Relative sensitivity of two marine bivalves for detection of genotoxic and cytotoxic effects: a field assessment in the Tamar Estuary, South West England.

The input of anthropogenic contaminants to the aquatic environment is a major concern for scientists, regulators and the public. This is especially relevant in areas such as the Tamar valley in SW England, which has a legacy of contamination from industrial activity in the nineteenth and twentieth centuries. Following on from previous laboratory validation studies, this study aimed to assess the relationship between genotoxic and cytotoxic responses and heavy metal concentrations in two bivalve species sampled from locations along the Tamar estuary. Adult cockles, Cerastoderma edule, and blue mussels, Mytilus edulis, were sampled from five locations in the Tamar and one reference location on the south Devon coast. Bivalve haemocytes were processed for comet and neutral red retention (NRR) assays to determine potential genotoxic and cytotoxic effects, respectively. Sediment and soft tissue samples were analysed for metal content by inductively coupled plasma mass spectrometry. Sediment concentrations were consistent with the physico-chemical nature of the Tamar estuary. A significant correlation (P = 0.05) was found between total metal concentration in sediment and C. edule soft tissues, but no such correlation was found for M. edulis samples. DNA damage was elevated at the site with highest Cr concentrations for M. edulis and at the site with highest Ni and Pb concentrations for C. edule. Analysis of NRR revealed a slight increase in retention time at one site, in contrast to comet data. We conclude that the comet assay is a reliable indicator of genotoxic damage in the field for both M. edulis and C. edule and discuss reasons for the apparent discrepancy with NRR.

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.

Metals and marine microplastics: Adsorption from the environment versus addition during manufacture, exemplified with lead.

There are two means by which metals associate with microplastics in the aquatic environment. Firstly, they may be adsorbed to the plastic surface or hydrogenous-biogenic accumulations thereon, and secondly, they may be present in the polymeric matrix as functional additives or as reaction or recyclate residues. In this study, the relative significance of these associations is evaluated with respect to Pb in beached marine microplastics. Thus, adsorbed Pb was determined in <5 mm, neutrally-coloured polyethylene pellets that contained no detectable Pb added during manufacture by digestion in dilute aqua regia, while the bioaccessibility of this association was evaluated using an avian physiologically-based extraction test (PBET). Here, up to about 0.1 µg g-1 of Pb was adsorbed to the plastic and between about 60 and 70% of the metal was accessible. Lead present as additive or residue was determined by x-ray fluorescence analysis of a wider range of beached plastics (polyolefins and polyvinyl chloride), with a selection of positive samples grated to mm-dimensions and subjected to the PBET. Here, total Pb concentrations up to 40,000 µg g-1 and bioaccessibilities up to 16% were observed, with bioaccessible concentrations exceeding equivalent values for adsorbed Pb by several orders of magnitude. Ingestive exposure to Pb, and potentially other toxic metals, is more important through the presence of additives in historical plastics and recyclate residues in contemporary plastics than from adsorption, and it is recommended that future studies focus more on the environmental impacts and fate of metals bound in this form.

Protective effects of selenium on mercury-induced DNA damage in mussel haemocytes.

Little is known of the antioxidant role of selenium (Se) in aquatic invertebrates. We investigated the effects of Se on mercury-induced DNA damage in haemocytes from Mytilus edulis using alkaline single cell gel electrophoresis, that is, the Comet assay. The basal percentage tail DNA value for mussel haemocytes was 9.8+/-0.2% (mean+/-S.E.M., n=70). Exposing mussels to Hg(2+) (nominal concentration 20 microgL(-1)) for three days led to an increase in tail DNA to 61.1+/-1.8% (n=10). With added Se (as selenite, nominal concentration 4 microgL(-1)), Hg-induced DNA damage was reduced to 39.5+/-3.1% (n=10). Se pre-exposure also provided some protection against Hg-induced DNA damage (% tail DNA=51.0+/-2.9%, n=10). Basal glutathione peroxidase (GPx) activity in cell-free haemolymph was 93.7+/-3.5 nmol min(-1)mg(-1) (mean+/-S.E.M., n=70). Increases in GPx activity were seen when Se was added during and/or after exposure to Hg. For example, a 3-4-fold increase was seen after three days exposure to Hg in the presence of added Se. Interestingly GPx activity doubled after three days in the presence of added Se alone, but was unchanged after exposure to HgCl(2) alone. These results suggest that the availability of Se in the natural environment could affect the antioxidant status of mussels, and consequently could affect levels of DNA damage.

Genotoxic, cytotoxic and ontogenetic effects of tri-n-butyltin on the marine worm, Platynereis dumerilii (Polychaeta: Nereidae).

The genotoxic, cytotoxic and ontogenetic (embryo-larval) or developmental effects of tri-n-butyltin (TBT), were investigated in Platynereis dumerilii. Following the determination of maximum tolerated dose with regard to ontogenetic effects and mortality, early life stages of P. dumerilii were exposed to a range of TBT concentrations. Subsequently, the embryo-larvae were analysed for evidence of genotoxicity and cytotoxicity. Genotoxicity was assessed using cytogenetic endpoints that included the frequency of sister chromatid exchanges and chromosomal aberrations from metaphase spreads. Cytotoxicity was evaluated by determining the proliferative rate index of the growing embryo-larval cells using 5-bromodeoxyuridine labelling of the chromosomes or fluorescence plus Giemsa staining technique. TBT-exposed embryo-larvae of P. dumerilii exhibited sensitivity similar to that of other invertebrates, indicating that P. dumerilii is a suitable ecotoxicity test species. The results also suggested dose-dependent effects for genotoxic and cytotoxic end points in relation to TBT exposure. The present study highlights the need to elucidate the relative importance of direct genotoxic and indirect effects through production of genotoxic hormonal derivatives.

Enhanced toxicity of 'bulk' titanium dioxide compared to 'fresh' and 'aged' nano-TiO2 in marine mussels (Mytilus galloprovincialis).

Marine bivalves (Mytilus galloprovincialis) were exposed to titanium dioxide (10 mg L(-1)) either as engineered nanoparticles (nTiO2; fresh, or aged under simulated sunlight for 7 days) or the bulk equivalent. Inductively coupled plasma-optical emission spectrometry analyses of mussel tissues showed higher Ti accumulation (>10-fold) in the digestive gland compared to gills. Nano-sized TiO2 showed greater accumulation than bulk, irrespective of ageing, particularly in digestive gland (>sixfold higher). Despite this, transcriptional expression of metallothionein genes, histology and histochemical analysis suggested that the bulk material was more toxic. Haemocytes showed significantly enhanced DNA damage, determined by the modified comet assay, for all treatments compared to the control, but no significant differences between the treatments. Our integrated study suggests that for this ecologically relevant organism photocatalytic ageing of nTiO2 does not significantly alter toxicity, and that bulk TiO2 may be less ecotoxicologically inert than previously assumed.

Gills are an initial target of zinc oxide nanoparticles in oysters Crassostrea gigas, leading to mitochondrial disruption and oxidative stress.

The increasing industrial use of nanomaterials during the last decades poses a potential threat to the environment and in particular to organisms living in the aquatic environment. In the present study, the toxicity of zinc oxide nanoparticles (ZnONP) was investigated in Pacific oysters Crassostrea gigas. The nanoscale of ZnONP, in vehicle or ultrapure water, was confirmed, presenting an average size ranging from 28 to 88 nm. In seawater, aggregation was detected by TEM and DLS analysis, with an increased average size ranging from 1 to 2 µm. Soluble or nanoparticulated zinc presented similar toxicity, displaying a LC50 (96 h) around 30 mg/L. High zinc dissociation from ZnONP, releasing ionic zinc in seawater, is a potential route for zinc assimilation and ZnONP toxicity. To investigate mechanisms of toxicity, oysters were treated with 4 mg/L ZnONP for 6, 24 or 48 h. ZnONP accumulated in gills (24 and 48 h) and digestive glands (48 h). Ultrastructural analysis of gills revealed electron-dense vesicles near the cell membrane and loss of mitochondrial cristae (6 h). Swollen mitochondria and a more conspicuous loss of mitochondrial cristae were observed after 24 h. Mitochondria with disrupted membranes and an increased number of cytosolic vesicles displaying electron-dense material were observed 48 h post exposure. Digestive gland showed similar changes, but these were delayed relative to gills. ZnONP exposure did not greatly affect thiol homeostasis (reduced and oxidized glutathione) or immunological parameters (phagocytosis, hemocyte viability and activation and total hemocyte count). At 24 h post exposure, decreased (-29%) glutathione reductase (GR) activity was observed in gills, but other biochemical responses were observed only after 48 h of exposure: lower GR activity (-28%) and levels of protein thiols (-21%), increased index of lipid peroxidation (+49%) and GPx activity (+26%). In accordance with ultrastructural changes and zinc load, digestive gland showed delayed biochemical responses. Except for a decreased GR activity (-47%) at 48 h post exposure, the biochemical alterations seen in gills were not present in digestive gland. The results indicate that gills are able to incorporate zinc prior (24 h) to digestive gland (48 h), leading to earlier mitochondrial disruption and oxidative stress. Our data suggest that gills are the initial target of ZnONP and that mitochondria are organelles particularly susceptible to ZnONP in C. gigas.

Selenium in water enhances antioxidant defenses and protects against copper-induced DNA damage in the blue mussel Mytilus edulis.

Selenium and copper are naturally occurring elements in the environment that have important roles in cellular function. Selenium is known for its role in antioxidant defense, whereas copper is a redox-active metal capable of acting as a pro-oxidant. We investigated the effects of short term selenium (Na(2)SeO(3)) supplementation (4 µg/L for 3 days) on antioxidant parameters of the blue mussel, Mytilus edulis, and its possible protective effects against a subsequent copper (CuSO(4)) exposure (56 µg/L for 3 days). Selenium supplementation caused a 4-fold increase in glutathione levels in gills. The activity of selenium-dependent glutathione peroxidase was modulated by selenium in gills (2-fold increase) and also in cell-free haemolymph (40% increase). Copper exposure produced decreases in protein thiol levels (35%) and in thioredoxin reductase activity (60%) in gills and induced an increase in DNA damage in haemocytes (70% increase in % tail DNA observed using the comet assay). The decrease in thioredoxin reductase activity may constitute a mechanism of copper toxicity in bivalves, warranting further investigation. Pre-treatment with selenium largely prevented these deleterious effects of copper on protein thiols, thioredoxin reductase activity and DNA damage. The results suggest that induction of key antioxidant defenses such as glutathione and selenium-dependent glutathione peroxidase, as a result of selenium supplementation, may play an important role in protection of aquatic organisms against oxidative stress.

Adsorption kinetics of platinum group elements in river water.

The uptake of platinum group elements (PGE) by different preparations of estuarine sediment suspended in filtered river water has been examined. For a given PGE, adsorption time courses to untreated sediment and to sediment whose hydrous metal oxides or organic matter had been removed by appropriate chemical treatments were similar. Adsorption of Rh(lll) and Pt(IV) proceeded via a first-order reversible reaction. For Rh, forward rate constants were 1 order of magnitude greater than reverse rate constants, but for Pt, forward and reverse constants were comparable. Respective system response times, required to attain 63% of the new equilibrium, ranged from about 10 to 30 h and 2 to 20 h. In contrast, rapid, initial uptake of Pd(ll) was succeeded, in most instances, by a protracted period of desorption, requiring a more complex mechanistic interpretation. In all cases, adsorption was reduced following a period of PGE equilibration with filtered river water, suggesting that complexation with natural organic ligands exerts a significant control on the adsorption process by, for example, stabilizing PGE in solution. Exchangeability of adsorbed PGE, evaluated by ammonium acetate extraction, decreased in the order Pd > Pt > Rh, in qualitative agreement with the proposed or modeled adsorption mechanisms. Experimental results, together with independent assessments of PGE mobility from secondary sources (e.g. road dust), indicate that Pd has the greatest potential for long-range transport and bioaccumulation in the aquatic environment.