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.

Merging nano-genotoxicology with eco-genotoxicology: an integrated approach to determine interactive genotoxic and sub-lethal toxic effects of C(60) fullerenes and fluoranthene in marine mussels, Mytilus sp.

Whilst there is growing concern over the potential detrimental impact of engineered nanoparticles (ENPs) on the natural environment, little is known about their interactions with other contaminants. In the present study, marine mussels (Mytilus sp.) were exposed for 3 days to C(60) fullerenes (C(60); 0.10-1 mg l(-1)) and a model polycyclic aromatic hydrocarbon (PAH), fluoranthene (32-100 µg l(-1)), either alone or in combination. The first two experiments were conducted by exposing the organisms to different concentrations of C(60) and fluoranthene alone, in order to determine the effects on total glutathione levels (as a measure of generic oxidative stress), genotoxicity (DNA strand breaks using Comet assay in haemocytes), DNA adduct analyses (using (32)P-postlabelling method) in different organs, histopathological changes in different tissues (i.e. adductor muscle, digestive gland and gills) and physiological effects (feeding or clearance rate). Subsequently, in the third experiment, a combined exposure of C(60) plus fluoranthene (0.10 mg l(-1) and 32 µg l(-1), respectively) was carried out to evaluate all endpoints mentioned above. Both fluoranthene and C(60) on their own caused concentration-dependent increases in DNA strand breaks as determined by the Comet assay. Formation of DNA adducts however could not be detected for any exposure conditions. Combined exposure to C(60) and fluoranthene additively enhanced the levels of DNA strand breaks along with a 2-fold increase in the total glutathione content. In addition, significant accumulation of C(60) was observed in all organs, with highest levels in digestive gland (24.90 ± 4.91µg C(60) g(-1) ww). Interestingly, clear signs of abnormalities in adductor muscle, digestive gland and gills were observed by histopathology. Clearance rates indicated significant differences compared to the control with exposure to C(60), and C(60)/fluoranthene combined treatments, but not after fluoranthene exposure alone. This study demonstrated that at the selected concentrations, both C(60) and fluoranthene evoke toxic responses and genetic damage. The combined exposure produced enhanced damage with additive rather than synergistic effects.

A multiple biomarker approach to investigate the effects of copper on the marine bivalve mollusc, Mytilus edulis.

While copper (Cu) is considered to be an essential trace element for many organisms, overexposure to this metal can induce a wide spectrum of effects including DNA damage. Given that Cu is a highly relevant contaminant in the marine environment, we aimed to evaluate the induction of DNA strand breaks (using the comet assay) in haemocytes and concurrently also determined biological responses at higher levels of biological organisation in bivalve molluscs, Mytilus edulis, following exposure for 5 days to a range of environmentally realistic levels of Cu (18-56 µg l(-1)). Prior to evaluation of genetic damage, the maximum tolerated concentration (MTC) was also determined, which was found to be (100 µg l(-1)) above which complete mortality over the exposure period was observed. In addition to DNA damage, levels of glutathione in adductor muscle extracts, histopathological examination of various organs (viz., adductor muscle, gills and digestive glands) and clearance rates as a physiological measure at individual level were also determined. Furthermore, tissue-specific accumulation and levels of Cu in water samples were also determined using ICP-MS. There was a strong concentration-dependant induction for DNA damage and total glutathione levels increased by 1.8-fold at 56 µg l(-1) Cu. Histological examination of the organs showed qualitatively distinct abnormalities. Clearance rate also showed a significant decrease compared to controls even at the lowest concentration (i.e. 18 µg l(-1); P=0.003). Cu levels in adductor muscle (P=0.012), digestive gland (P=0.008) and gills (P=0.002) were significantly higher than in the control. The multi-biomarker approach used here suggests that in some cases clear relationships exist between genotoxic and higher level effects, which could be adopted as an integrated tool to evaluate different short and long-term toxic effects of pollutants.

Hypoxia-induced oxidative DNA damage links with higher level biological effects including specific growth rate in common carp, Cyprinus carpio L.

Both hypoxia and hyperoxia, albeit in different magnitude, are known stressors in the aquatic environment. Adopting an integrated approach, mirror carp (Cyprinus carpio L.), were exposed chronically (i.e. 30 days) to hypoxic (1.8 ± 1.1 mg O(2) l(-1)) and hyperoxic (12.3 ± 0.5 mg O(2) l(-1)) conditions and resultant biological responses or biomarkers were compared between these two treatments as well as with fish held under normoxic conditions (7.1 ± 1.04 mg O(2) l(-1)). The biomarkers determined included the activities of glutathione peroxidase (GPx), measurement of oxidative DNA damage (using modified Comet assay employing bacterial enzymes: Fpg and Endo-III), haematological parameters, histopathological and ultrastructural examination of liver and gills. Specific growth rate (SGR) of the fish, as an important ecotoxicological parameter was also determined over the exposure period. The study suggested that while the levels of hepatic GPx were unaffected, there was a significant difference in activity in the blood plasma under different exposure conditions; the hyperoxic group showed increased GPx activity by approximately 37% compared to normoxic group and the hypoxic group showed a decrease by approximately 38% than the normoxic group. Interestingly, oxidative DNA damage was significantly higher in both hypoxic and hyperoxic by approximately 25% compared to normoxic conditions, Fpg showing enhanced level of damage compared to the Endo-III treatment (P < 0.001). The haematological parameters showed enhanced values under hypoxic conditions. Transmission electron microscopic (TEM) studies revealed damage to liver and gill tissues for both the treatments. Interestingly, SGR of fish was significantly lowered in hypoxic by approx. 30% compared to normoxic condition and this was found to be correlated with DNA damage (R = -0.82; P = 0.02). Taken together, these results indicate that prolonged exposure to both hypoxic and hyperoxic conditions induce oxidative stress responses at both DNA and tissue levels, and hypoxia can result in compensatory changes in haematological and growth parameters which could influence Darwinian fitness of the biota with wider ecological implications.

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.

Contamination of bivalve haemolymph samples by adductor muscle components: implications for biomarker studies.

Haemolymph samples and haemocytes collected via the adductor muscles of bivalve molluscs are extensively used in ecotoxicological studies. Withdrawal of haemolymph from mussels, Mytilus edulis, via the posterior adductor muscle, may lead to contamination with the intracellular contents of adductor myocytes. Lysopine dehydrogenase (LyDH) activity, an adductor myocyte marker, was used to investigate the impact of this potential contamination on levels of total glutathione, glutathione peroxidase (GPx) and acetylcholinesterase (AChE) measured in cell-free haemolymph. The mean glutathione content of cell-free haemolymph from 28 mussels was 3.2 +/- 1.8 microM (mean +/- SD). There was a linear relationship (slope = 0.28 +/- 0.03 min; mean +/- SE; P < 0.0001, n = 28) with haemolymph LyDH levels suggesting that at least some of the glutathione measured in cell-free haemolymph had arisen from contamination. Haemolymph LyDH activity was significantly higher in samples extracted using larger diameter needles, and also in samples where there had been some difficulty in the extraction. Exposure of mussels to oxidative stress using 40 microg l(-1) Cu for 5 days resulted in a 1.7 fold increase in glutathione (P = 0.033), but no increase (P = 0.810) in LyDH activity in adductor muscle. This was reflected in a similar increase in the slope of a plot of cell-free haemolymph glutathione versus LyDH activity (P = 0.011), consistent with both of these having originated from the adductor muscle. Cell-free haemolymph GPx and AChE activities also correlated with LyDH activity (Spearman rank correlation coefficients of 0.531 (P = 0.0068) and 0.537 (P = 0.0062), respectively, n = 27) suggesting that these also arise from contamination of the haemolymph. For GPx there was a significant linear relationship (P = 0.025) with haemolymph LyDH levels consistent with both enzymes originating from the myocytes. However, there was hyperbolic relationship (P = 0.0004) between haemolymph AChE and LyDH activities. It appears that this is because the AChE originates from a different compartment to the LyDH, i.e. cholinergic neuromuscular junctions in the adductor muscle. We conclude that it would be prudent, when considering the possibility of using a biomarker in cell-free haemolymph from bivalve molluscs, to check whether contamination could be an issue.