obtained for non-extractable As (11.3-17.5 %) and other molecules such as arsenocholine and methyl-arsonate (<5 %). Inorganic arsenic was detectable in only two samples, at concentrations lower than <0.1 µg/g. These As data are the first generated for these mollusks in NW Mexico and indicate that C. gigas and C. corteziensis farmed in this area are safe for human consumption in terms of arseno-compounds.

A multidisciplinary weight of evidence approach for classifying polluted sediments: Integrating sediment chemistry, bioavailability, biomarkers responses and bioassays.

Evaluation of chemical bioavailability and onset of biological alterations is fundamental to assess the hazard of environmental pollutants, particularly when associated to sediments which need to be removed. In the present work, five sediment samples were collected from the Venice Lagoon and data from sediment chemistry were integrated with those of bioaccumulation of chemicals in European eel (Anguilla anguilla) exposed under laboratory conditions, responses of a wide battery of biomarkers, and standardized ecotoxicological bioassays. The overall results were elaborated within a recently developed, software-assisted weight of evidence (WOE) model which provides synthetic indices for each of considered line of evidence (LOE), before a general evaluation of sediment hazard. Levels of chemicals in sediments were not particularly elevated when compared to sediment quality guidelines of Venice Protocol. On the other hand, bioavailability was evident in some samples for Cd, Cu, Zn and, especially, polycyclic aromatic hydrocarbons. The ecotoxicological approach provided further evidence on the biological and potentially harmful effects due to released contaminants, and oxidative-mediated responses appeared of primary importance in modulating sublethal responses and the onset of cellular alterations. Biomarkers variations were sensitive, and more evident variations included significant changes of cytochrome P450 biotransformation pathway, antioxidant responses, onset of oxidative damages, lysosomal membrane stability and genotoxic effects. The results obtained from the battery of bioassays indicated that responses measured at organism level were in general accordance but less marked compared to the onset of sublethal changes measured through biomarkers. Overall this study revealed differences when comparing evaluations obtained from different LOEs, confirming the importance of considering synergistic effects between chemicals in complex mixtures. Compared to a qualitative pass-fail approach toward normative values, the proposed WOE model allowed a quantitative characterization of sediment hazard and a better discrimination of on the basis of various types of chemical and biological data.

Hyperaccumulation of vanadium in the Antarctic polychaete Perkinsiana littoralis as a natural chemical defense against predation.

BACKGROUND, AIM, AND SCOPE: Exceptionally high levels of trace metals have been reported in specific tissues of certain polychaetes. In the present study, the Antarctic fan worm Perkinsiana littoralis was shown to hyperaccumulate vanadium in the branchial tissues, and the hypothesis of an antipredatory strategy has been investigated. MATERIALS AND METHODS: Trace metals (Ag, Al, As, Ba, Cd, Co, Cr, Cu, Fe, Hg, Mn, Ni, Pb, Se, V, Zn) were measured by atomic absorption spectrophotometry in tissues of P. littoralis and, only for V, in two Antarctic bivalves and in various Mediterranean polychaetes. Subcellular distribution of vanadium was investigated in P. littoralis after differential centrifugations; feeding trials with the Antarctic rock cod Trematomus berancchii were performed to test the palatability of P. littoralis. RESULTS AND DISCUSSION: Analyses of trace metals in tissues of P. littoralis confirmed the naturally high bioavailability of cadmium due to upwelling phenomena in the investigated area and revealed extremely high concentrations of vanadium up to 10,000 microg/g, in the branchial crowns; much lower concentrations were measured in the body portions and even less in the Antarctic bivalves and in Mediterranean polychaetes. The subcellular distribution indicated that this metal was associated in branchial crowns with both heavy components and vanadium binding proteins; the latter predominated in body tissues, although with a different pattern of molecular weight. Feeding trials suggested that the elevated levels of vanadium in branchial crown of P. littoralis act as chemical deterrents against predation in more exposed tissues. RECOMMENDATIONS AND PERSPECTIVES: The hyperaccumulation of toxic metals might represent a common antipredatory strategy for unpalatable branchial crowns of sabellid polychaetes, as recently hypothesized also for the high concentrations of arsenic in the Mediterranean Sabella spallanzanii. The evolution of such adaptation and the reasons behind the possibility for different species to accumulate different metals represent a stimulating field of investigation for future studies.

Seasonal, spatial and inter-annual variations of trace metals in mussels from the Adriatic sea: a regional gradient for arsenic and implications for monitoring the impact of off-shore activities.

Mussels are widely used as bioindicator organisms for monitoring chemical pollutants including trace metals. These elements are natural constituents in the marine environment and their basal concentrations in the organisms can be influenced by several environmental and biological factors. The aim of this work was to extend our knowledge on the natural variability of trace metals in mussels tissues, focusing on seasonal and inter-annual fluctuations in a coastal reference site of the Adriatic coast (Portonovo); a total of 39 samplings were performed during 5 years, providing an extended data-set for tissue levels of As, Ba, Cd, Cr, Cu, Fe, Hg, Mn, Ni, Pb, Se, V and Zn. Concentrations of trace metals in mussels tissues revealed marked seasonal fluctuations with significant differences between various sampling years. Such fluctuations appeared mostly related to phytoplanctonic blooms and especially to reproductive cycle which exhibited a certain inter-annual shift of the gametogenesis period. Lower concentrations were measured in summer months for the majority of elements while a different seasonal cycle was observed for arsenic, not correlated with gonadic development, neither with other elements. Chemical speciation of arsenic was characterized to distinguish compounds of natural origin from those potentially reflecting an anthropogenic impact. Arsenobetaine and arsenocholine were always the predominant forms (up to 85% of total arsenic), while a significant increase of dimethylarsine and trimethylarsine oxide in spring (24% of total arsenic) might reflect the effect of phytoplanctonic bloom on both geochemistry and trophic transfer of this element. A significant inter-annual variability was observed for both the seasonal cycle and the range of values measured for all the elements, with particularly marked differences for arsenic, ranging from less than 10 to more than 40 microg/g in summer periods of different years. Data obtained on reference mussels were used to assess the impact of 41 off-shore platforms distributed along 5 nautical districts in the Northern and Central Adriatic Sea. Organisms sampled on these structures from 2001 to 2005 exhibited a certain enrichment of cadmium and zinc, probably associated to the use of anodic electrodes. Marked annual and geographical variations were measured for concentrations of arsenic in mussels of different platforms. However, the comparison with results of Portonovo, allowed to exclude the anthropogenic impact of exploitation activities and revealed a natural regional gradient of arsenic levels in mussels tissues associated to the changing influence of the Po river runoff on seawater salinity. In particular the higher concentrations measured in organisms sampled from platforms in the Central compared to Northern Adriatic confirmed a significant relationship between salinity and arsenic bioaccumulation, consistent with a role of arsenobetaine as an acquired osmolyte for mussels. The overall results confirmed the importance of natural variability when assessing the potential impact of anthropogenic activities.

Antioxidant responses in the nereidid Laeonereis acuta (Annelida, Polychaeta) after cadmium exposure.

The objective of this study was to evaluate the effects of the exposure to cadmium on the antioxidant responses in the polychaeta Laeonereis acuta. The worms were submitted to 0, 5, and 100 microg of Cd/L during a period of test of 7 days. Cadmium was significantly (p<0.05) accumulated in L. acuta in both concentrations assayed, but the concentration of reactive oxygen and nitrogen species (RONS) increased (p<0.05) only in the group submitted to the highest concentration of cadmium (100 microg/L). At this concentration, a decrease in the activity of the superoxide dismutase and an increase of glutathione-S-transferase activity (p<0.05) was observed. The levels of both lipid peroxides and the activities of catalase and glutathione peroxidase were not affected (p>0.05) by the exposition to cadmium. Thus, cadmium can augment RONS levels and can interfere with the antioxidant defense system of the polychaete L. acuta, although cadmium does not directly induce oxidative stress unlike copper and iron.

An ecotoxicological protocol with caged mussels, Mytilus galloprovincialis, for monitoring the impact of an offshore platform in the Adriatic Sea.

An ecotoxicological protocol with caged mussels, Mytilus galloprovincialis, was developed to evaluate the potential impact of an offshore gas platform in the central Adriatic Sea. Reference organisms were collected on a seasonal basis from an unpolluted site and transplanted for four weeks in both the sampling area and to the investigated platform. Chemical analyses of trace metals in mussel tissues were integrated with a multi-biomarker approach for the early detection of biological responses at several cellular targets. Induction of metallothioneins, peroxisomal proliferation and activity of acetylcholinesterase were measured as markers for specific classes of chemicals. Special attention was given to oxyradical metabolism and appearance of oxidative-mediated toxicity to reveal a more general onset of cellular disturbance. In addition to individual antioxidants (superoxide dismutase, catalase, glutathione S-transferases, glutathione reductase, Se-dependent and Se-independent glutathione peroxidases, and levels of total glutathione), the total oxyradical scavenging capacity (TOSC) allowed a quantification of the overall capability to neutralize specific forms of intracellular reactive oxygen species (ROS; i.e. peroxyl and hydroxyl radicals). Cellular damages were evaluated as lysosomal destabilization (membrane stability, accumulation of lipofuscin and neutral lipids), lipid peroxidation products (malondialdehyde) and DNA integrity (strand breaks and micronuclei); the air survival test was finally applied to evaluate the overall physiological condition of mussels. Concentration of trace metals (As, Ba, Cd, Cr, Cu, Fe, Hg, Mn, Ni, Pb, Zn) revealed only limited variations in transplanted mussels during various experimental periods and such changes appeared partly related to natural fluctuations. Among biological responses, variations of antioxidants and lysosomal stability were confirmed as sensitive early warning signals for biological disturbance of both natural and anthropogenic origin. The presented protocol with caged mussels allowed marked biological effects caused by the investigated platform to be excluded, and represented a useful approach that is easy to extend for monitoring the impact of offshore activities in the Adriatic sea.

Bioaccumulation and biotransformation of arsenic in the Mediterranean polychaete Sabella spallanzanii: experimental observations.

The Mediterranean fan worm Sabella spallanzanii is characterized by elevated basal levels of arsenic in branchial crowns (>1000 microg/g) and an unusual prevalence of dimethylarsinic acid (DMA), a relatively toxic compound with a possible antipredatory role. The aim of this work was to obtain further insights on the capability of this polychaete to accumulate arsenic from different compounds and to operate biotransformation reactions. Laboratory exposures to arsenate (As(V)), dimethylarsinic acid (DMA), trimethylarsine (TMA), and arsenobetaine (AsB) revealed significant differences among tissues and kind of experiments. The highest increases of arsenic content were observed in branchial crowns of organisms treated with arsenate, which can enter the cell through the phosphate carrier system; lower variations were measured with DMA and TMA, while not-significant changes of total As occurred after treatments with AsB. In body tissues, exposure to As(V), DMA, and TMA confirmed a progressively lower accumulation of total arsenic, while a marked increase was caused by AsB. Obtained results suggested that accumulated arsenic could be chemically transformed, thus explaining the elevated basal levels of DMA typical of S. spallanzanii; during all the experiments, DMA was the most accumulated molecule, suggesting that this species possesses the enzymatic pathways for methylation and demethylation reactions of inorganic and trimethylated arsenicals. Only arsenobetaine was not converted into DMA, which would confirm a microbial pathway for degradation for this molecule, particularly important in body tissues of S. spallanzanii for the presence of bacteria associated to digestive tracts. Overall, the present study suggests future investigations on the biological role of arsenic and DMA in S. spallanzanii as a potential adaptive mechanism against predation in more vulnerable tissues.

Toxicological responses in Laeonereis acuta (annelida, polychaeta) after arsenic exposure.

Several environmental pollutants, including metals, can induce oxidative stress. So, the objective of this study was to evaluate the effects of arsenic (As(III), as As(2)O(3)) on the antioxidant responses in the polychaete Laeonereis acuta. Worms were exposed to two environmentally relevant concentrations of As, including the highest previously allowed by Brazilian legislation (50 microg As/l). A control group was kept in saline water (10 per thousand) without added metal. It was observed that: (1) a peak concentration of lipid peroxide was registered after 2 days of exposure to 50 microg As/l (61+/-3.2 nmol CHP/g wet weight) compared to the control group (43+/-4.5 nmol CHP/g wet weight), together with a lowering of the activity of the antioxidant enzyme catalase (-47 and -48%, at 50 or 500 microg As/l respectively) and a higher superoxide dismutase activity (+305% at 50 microg As/l with respect to the control group); (2) a lower conjugation capacity through glutathione-S-transferase activity was observed after 7 days of exposure to 50 microg As/l (-48% compared to the control group); (3) a significant increase in As concentration was verified after 1 week of exposure to both As concentrations (50 and 500 microg/l); (4) worms exposed to As showed a limited accumulation of related methylated As species and the levels of non-toxic As species like arsenobetaine (AsB) and arsenocholine (AsC) remained unchanged during the exposure period when compared with the controls. Overall, it can be concluded that As interfered in the antioxidant defense system of L. acuta, even at low concentrations (50 microg/l) that Brazilian legislation previously considered safe. The fact that worms exposed to As showed high levels of methylated As species indicates the methylation capability of L. acuta, although the high levels of inorganic As suggest that not all the administered As(III) (as As(2)O(3)) is completely removed or biotransformed after 7 days of exposure.

Short-term responses to cadmium exposure in the estuarine polychaete Laeonereis acuta (polychaeta, Nereididae): subcellular distribution and oxidative stress generation.

Some effects of cadmium exposure (100 microg/L for 4, 8, 12, and 24 h) on the estuarine polychaete Laeonereis acuta (Nereididae) were evaluated. This polychaete was able to accumulate cadmium in the body, with the metal stored mainly in the cytosolic fraction (>10 kDa). Activity of the antioxidant enzymes superoxide dismutase, glutathione S-transferase, and glutathione reductase (GR) as well as the total oxyradical scavenger capacity, the glutamate cysteine ligase catalytic subunit gene expression, and the metallothionein-like proteins content were not affected by cadmium at any exposure time tested. Catalase (CAT) activity, however, was significantly lower (p < 0.05) in worms treated with cadmium compared with that in controls after 8 h of exposure. At the same exposure time, lipid peroxide levels were increased (p < 0.05) in worms exposed to cadmium compared with those in control worms. Interestingly, CAT and GR activities decreased over time (p < 0.05) independent of cadmium treatment, which is a result that could be attributed to starvation. The effects caused by cadmium in the present study were observed only after 8 h of exposure, demonstrating that cadmium can generate oxidative stress.

Use of the land snail Helix aspersa as sentinel organism for monitoring ecotoxicologic effects of urban pollution: an integrated approach.

Atmospheric pollution from vehicular traffic is a matter of growing interest, often leading to temporary restrictions in urban areas. Although guidelines indicate limits for several parameters, the real toxicologic impacts remain largely unexplored in field conditions. In this study our aim was to validate an ecotoxicologic approach to evaluate both bioaccumulation and toxicologic effects caused by airborne pollutants. Specimens of the land snail Helix aspersa were caged in five sites in the urban area of Ancona, Italy. After 4 weeks, trace metals (cadmium, chromium, copper, iron, manganese, nickel, lead, and zinc) and polycyclic aromatic hydrocarbons (PAHs) were measured and these data integrated with the analyses of molecular and biochemical responses. Such biomarkers reflected the induction of detoxification pathways or the onset of cellular toxicity caused by pollutants. Biomarkers that correlated with contaminant accumulation included levels of metallothioneins, activity of biotransformation enzymes (ethoxyresorufin O-deethylase, ethoxycoumarin O-deethylase), and peroxisomal proliferation. More general responses were investigated as oxidative stress variations, including efficiency of antioxidant defenses (catalase, glutathione reductase, glutathione S-transferases, glutathione peroxidases, and total glutathione) and total oxyradical scavenging capacity toward peroxyl and hydroxyl radicals, onset of cellular damages (i.e., lysosomal destabilization), and loss of DNA integrity. Results revealed a marked accumulation of metals and PAHs in digestive tissues of organisms maintained in more traffic-congested sites. The contemporary appearance of several alterations confirmed the cellular reactivity of these chemicals with toxicologic effects of potential concern for human health. The overall results of this exploratory study suggest the utility of H. aspersa as a sentinel organism for biomonitoring the biologic impact of atmospheric pollution in urban areas. Key words: atmospheric pollutants, bioindicators, biomarkers, DNA integrity, lysosomes, metallothioneins, oxidative stress, peroxisomes, polycyclic aromatic hydrocarbons, trace metals.

Pro-oxidant effects of extremely low frequency electromagnetic fields in the land snail Helix aspersa.

Pro-oxidant effects of extremely low frequency (ELF) 50-Hz magnetic fields were investigated in the land snail Helix aspersa exposed both in short-term laboratory treatments and under field conditions by maintaining the organisms in the proximity of a power line for up to 2 months. Oxidative perturbations were investigated as individual antioxidants (catalase, glutathione reductase, glutathione S-transferases, and total glutathione) and total scavenging capacity toward peroxyl radicals and hydroxyl radicals. Accumulation of lipid peroxidation products, destabilization of lysosomal membranes, and loss of DNA integrity were also evaluated as markers of cell damage. The overall results indicated an oxidative challenge caused by ELF magnetic fields with particularly prompt and sensitive responses for catalase, glutathione reductase, and the overall capability to neutralize peroxyl radicals. Cell injuries occurred to different extents according to duration and intensity of electromagnetic exposure and confirmed complex cause-effect relationships between pro-oxidant factors, efficiency of antioxidant defenses, and the onset of oxidative toxicity. This study highlights the importance of a multimarker approach for detecting a wide panel of biological responses, the necessity of investigating the long-term effects of early oxidative responses, and the role of ELF in enhancing susceptibility to other forms of pathologies or diseases.