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.     

Effects of long-term exposure to silver or copper on growth, bioaccumulation and histopathology in the blue mussel Mytilus edulis

The purpose of this study was to determine the long-term accumulation of either silver or copper from low concentrations in seawater by blue mussels, Mytilus edulis. Mussels raised from eggs in the laboratory to the age of 2·5 months (approximately 4·5 mm in length) were continuously exposed to 0, 1, 5 and 10μg/liter of either silver (nitrate) or copper (chloride) and sampled at 12, 18 and 21 months for growth studies, measurements of metal accumulation and histopathological examination.Whole-body soft tissues were analyzed for the presence of both silver and copper, as background levels of copper in the incoming seawater averaged 2–4 μg/liter. Mussels exposed to silver had accumulated significant amounts of silver only at the highest test concentration (10 μg/liter Ag) after 12 months, but at 18 and 21 months significant levels were accumulated at all three test concentrations. Mussels exposed to copper accumulated significant amounts of copper at 5 and 10 μg/liter Cu after all three sampling periods, but not at 1μg/liter. Silver-exposed animals also accumulated significantly greater amounts of copper than control animals.In a comparative study, field-collected juvenile mussels (approximately 16·1 mm in shell length) and adult mussels (approximately 53·4 mm in shell length) were exposed for 12 months to 0, 5, 25 and 50 μg/liter silver only and subsequently sampled for metal-accumulation analyses and growth measurements. Juvenile mussels accumulated significant amounts of silver at all test concentrations, with the exception of mussels exposed to 5 μg/liter Ag for 6 months. Copper accumulation in the silver-exposed juveniles was significant only at 50 μg/liter Ag after 6 months, but at all test concentrations after 12 months. Adult mussels exposed to silver accumulated significant levels of both silver and copper, but at somewhat lower levels than juveniles.In the growth study, silver had no effect on laboratory reared mussels at the highest concentration of 10 μg/liter tested, whereas copper at 10 μg/liter did appear to affect growth as early as 4 months after the start of experimental exposure. Field-collected juvenile mussels did show inhibition in growth after 6 months' exposure to 25 and 50 μg/liter Ag, with some growth occurring after 12 months. Adults also showed inhibition in growth after 6 months but not at 12 months.Histopathological examination of mussels exposed to either 5 or 10 μg/liter of copper for 18 months showed changes in the digestive diverticula, gastrointestinal tract, reproductive tract and muscle tissues. These changes were more noticeable in mussels exposed to 5 μg/liter Cu than in those exposed to 10 μg/liter. Mussels exposed to silver for 21 months showed yellowish to black particulate deposition in the basement membrane and connective tissue of the various organs and tissues. Silver deposition increased with increasing test concentration.     

The role of oxyradicals in intracellular proteolysis and toxicity in mussels

Studies were performed on the common mussel, M. edulis L., to determine whether copper (Cu) exposure can affect the extent to which digestive cell proteins are oxidised and whether such oxidative damage is mediated by free radicals. Three age groups of mussels were exposed for 6 ^-days to environmentally realistic concentrations of Cu and then digestive gland homogenates were examined for evidence of protein carbonyl formation. Significant increases in carbonyls relative to untreated control mussels were seen for the youngest (2–4 year-old) and oldest (≥ 10 year-old) mussels only after exposure for 6 days, followed by recovery from exposure for a further 6 days. Untreated mussels also showed an age-related difference in protein oxidation, with a significantly lower concentration in the youngest animals (2–4 year olds). Copper did not affect the levels of modified tryptophan or tyrosine residues or the extent of total lipid peroxidation in digestive gland homogenate. Significant depletion of total vitamin E (a-tocopherol) was seen only in young and medium-aged mussels following exposure for 6 days. The levels of protein carbonyl groups were increased in digestive cell cytosol and lighter lysosomes but not in heavier lysosomes or digestive gland microsomes following 5 days exposure to Cu. Dihydrohodamine-123 was converted to fluorescent rhodamine-123 following sequestration into digestive cell lysosomes. The results suggest a link between the lysosomal sequestration of copper, a concomitant increase in the production of oxyradicals and the potential for intracellular oxidative damage, as well as an increased capacity for oxidative damage in older animals.     

Use of the Comet assay to assess DNA damage in hemocytes and digestive gland cells of mussels and clams exposed to water contaminated with petroleum hydrocarbons

Oil spills can result in the deposition of large quantities of petroleum hydrocarbons into intertidal and shallow waters seriously impacting bivalve populations. Petroleum hydrocarbons are enriched in polycyclic aromatic hydrocarbons (PAH) and PAH analogs many of which may have potential to damage DNA. The Comet assay is useful for assessing DNA damage and has been used to a limited degree with aquatic organisms, but mostly with studies in vitro. We have carried out studies with the Comet assay to assess the DNA damaging potential of complex mixtures of petroleum hydrocarbons for bivalves. Experiments were carried out with mussels (Mytilus edulis) and clams (Mya arenaria) with dispersions and water soluble fractions of an Arabian crude oil which was also chemically characterized in detail by GC-MS. Pilot studies were first conducted to evaluate test performance and reproducibility. An interindividual coefficient of variation ranging from 17 to 30% was established for the assay with hemocytes and digestive gland cells of both species. Exposure to hydrocarbon fractions had no significant impact on clams. However, an increase in DNA damage was observed at P < 0.1 with digestive gland cells of mussels exposed to aqueous fractions of a light crude oil. These studies have demonstrated a potential for DNA damage in bivalves exposed to oil spills in inshore waters as well as potential for interspecies sensitivity.     

Metal pollution assessment of the marine environment by determination of metal-binding proteins in Mytilus sp.

Adult mussels (Mytilus galloprovincialis) (shell length 5–7 cm) exposed to elevated concentrations of Cd (0.1–1.3 mg l⁻¹), in a flowing seawater aquarium under laboratory conditions, responded by elevated synthesis of metal-binding proteins in a digestive gland. Response depended on the Cd concentration level as well as on duration of the exposure time. Results obtained show that inducible proteins (isolated by gel-filtration column chromatography and characterized by electrochemical and spectrophotometric methods) should be considered metallothionein-like proteins.The induced metal-binding proteins of mussels in specific organs or tissues are considered to be a potential biological indicator of metal pollution, taking into account specific qualities of the mussel response to different metals. The advantages of the analytical methods applied (gel-chromatography and polarography) and the relevance of the laboratory data when transferred to environmental conditions are discussed. Previously reported results dealing with metal-binding proteins of Mytilus species are also summarized.     

Survival,growth and shell deformities of copper- and cadmium-exposed mussels (Mytilus edulis L.) in brackish water

Mussels, Mytilus edulis L., were exposed to elevated concentrations of copper or cadmium in the laboratory, then placed in cages in the sea (salinity 7‰). One year later maximum lengths of the mussels were measured and shells screened for deformities. Growth was 0·6 cm year^?1 in the control cages and retarded in cages of exposed mussels. A total of 63% of cadmium-exposed and 46% of copper-exposed mussels had shell deformities. In the control cages 26% were deformed while in a natural population only 3% were deformed. The proportion of deformities to growth was inversely related. Low concentrations showed their injuriousness in this long-term test, probably due to the closing of the valves during exposure to high concentrations.     

Total thiol redox status as a potent biomarker of PAH-mediated effects on mussels

This laboratory study describes phenanthrene (Ph) and/or anthracene (An) ability to alter the total thiol redox status (TRS), via depletion of protein free thiols (PSH) and glutathione (GSH) levels, in gills of mussel Mytilus galloprovincialis, after a short-term (7 days) exposure period to each contaminant (at a final concentration of 0.1 mg L⁻¹) or in a mixture (ration 1:1, at a final concentration of 0.2 mg L⁻¹). A number of observable changes, like lysosomal membrane impairment (as detected via the neutral red retention time assay, primarily performed in haemocytes), enhancement of lipid peroxidation byproducts, increased nuclear abnormalities, inhibition of AChE and ALP activity, as well as a significant depletion of PSH and GSH were detected in gills of exposed mussels, in any case. Significant relationships occurred among TRS parameters with each change/stress indices measured in tissues of mussels, could reinforce the use of PSH as a potent biomarker.     

Shore exposure affects mussel population structure and mediates the effect of epibiotic algae on mussel survival in SW Ireland

On rocky shores, the relative importance of abiotic and biotic processes that regulate community structure are thought to vary with levels of shore exposure. This can lead to characteristic features found on sheltered and exposed shores. This study identified differences in the population structure of mussels on exposed and sheltered rocky shores on Atlantic coasts of south-west Ireland. Direct interactions between epibiotic algae and their host mussels were also examined to test if potential effects varied with shore exposure. Mussel beds on sheltered shores were less dense and comprised larger mussels with greater rates of individual survival and growth than those on exposed shores. The results of a field experiment showed that algal epibionts had a negative effect on mussel survival on sheltered shores but not on exposed shores. Surprisingly, the presence of algal epibionts had no effect on mussel growth on either shore type. These findings contrast with those of previous studies. The effects of shore exposure and algal epibionts on mussels may be species-specific and may interact with other factors across different regions. This study shows that predictions of effects of exposure on mussel populations and their epibionts should only be based on specific experimental evidence and cannot be generalised across regions.     

Development of two novel CYP-antibodies and their use in a PCB exposure experiment with Mytilus edulis

In an attempt to learn more about the cytochrome P450 (CYP) system of mussels, we used protein databases and alignment software to extract highly conserved CYP sequences. From these alignments synthetic peptides were produced and used for rabbit immunisation, which yielded polyclonal antibodies against the CYP families 2 and 4. The antibodies were evaluated with Western Blot and ELISA assays, using digestive gland microsomal samples from the mussel Mytilus edulis. Western Blots revealed immunoreactions for both antibodies. The anti-CYP2 sequence rendered one major immunopositive protein of ≈49 kDa size, and weak signals for proteins of ≈41 and 56 kDa size. The anti-CYP4 sequence rendered two major bands of ≈56 and 59 kDa size, and also a weak immunoreaction with a protein of ≈43 kDa size. ELISA rendered only weak signals even with a 1:50 dilution of IgG-purified serum. A 10-day exposure to Aroclor 1254 did not appear to affect any of the immunopositive proteins, while total PCBs in soft bodies increased from 14–40 ng/g DW in controls to 373–638 ng/g DW in exposed mussels.     

Partitioning of copper among copper-binding proteins in the mussel Mytilus edulis exposed to soluble copper

Partitioning of copper among copper-binding proteins was evaluated in digestive glands of Mytilus edulis exposed to soluble copper. Groups of mussels were held in flow-through bioassay systems and exposed to 25 μg Cu liter^?1 for up to 21 weeks. At three-week intervals, groups of 25 mussels were removed and the digestive glands were analyzed for copper-binding proteins by gel-permeation chromatography and atomic absorption spectrometry.Chronic exposure to copper resulted in increased amounts of copper in the low molecular weight (LMW) protein fraction, which contains metallothioneins, and in the high molecular weight (HMW) protein fraction, which contains metalloenzymes. Concentrations of copper in the LMW protein fraction increased and then appeared to plateau with long exposure times, whereas those in the HMW protein fraction continued to increase with exposure time.     

Recovery of Mytilus edulis L. from chronic oil exposure

Previous laboratory studies¹ have shown that physiological and cellular processes of Mytilus edulis are affected by exposure to low and environmentally realistic concentrations of oil. However, there is little information concerning the rate of recovery from oil exposure and the extent to which physiological recovery may be related to the depuration of hydrocarbons from the tissues. The present study has shown a marked reduction in the feeding rate and scope for growth of mussels exposed to two concentrations of diesel oil (30 and 130 μg/litre) for 8 months. During recovery from oil exposure the depuration of hydrocarbons from the tissues was concomitant with the recovery of physiological performance. Mussels exposed to high oil concentrations (‘high-oil’ mussels) were found to recover more rapidly than those exposed to low oil concentrations (‘low-oil’ mussels), both in terms of depuration and scope for growth, and there was evidence of ‘catch-up’ growth. Recovery of both low- and high-oil mussels was complete after approximately 55 days.     

Assessing site-specific effects of TBT contamination with mussel growth rates

During nine field transplant tests in San Diego Bay (1987–1990), juvenile mussels were exposed to mean concentrations of tributyltin (TBT) in ambient seawater ranging from 2 to 530 ng liter⁻¹ for 12 weeks under natural conditions. A total of 79 cages with 18 mussels each were monitored at 18 different sites. Growth and seawater TBT concentrations were measured weekly or on alternate weeks (biweekly). Mean growth rates ranged from 17 to 505 mg week⁻¹ (0·2 to 2·5 mm week⁻¹). Accumulation of TBT in mussel tissues was measured at the end of each 12-week test exposure and ranged from 0·1 to 3·2 μg g⁻¹ TBT wet weight. The frequency of the measurements and the integration of chemical and biological measurements improved the accuracy of the assessment over more traditional approaches. Growth was significantly related to seawater and tissue TBT. The statistical relationships with growth effects were used to estimate chemical effect zones for TBT in San Diego Bay. Site-specific differences were distinguished by additional statistical analyses and consideration of environmental significance.     

A 15-month study of zooplankton ingestion by farmed mussels (Mytilus edulis) in Bantry Bay, Southwest Ireland

There is a growing body of evidence to suggest that bivalve molluscs routinely ingest zooplankton. To elucidate further these observations, a 15-month study of zooplankton ingestion by farmed mussels was conducted using mussel long-lines in Bantry Bay, Ireland. Stomach content analysis of the mussels showed that there was evidence of zooplankton ingestion throughout the sampling period, but that highest mean numbers of zooplankters were ingested by mussels in the spring and summer months. Various zooplankton species were present in mussel stomachs. Harpacticoid copepods were found more often in stomach contents than calanoid copepods, probably due to their proximity to the bivalves' inhalent siphons. Barnacle cyprids featured in large numbers in stomach contents, but only for a period of 3 months which broadly corresponded with their pelagic phase. Sizes of ingested zooplankton ranged from 126 μm to 6 mm, but more of the smaller zooplankters (e.g. crustacean nauplii) were ingested. When lengths of ingested copepods were compared with those found in plankton net samples, it was found that the net-sampled copepods were significantly larger than those found in mussel stomachs, suggesting that mussels select for smaller categories within the zooplankton available to them. Soft bodied zooplankton was rarely found in mussel stomachs but their absence may be due to rapid digestion or they may have been destroyed in the preservation process. Ingestion of zooplankton by bivalves is discussed in the context of the impacts mussel farms have on resident zooplankton populations.     

Multiple forms of lysozyme in copper stressed mussels (Mytilus edulis)

In a previous study we monitored fluctuations in the concentration of lysozyme in the hemolymph of copper stressed bay mussels, Mytilus edulis, concurrently by measuring enzymatic activity and by an enzyme-linked immunosorbent assay (ELISA).¹ In a number of instances the data collected by the two techniques from copper stressed mussels yielded contradictory results. In these instances the quantity of lysozyme, determined enzymatically, was significantly greater than the quantities determined by ELISA. These results prompted subsequent investigation into the cause of the discrepancies. In the course of the study a high molecular weight protein fraction (35 000 MW) from mussel hemocytes was isolated. Decreasing the ionic strength and concentration of the fraction caused dissociation into a 14 600 MW fraction characteristically identical to lysozyme.² Association to the 35 000 MW form is reversible upon an increase in ionic strength and concentration, and is presumed to be a dimer of lysozyme.^3,4 The ionic strength and pH optima for hydrolysis of suspensions of Micrococcus luteus were determined for the dimer. Polyclonal anti-lysozyme gammaglobulin preparations¹ were used in determinations of the relative immunological reactivity of various lysozyme preparations by immunodiffusion.⁵ The monomer form was more favorably precipitated or detectable than the lysozyme in hemocytes. disturbed hemocytes indicate an apparent increase in the extracellular concentration of the dimer as a result of cell damage. The observed low immunological reactivity would allow for the lack of detection by ELISA, and may have interfered with antibody-monomer interactions.     

Antioxidant and pro-oxidant challenge of tannic acid in mussel hemocytes exposed to cadmium

The present study investigates the antioxidant and pro-oxidant behavior of tannic acid (TA) in hemocytes of mussel Mytilus galloprovincialis, in the presence or the absence of cadmium (Cd). TA at concentrations up to 20 μM, primarily found to be no toxic (in terms of cell viability, superoxide anions, nitric oxide and lipid peroxidation products currently estimated), significantly diminished the cytotoxic and oxidative effects induced by the metal (50 and/or 100 μM) in all cases. On the other hand, higher concentrations of TA (40 and 60 μM) were toxic, thus enhancing Cd-mediated cytotoxic and oxidative effects. The present study showed TA beneficiary properties in hemocytes of mussels, at least at low concentrations, while TA at concentrations higher than 20 μM could serve as an excellent oxidized substrate, thus enhancing toxic effects either alone or with the presence of micromolar concentrations of non transition metals, such as Cd.     

In vitro mechanistic differences in benzo[a]pyrene–DNA adduct formation using fish liver and mussel digestive gland microsomal activating systems

Turbot (Scophthalmus maximus) and mussel (Mytilus edulis) microsomes were incubated with DNA to examine if microsomal in vitro metabolism of BaP could result in DNA adducts detected by ³²P-postlabelling. Turbot DNA was incubated with benzo[a]pyrene (BaP), NADPH and microsomal activating systems prepared from either livers of unexposed turbot, turbot exposed to BaP or β-naphthoflavone (ß-NF) or digestive glands from mussels. The β-NF activating system generated the highest levels of DNA adducts detected in this study (451.7 adducts per 10⁸ nucleotides) and were distributed in three discrete adduct TLC spots, one of which (97% of the total adducts) co-migrated with the ³²P-postlabelled BaP 7,8-diol, 9,10-epoxide-N²-guanine adduct. Fewer adducts (P <0.05) were generated by BaP-induced microsomes (9.4–30.6 adducts per 10⁸ nucleotides) but levels were higher (P <0.05) than those generated from untreated fish (3.5 adducts per 10⁸ nucleotides). Co-incubation with 500 μM α-naphthoflavone (α-NF) resulted in 97–99% inhibition in adduct formation implicating cytochrome P450-dependent (CYP) bioactivation however there was some evidence for carry over of BaP in the liver microsomal preparations from BaP injected fish. In contrast to the fish activating systems, no DNA adducts were observed when mussel microsomes were incubated with BaP, DNA and NADPH.     

Molecular investigations into pollutant impact on roundnose grenadier (C. rupestris) and transplanted common mussel (M. edulis) in Skagerrak, the North Sea

The use of a suite of different biochemical markers in the deep sea fish Roundnose grenadier and caged common mussels, and analyses of contaminant levels in the mussels, indicate that Skagerrak are more polluted than waters around the Faroe Islands. In Roundnose, induction of ethoxyresorufin-O-deethylase (EROD) is consistent with increased benzo(a)pyrene hydroxylase (BPH) in mussels and elevated levels of polynuclear aromatic hydrocarbons (PAHs) in mussels and sediment from Skagerrak. This evidence of a large-scale environmental impact of pollutants should lead to further efforts to decrease transport into, and deposition of, waste compounds in the Skagerrak area.     

U.S. Mussel Watch: 1977–1978 results on trace metals and radionuclides

The results of the U.S. Mussel Watch Monitoring Program for the period 1976–1978 for trace metals and artificial radionuclides in bivalves are presented. The substances analysed included Ag, Cu, Zn, Cd, Ni, Pb, ²³⁸Pu, ^239+240Pu and ²⁴¹Am. The analyses of organic substances will be presented elsewhere. The concentrations of these substances in the bivalves may reflect upwelling processes, anthropogenic inputs or natural levels. Off the California coast, mussels show markedly elevated Pu and Cd concentrations in coastal areas adjacent to the most intensive upwelling zones. Elevated levels of Pb, for example, are found in organisms living adjacent to highly urbanized places. The general patterns of distribution repeat themselves year after year at a given site. Thus, it is concluded that annual monitoring activities may not be necessary and that a frequency of sampling of several or so years may be more appropriate to identify pollution problems. Finally, national or regional baselines for metal concentrations in bivalves from unpolluted waters are proposed. National baselines for Pb in the west coast mussels of 1·0 parts 10^?6 and for Ag in east coast mussels of 0·05 parts 10^?6 are suggested.     

Reproductive success as an indicator of genotoxicity in the polychaete worm, Neanthes arenaceodentata

The effects on reproduction of single or chronic exposure to the direct-acting mutagen, radiation, were evaluated using Neanthes arenaceodentata as the model animal. In the first set of experiments, mated pairs were irradiated when oocytes were visible in the female and were given a single dose of either 0·5, 1·0, 2·0, 5·0, 10 or 50 Gy. In the second set of experiments, lifetime exposure was initiated upon the spawning of the P₁ female and was terminated upon the spawning of the F₁ female; the average total doses (0·55, 6·5 and 54 Cy) were delivered at a rate of 0·19, 2·1 or 17 mGyH⁻¹. Our results on embryo abnormalities and mortalities indicated that lethal mutations were most likely induced in the germ cells and had an adverse effect on reproductive success by reducing the survival of early life stages. Except for those mated pairs exposed acutely to 10 or 50 Gy or exposed chronically to 17 mGyh⁻¹, there was no evidence of gamete killing. However, evidence was obtained for mutagen-damage accumulation in developing gametes from continuous exposure to radiation.     

Effects of linear alkylbenzene sulphonate (LAS) and cadmium in the digestive gland of mussel, Mytilus sp.

A laboratory study was carried out exposing mussels (Mytilus sp.) to linear alkylbenzene sulphonate (LAS) (6 mg litre⁻¹), Cd (0.05 mg litre⁻¹) and LAS plus Cd at the same concentrations. The aim was to assess the use of several histopathological and biochemical indices as potential biomarkers of the impact of these xenobiotics in the digestive gland of molluscs. Treated mussels actively accumulated Cd in the digestive gland compared with controls (p 0.01), the highest levels occurring after 30 days of exposure in the group treated with Cd plus LAS. Among several histological alterations screened in digestive gland tissues the thickness of digestive tubules in Cd treated animals decreased more markedly (p 0.01) than in LAS exposed mussels. As for biochemical parameters, the investigated antioxidant enzyme activities, superoxide dismutase (SOD), catalase, DT-diaphorase and glutathione peroxidase (GPX) did not show any significant induction due to these xenobiotics. However, a slight decrease of the antioxidant defences of the animals was detected after 30 days of exposure to contaminants.