Stabilization of engineered zero-valent nanoiron with Na-acrylic copolymer enhances spermiotoxicity.

Studies were carried out to assess the effects of stabilized (i.e., coated with organic polyacrylic stabilizer) and nonstabilized forms of zero-valent nanoiron (nZVI) on the development of Mytilus galloprovincialis embryos following 2 h exposure of the sperm prior to in vitro fertilization. Both forms of nZVI caused serious disruption of development, consisting of 30% mortality among spermatozoa with subsequent 20% decline in fertilization success, and delay in development, i.e., over 50% of the larvae were suspended in the trochophore stage. Significant DNA damage was also detected in sperm exposed to the highest exposure concentrations (10 mg L(-1)). Distinct dose response to the two different types of nZVI observed are linked to aggregation behavior that is controlled by the surface stabilizers. This work reports on conventional biomarkers (for membrane integrity, genotoxicity, and developmental toxicity) applied for the rapid assessment of toxicity of nZVI, which are able to detect surface property-related effects to meet the requirements of risk assessments for nanotechnology. The study highlights the potential ecotoxicological impact of an environmentally relevant engineered nanoparticle. Implications of the NOM-nZVI interactions regarding soil and groundwater remediation and wastewater treatment are discussed.

Developmental toxicity of benzotriazole in the protochordate Ciona intestinalis (Chordata, Ascidiae).

Benzotriazoles (BT) are applied as anticorrosive and de-icing agents and have been detected in a variety of aquatic ecosystems and municipal wastewater effluents. We have assessed the developmental effects of benzotriazole (CAS number 95-14-7) to the marine invertebrate Ciona intestinalis (Chordata, Ascidiae). At 15 +/- 1 degrees C, the 24 h No-Observed Effect Concentration (NOEC) and Lowest Observed Effect Concentration (LOEC) values based on embryo morphological development were 100 and >100 mg L(-1), respectively (nominal concentration under static conditions). After 48 h, the NOEC and LOEC values were 10 and 32 mg L(-1), respectively. Light and electron microscopy studies on benzotriazole-exposed larva indicated that the primary target cells were the extra-embryonic test cells, which are known to play a significant apoptotic role during ascidian metamorphosis. The visible decline of test cells in benzotriazole-exposed larvae raises the possibility that the compound interferes with the regulation of embryo development in protochordates such as C. intestinalis. Further research is warranted to assess the potential longer term sublethal impacts of benzotriazole in aquatic organisms.

Uptake and biological responses to nano-Fe versus soluble FeCl3 in excised mussel gills.

Nano-Fe particle uptake was experimentally examined in vitro using excised gills and blood cells of the edible blue mussel Mytilus sp. Whole gills were exposed to both Fe(2)O(3) nanoparticles and a solution of the hydrated FeCl(3) salt, for up to 12 h, and blood cells for 30 min. Equimolar Fe(+3) in the nano- and the soluble form was estimated under the assumption of dense spherical particles accommodating the same number of Fe(+3) as in the dissolved salt solution, namely: 1,000 microg L(-1) Fe(2)O(3) equivalent to 100 microg L(-1) FeCl(3).6H(2)O. Putative toxic impact of nano-Fe in gill epithelia and blood cells was assessed by an array of techniques including light- and electron microscopy, biomarkers for oxidative stress (lipid peroxidation levels), neurotoxic effects (acetylcholinesterase activity) and cytotoxicity (neutral red retention). Total and filtered fractions (20 and 200 nm, respectively) of Fe were analysed by ICP-OES. Our results provide evidence for the following: (1) much of both the soluble (95%) and the nano-Fe (90%) were removed from the water column within 12 h; (2) dissolved- and nano-Fe seemed to follow different routes of uptake within the gill epithelium; (3) both nano-Fe and soluble FeCl(3) caused similar impairment of lysosomal stability in circulating blood cells; (4) lipid peroxidation in gills exposed to the two distinct forms of Fe was increased, while acetylcholinesterase activity was unaffected. In these short-term in vitro studies, there appears to be little difference in toxic response between exposure to the Fe salt and the nano-Fe indicating that, in this case, the nanoparticles do not invoke special properties affecting biological function in gills. However, with the use of nano-Fe as a food additive, clearly longer-term in vivo studies are warranted.

Metal concentrations in digestive gland and mantle of Sepia officinalis from two coastal lagoons of Portugal.

Concentrations of both essential (Fe, Cu, Zn) and non essential (Cd, Hg and Pb) metals were measured in the digestive gland and mantle of female cephalopods Sepia officinalis captured in two distinct lagoons in Portugal: Aveiro Lagoon, with a history of anthropogenic and industrial pollution, and Formosa Lagoon receiving urban effluents. We provide evidence for the following: (1) the digestive gland is the main target organ for both essential and non essential metals, frequently containing concentrations few orders of magnitude higher as compared to mantle; the sole exception from this was the Hg that is equally distributed in the two tissues; (2) unexpectedly, the higher levels of metals were found in animals captured in the less polluted lagoon, except for Cd whose bioavailability in Aveiro lagoon might be related to industrial sources, while the influence of Cd speciation in local pray composition should not be ruled out (3) size influenced metal concentration in different way: smaller individuals accumulated significantly more Cu, while Hg concentrations showed the opposite trend; (4) Cd is positively correlated to Zn and Cu in digestive gland of specimens collected in spring in Aveiro Lagoon, and no relationship was found in Formosa Lagoon; (5) the molar ratios Cd:Zn and Cd:Cu in digestive gland increased with body weight in specimens from Aveiro area, both ratios becoming particularly higher in older individuals. Metal-specific accumulation patterns in both mantle and digestive gland at the two sites are discussed in the light of their toxicological implications.

Trophic influences of metal accumulation in natural pollution laboratories at deep-sea hydrothermal vents of the Mid-Atlantic Ridge.

This study reports on the concentration of both, toxic (Hg) and essential (Fe, Cu, Zn) metals in representative species, at four hydrothermal vents along the Mid-Atlantic Ridge (MAR) corresponding to the different exposure conditions that are the result of contrasted geological setting and different depths along the ridge axis. Macro fauna collected from these vent sites showed markedly different whole tissue concentration of metals reflecting exposure levels. Bio-transfer of metals within a typical hydrothermal food chain was investigated. There were trophic level-specific variations in essential metal accumulation showing a general trend within which the secondary consumers (predators/scavengers) and primary consumers (filter-feeder/symbiont reliant species like the mixotrophic mollusks and sponges all having metal concentrations above those in passive suspension-feeders such as echinoderms, gorgonians) accumulated highest concentrations of all elements followed by primary producers such as endosymbiont bacteria. Mercury accumulation followed a somewhat different sequence among the same taxonomic groups: top concentrations in the symbiont-bearing filter-feeder bivalves >crustaceans> sponges > bacteria > echinoderms, gorgonians and tunicates. There were no indications of biomagnification of Hg, Cu and Zn unlike Fe that showed positive transference factors (TF) along two trophic levels of a typical chain at Menez Gwen.

Distribution of micro-essential (Fe, Cu, Zn) and toxic (Hg) metals in tissues of two nutritionally distinct hydrothermal shrimps.

Hydrothermal ecosystems of the Mid Atlantic Ridge (MAR) are dominated by shrimps and mussels that are naturally exposed to elevated levels of heavy metals providing unique in situ laboratories for ecotoxicological investigations. This study reports on the tissue compartmentalization of both micro-essential (Fe, Zn, Cu) and toxic metals (Hg) in two nutritionally distinct cariddean vent shrimps: Rimicaris exoculata and Mirocaris fortunata, in order to shed light on organism-biota interactions at hydrothermal vents. High metal concentrations in shrimps confirmed extreme exposure levels at both geochemically different hydrothermal vents (Rainbow and Lucky Strike). However, Hg concentrations were below those reported in species for human consumption that may either suggest low bioavailability of the metal, or its effective detoxification/depuration by the hydrothermal shrimp that needs to be confirmed by post-capture toxicological investigations. Distribution of metals in different tissues had very similar patterns in both shrimp species, the target organs being gill and pylorus. Tissue levels correlated well with end-member fluid composition with regard to element ratios, i.e. Fe/Zn and Fe/Cu ratios in end-member fluids reported for Rainbow and L. Strike were conserved in the gills of M. fortunata. Moreover, R. exoculata that lives closer to venting exits as compared to M. fortunata, had similar or often less metals accumulated in selected organs, possibly owing to its higher degree of adaptation to hydrothermal conditions. Despite of high concentrations in tissues micro essential metals only represented a small fraction (14-36%) of the whole body burden indicating preponderance of minerals on the surface and/or in the gut, and thus points to improper use of whole body concentration in metal bioavailability interpretations at hydrothermal vents.

Effect of sub-lethal concentrations of aluminium on the filtration activity of the freshwater mussel Anodonta cygnea L. at neutral pH.

Significant amounts of aluminium (Al) are commonly present in rivers and lakes, largely in particulate form in neutral waters. Freshwater bivalves, as filter feeders are therefore exposed to both particulate and dissolved metal and are potentially vulnerable to Al. The effect of Al on filtering behaviour of the freshwater mussel Anodonta cygnea L. was investigated during short (1 hour) and long-term (15 days) exposure to environmentally relevant concentrations (250 and 500 microg l(-1)) at neutral pH. Water flow through the outflow siphon was monitored as an indicator of pumping capacity. Short-term (1 hour) exposure to 500 microg l(-1) added Al produced an irreversible decrease in the duration of filtering periods, presumably as an avoidance response to the toxicant. One-hour exposure 250 microg l(-1) Al had no detectable effect. When mussels were exposed to 250 or 500 microg l(-1) added Al for 15 days, siphon activity measured in days 11-15 of exposure was inhibited by 50% and 65%, respectively, compared to pre-exposure levels. Recovery occurred following transfer of mussels to uncontaminated water. Interaction between Al and freshwater bivalves at neutral pH may affect both the performance of the mussels and the chemical speciation of the metal in the natural environment.

The interaction of polymer-coated magnetic nanoparticles with seawater.

Laboratory studies were conducted to evaluate the interaction between bare and polymer-coated magnetic nanoparticles (MNPs) with various environmentally relevant carrying solutions including natural oceanic seawater with and without addition of algal exopolymeric substances (EPS). The MNPs were coated with three different stabilising agents, namely gum Arabic (GA-MNP), dextran (D-MNP) and carboxymethyl-dextran (CMD-MNP). The colloidal stability of the suspensions was evaluated over 48 h and we demonstrated that: (i) hydrodynamic diameters increased over time regardless of carrying solution for all MNPs except the GA-coated ones; however, the relative changes were carrying solution- and coat-dependent; (ii) polydispersity indexes of the freshly suspended MNPs are below 0.5 for all coated MNPs, unlike the much higher values obtained for the uncoated MNPs; (iii) freshly prepared MNP suspensions (both coated and uncoated) in Milli-Q (MQ) water show high colloidal stability as indicated by zeta-potential values below -30 mV, which however decrease in absolute value within 48 h for all MNPs regardless of carrying solution; (iv) EPS seems to "stabilise" the GA-coated and the CMD-coated MNPs, but not the uncoated or the D-coated MNPs, which form larger aggregates within 48 h; (v) despite this aggregation, iron (Fe)-leaching from MNPs is sustained over 48h, but remained within the range of 3-9% of the total iron-content of the initially added MNPs regardless of suspension media and capping agent. The environmental implications of our findings and biotechnological applicability of MNPs are discussed.

Chemical interaction of atmospheric mineral dust-derived nanoparticles with natural seawater--EPS and sunlight-mediated changes.

Laboratory studies were conducted to investigate the interactions of nanoparticles (NPs) formed via simulated cloud processing of mineral dust with seawater under environmentally relevant conditions. The effect of sunlight and the presence of exopolymeric substances (EPS) were assessed on the: (1) colloidal stability of the nanoparticle aggregates (i.e. size distribution, zeta potential, polydispersity); (2) micromorphology and (3) Fe dissolution from particles. We have demonstrated that: (i) synthetic nano-ferrihydrite has distinct aggregation behaviour from NPs formed from mineral dusts in that the average hydrodynamic diameter remained unaltered upon dispersion in seawater (~1500 nm), whilst all dust derived NPs increased about three fold in aggregate size; (ii) relatively stable and monodisperse aggregates of NPs formed during simulated cloud processing of mineral dust become more polydisperse and unstable in contact with seawater; (iii) EPS forms stable aggregates with both the ferrihydrite and the dust derived NPs whose hydrodynamic diameter remains unchanged in seawater over 24h; (iv) dissolved Fe concentration from NPs, measured here as <3 kDa filter-fraction, is consistently >30% higher in seawater in the presence of EPS and the effect is even more pronounced in the absence of light; (v) micromorphology of nanoparticles from mineral dusts closely resemble that of synthetic ferrihydrite in MQ water, but in seawater with EPS they form less compact aggregates, highly variable in size, possibly due to EPS-mediated steric and electrostatic interactions. The larger scale implications on real systems of the EPS solubilising effect on Fe and other metals with the additional enhancement of colloidal stability of the resulting aggregates are discussed.

Colloidal stability of nanoparticles derived from simulated cloud-processed mineral dusts.

Laboratory simulation of cloud processing of three model dust types with distinct Fe-content (Moroccan dust, Libyan dust and Etna ash) and reference goethite and ferrihydrite were conducted in order to gain a better understanding of natural nanomaterial inputs and their environmental fate and bioavailability. The resulting nanoparticles (NPs) were characterised for Fe dissolution kinetics, aggregation/size distribution, micromorphology and colloidal stability of particle suspensions using a multi-method approach. We demonstrated that the: (i) acid-leachable Fe concentration was highest in volcanic ash (1 m Mg(-1) dust) and was followed by Libyan and Moroccan dust with an order of magnitude lower levels; (ii) acid leached Fe concentration in the<20 nm fraction was similar in samples processed in the dark with those under artificial sunlight, but average hydrodynamic diameter of NPs after cloud-processing (pH~6) was larger in the former; iii) NPs formed at pH~6 were smaller and less poly-disperse than those at low pH, whilst unaltered zeta potentials indicated colloidal instability; iv) relative Fe percentage in the finer particles derived from cloud processing does not reflect Fe content of unprocessed dusts (e.g. volcanic ash>Libyan dust). The common occurrence of Fe-rich "natural nanoparticles" in atmospheric dust derived materials may indicate their more ubiquitous presence in the marine environment than previously thought.

Are reproduction impairments of free spawning marine invertebrates exposed to zero-valent nano-iron associated with dissolution of nanoparticles?

Studies were carried out to assess the effects of coating applied to zero-valent nano-iron (nZVI) on early life stage development of three key marine invertebrate species Mytilus galloprovincialis, Ciona intestinalis and Psammechinus milliaris. Embryo development was assessed following a 2-h exposure of the sperm to concentrations of two nZVIs of up to 10 mg l(-1) followed by in vitro fertilisation. Disruption of embryo development was most severe in sea squirts followed by mussel, while the urchin embryos were not significantly affected as compared with controls. An over twofold decrease in fertilisation success alongside significant delay in the embryo development was observed, and the effect was more severe with the coated form, possibly owing to its better colloidal stability. We provide in vitro evidence for the rapid dissolution (within 2 h) of nZVI in seawater to a degree that concentration of total solute Fe released from the coated ZVI particles exceeds safe limits of NOECs established for dissolved Fe.

The effect of engineered iron nanoparticles on growth and metabolic status of marine microalgae cultures.

Synthetic zero-valent nano-iron (nZVI) compounds are finding numerous applications in environmental remediation owing to their high chemical reactivity and versatile catalytic properties. Studies were carried out to assess the effects of three types of industrially relevant engineered nZVI on phytoplankton growth, cellular micromorphology and metabolic status. Three marine microalgae (Pavlova lutheri, Isochrysis galbana and Tetraselmis suecica) were grown on culture medium fortified with the nano-Fe compounds for 23 days and subsequent alterations in their growth rate, size distribution, lipid profiles and cellular ultrastructure were assessed. The added nano Fe concentrations were either equimolar with the EDTA-Fe conventionally added to the generic f/2 medium (i.e. 1.17 × 10(-5)M), or factor 10 lower and higher, respectively. We provide evidence for the: (1) broad size distribution of nZVI particles when added to the nutrient rich f/2 media with the higher relative percentage of the smallest particles with the coated forms; (2) normal algal growth in the presence of all three types of nZVIs with standard growth rates, cellular morphology and lipid content comparable or improved when compared to algae grown on f/2 with EDTA-Fe; (3) sustained algal growth and normal physiology at nZVI levels 10 fold below that in f/2, indicating preference to nanoparticles over EDTA-Fe; (4) increased total cellular lipid content in T. suecica grown on media enriched with uncoated nZVI25, and in P. lutheri with inorganically coated nZVI(powder), when compared at equimolar exposures; (5) significant change in fatty acid composition complementing the nZVI(powder)-mediated increase in lipid content of P. lutheri; (6) a putative NP uptake mechanism is proposed for I. galbana via secretion of an extracellular matrix that binds nZVIs which then become bioavailable via phagocytotic membrane processes.

Ultrastructural and molecular evidence for potentially symbiotic bacteria within the byssal plaques of the deep-sea hydrothermal vent mussel Bathymodiolus azoricus.

This study reports on the presence of a putatively symbiotic bacterial flora within the byssus plaque of the deep sea hydrothermal mussel Bathymodiolus azoricus, contributing to metal sequestration/deposition and testing positive to methane oxidizing symbiont-specific fluorescent probes. Combining an array of approaches including histology, electron microscopy, X-ray microanalysis, analytical chemistry, and microbiology we provide evidence for the frequently assumed, but rarely shown influence of prokaryotes on the biogeochemical cycling of metals as well as inorganic C sources (i.e., methane) at deep sea hydrothermal vents. Our results indicate that in spite of its antibacterial protective sheath, the byssus plaque gives access to a whole range of prokaryotic organisms which may be responsible for the extremely high concentration of metallic elements (Fe, Cu, Zn, Mn, Co, Mo, Cd, Pb and Hg) measured in this attachment organ. The very high levels of metals in byssus, together with its frequent renewal rate due to the dynamic nature of the habitat, suggest that intra-byssal bacteria may have a major influence on biomineralisation/deposition of metals. The presence of a methanotroph morphotype within the byssus plaque was confirmed by FISH and TEM. The implications of the biogeochemical cycling of metals and methane at hydrothermal vents are discussed.

Shell nacre ultrastructure and depressurisation dissolution in the deep-sea hydrothermal vent mussel Bathymodiolus azoricus.

This study describes the micro-morphological features of the shell nacre in the vent mytilid Bathymodiolus azoricus collected along a bathymetric gradient of deep-sea hydrothermal vents of the mid-Atlantic ridge (MAR). Pressure-dependent crystallisation patterns were detected in animals subjected to post-capture hydrostatic simulations. We provide evidence for the following: (1) shell micro morphology in B. azoricus is similar to that of several vent and cold-seep species, but the prismatic shell layers may vary among bathymodiolids; (2) nacre micro-morphology of mussels from three vent sites of the MAR did not differ significantly; minor differences do not appear to be related to hydrostatic pressure, but rather to calcium ion availability; (3) decompression stress may cause drop off in pH of the pallial fluid that damages nascent crystals, and in a more advanced phase, the aragonite tablets as well as the continuous layer of mature nacre; and (4) adverse effects of decompression on calcium salt deposition in shells was diminished by re-pressurisation of specimens. The implications of the putative influence of hydrostatic pressure on biomineralisation processes in molluscs are discussed.

Biological factors influencing tissue compartmentalization of trace metals in the deep-sea hydrothermal vent bivalve Bathymodiolus azoricus at geochemically distinct vent sites of the Mid-Atlantic Ridge.

In this study, we investigated on concentrations of trace metals (Al, Cd, Mn, Co, and Hg) in the hydrothermal bivalve Bathymodiolus azoricus, a dominant species at most vent sites along the Mid-Atlantic Ridge (MAR), and in its endosymbiont bacteria and commensal parasite Branchipolynoe seepensis. Comparison of our results with data from the literature on non-hydrothermal bivalves suggests lack of "extreme" uptake of trace metals by B. azoricus, except for Hg concentration which exceeded manyfold previously reported values. Mussels collected from three geochemically distinct vent sites, Menez Gwen, Lucky Strike, and Rainbow, along the MAR showed significant differences in tissue concentration of metals. Proportionality of metals in soft tissues of mussels reflected variation of water chemistry at different vents, which in turn conserved the order of trace metal prevalence in undiluted fluids. There were significant tissue-specific differences in trace metal compartmentalization for all metals investigated. Byssus thread contained the highest metal concentration among examined tissues, and thus it is suggested to be an important detoxification route. Size-dependent differences in metal concentrations were detected only for Hg, revealing a general trend of small mussels accumulating more metal than big mussels. Endosymbiont bacteria are shown to exclusively sequester Al from the host gill and contribute to removal of other toxic metals in mussels from Menez Gwen. The commensal parasite present in all mussels from Lucky Strike had higher tissue concentrations of Mn, Al, and Co than the host gill, unlike Cd and Hg which were considerably lower in the former, and thus its role in detoxification remains unclear. Bioaccumulation potential of vent bivalves and associated organisms are quantified as concentration factors and compared to make inferences on the putative role of the endosymbiont bacteria and the commensal parasite in detoxification of trace metals.

Behavioural response to the bioavailability of inorganic mercury in the hydrothermal mussel Bathymodiolus azoricus.

The hydrothermal vent bivalve Bathymodiolus azoricus is naturally exposed to putatively elevated levels of mercury (Hg), exposure that dates back to the geological occurrence of vent ecosystems, and thus may have evolved evolutionary detoxification mechanisms. Therefore, it was used as a model organism in the present investigation to study the Hg-animal interaction. Mussels were exposed to inorganic Hg by daily administration of 20 microg l(-1) Hg for 21 days (cumulative added concentration was 420 microg l(-1), i.e. approximately 2 mmol l(-1)) under controlled laboratory conditions, and consequent bioaccumulation and detoxification patterns were investigated, while shell gaping behaviour indicative of filtering activity was monitored. As a result of Hg exposure, significant increase in duration, as well as decline in frequency of shell gaping occurred, which did not recover to pre-exposure levels following 21 days of Hg-free treatment. An increase in the duration of open-shelled status may indicate the absence of an avoidance reaction in the vent mussel coming in contact with Hg, unlike other bivalves that normally close their shells in response to stress compounds. Alternatively, it may suggest that Hg had an inhibitory effect on the adductor muscle function that is responsible for closing the shells. As a result, elevated Hg levels were measured in the soft tissues (270+/-71 microg g(-1) in gills, 245+/-52 microg g(-1) in digestive glands, 93+/-25 microg g(-1) in the mantle and 46+/-9 microg g(-1) in the foot), in byssus threads (peak levels of 442+/-89 microg g(-1)) and in pseudofaeces (reaching levels as high as 1000 microg g(-1)). Overall, gills contributed 75% to the total Hg body burden followed by mantle (13%), digestive gland (7%), byssus (3%) and foot (2%). Tissue Hg levels remained elevated in mussels transferred to Hg-free seawater even after 21 days, despite the high concentrations persistently eliminated with pseudofaeces both, during and after, exposure. This potential for bioaccumulation of inorganic Hg (concentration factors reached the order of magnitude of 10(4)) by the vent mussel, which does not seem to prevent uptake by shell closure, suggests that the main Hg-handling strategy is elimination via mucus.