Copper accumulation and toxicity in earthworms exposed to CuO nanomaterials: Effects of particle coating and soil ageing.

Engineered nanomaterials (ENMs) may be functionalised with a surface coating to enhance their properties, but the ecotoxicity of the coatings and how hazard changes with ageing in soil is poorly understood. This study determined the toxic effect of CuO ENMs with different chemical coatings on the earthworm (Eisenia fetida) in fresh soil, and then after one year in aged soil. In both experiments, earthworms were exposed for 14 days to the CuO materials at nominal concentrations of 200 and 1000 mg Cu kg-1 dry weight and compared to CuSO4. In the fresh soil experiment, CuO-COOH was found to be the most acutely toxic of the nanomaterials (survival, 20 ±â€¯50%), with tenfold increase of total Cu in the earthworms compared to controls. Sodium pump activity was reduced in most CuO ENM treatments, although not in the CuSO4 control. There was no evidence of glutathione depletion or the induction of superoxide dismutase (SOD) activity in any treatment. Histology showed a mild hypoplasia of mucous cells in the epidermis with some nanomaterials. In the aged soil, the CuO-NH4+ was the most acutely toxic ENM (survival 45 ±â€¯3%) and Cu accumulation was lower in the earthworms than in the fresh soil study. Depletion of tissue Mn and Zn concentrations were seen in earthworms in aged soil, while no significant effects on sodium pump or total glutathione were observed. Overall, the study showed some coating-dependent differences in ENM toxicity to earthworms which also changed after a year of ageing the soil.

Low hazard of silver nanoparticles and silver nitrate to the haematopoietic system of rainbow trout.

Silver nanoparticles (Ag NPs) are known for their antibacterial properties and are used in a growing number of nano-enabled products, with inevitable concerns for releases to the environment. Nanoparticles may also be antigenic and toxic to the haematopoietic system, but the immunotoxic effect of Ag NPs on non-target species such as fishes is poorly understood. This study aimed to assess the effect of Ag NP exposure via the water on the haematopoietic system of rainbow trout, Oncorhynchus mykiss, and to determine whether or not the hazard from Ag NPs was different from that of AgNO3. Fish were exposed for 7 days to a control (dechlorinated Plymouth freshwater), dispersant control, 1µgl-1 Ag as AgNO3 or 100µgl-1 Ag NPs. Animals were sampled on days 0, 4 and 7 for haematology, tissue trace metal concentration, biochemistry for evidence of oxidative stress/inflammation in the spleen and histopathology of the blood cells and spleen. The Ag NP treatment significantly increased the haematocrit, but the haematological changes were within the normal physiological range of the animal. Thrombocytes in spleen prints at day 4, and melanomacrophage deposits at day 7 in the spleen, of Ag NP exposed-fish displayed significant increases compared to all the other treatments within the time point. A dialysis experiment confirmed that dissolution rates were very low and any pathology observed is likely from the NP form rather than dissolved metal released from it. Overall, the data showed subtle differences in the effects of Ag NPs compared to AgNO3 on the haematopoietic system. The lack of pathology in the circulating blood cells and melanomacrophage deposits in the spleen suggests a compensatory physiological effort by the spleen to maintain normal circulating haematology during Ag NP exposure.

Sub-lethal effects of waterborne exposure to copper nanoparticles compared to copper sulphate on the shore crab (Carcinus maenas).

The toxicity of soluble copper (Cu) to marine organisms is reasonably well described. However, the hazard of Cu engineered nanomaterial (ENMs) is poorly understood. The aim of the present study was to compare the toxicity of Cu ENMs to Cu as CuSO4 in the shore crab, Carcinus maenas. The crabs were exposed via the water using a semi-static approach to 0.2 or 1mgL-1 of Cu ENMs or 1mgL-1 of Cu as CuSO4. Gills, hepatopancreas, chela muscle and haemolymph were collected at days 0, 4 and 7 for the body burden of Cu, histology and biochemical analysis [thiobarbituric acid reactive substances (TBARS) and total glutathione (GSH)]. Nominal exposure concentrations of both the ENMs and the metal salt were maintained at over 80% in each treatment throughout the experiment. By day 7, 54% mortality was recorded in the 1mgL-1 CuSO4 treatment, compared to just 21% in the 1mgL-1 Cu ENM-exposed crabs. The target organs for Cu accumulation were similar for both forms of Cu with highest concentrations in the gills, particularly the posterior gills; followed by the hepatopancreas, and with the lowest concentrations in the chela muscle. No changes were observed in the osmolarity of the haemolymph (ANOVA, P>0.05). TBARS were measured as an indicator of lipid peroxidation and showed the greatest change in the anterior and posterior gills and hepatopancreas of animals exposed to 1mgL-1 Cu ENMs (ANOVA or Kruskal-Wallis, P<0.05). No statistically significant changes in total GSH were observed (ANOVA, P>0.05; n=6 crabs per treatment). Histological analysis revealed organ injuries in all treatments. The types of pathologies observed in the Cu ENM treatments were broadly similar to those of the Cu as CuSO4 treatment. Overall, the target organs and Cu accumulation from Cu ENMs were comparable to that following exposure to Cu as CuSO4, and although there were some differences in the sub-lethal effects, the metal salt was more acutely toxic.

Effects of waterborne copper nanoparticles and copper sulphate on rainbow trout, (Oncorhynchus mykiss): physiology and accumulation.

Emerging data suggests that some types of nanoparticles (NPs) are toxic to fish, and given the well-known toxicity of dissolved metals, there are also concerns about whether metal-containing NPs present a similar or different hazard to metal salts. In this study, juvenile rainbow trout were exposed in triplicate to either a control, 20 or 100 µg l⁻¹ of either Cu as CuSO4 or Cu-NPs (mean primary particle size, 87±27 nm) in a semi-static aqueous exposure regime. Fish were sampled at days 0, 4, and 10 for tissue trace elements, haematology, and biochemistry. By day 4, fish from the 100 µg l⁻¹ Cu as CuSO4 treatment showed 85% mortality (treatment subsequently terminated) compared to 14% in the 100 µg l⁻¹ Cu-NP exposed fish. Mortality at day 10 was 4, 17, 10, and 19% in the control, 20 µg l⁻¹ Cu as CuSO4, 20 and 100 µg l⁻¹ Cu-NP treatments, respectively. Copper accumulation was seen in the gills of fish from all Cu treatments, and was statistically significant in both CuSO4 treatments at day 4 and all Cu treatments at day 10 compared to controls. No statistically significant Cu accumulation was seen in the spleen, brain or muscle of fish from any treatment, although an elevation in intestinal Cu was seen in the high Cu-NP treatment throughout. There were some transient changes in haematology and depletion of plasma Na⁺ that was treatment-related, with some differences between the nano form and metal salt, but Cu-NPs were not overtly haemolytic. A 6-fold decrease in branchial Na⁺/K⁺-ATPase activity in all Cu treatments (compared to controls), depletion of plasma and carcass ion concentrations suggest that Cu-NPs are an ionoregulatory toxicant to rainbow trout. Statistically significant decreases in Na⁺/K⁺-ATPase activity were also seen in the brains and intestine, and whilst there was no material-type effect in the former, this was only observed in the gut of fish exposed to 100 µg l⁻¹ Cu-NPs. There were material-dependent changes in tissue thiobarbituric acid reactive substances (TBARS), and in the gill the Cu-NPs caused a larger (though non-significant compared to control) increase in TBARS than the equivalent metal salt treatment (the latter actually being significantly reduced compared to all other treatments). Overall, these data show that Cu-NPs have similar types of toxic effects to CuSO4, which can occur at lower tissue Cu concentrations than expected for the dissolved metal.

Physiological effects of nanoparticles on fish: a comparison of nanometals versus metal ions.

The use of nanoscale materials is growing exponentially, but there are also concerns about the environmental hazard to aquatic biota. Metal-containing engineered nanoparticles (NPs) are an important group of these new materials, and are often made of one metal (e.g., Cu-NPs and Ag-NPs), metal oxides (e.g., ZnO and TiO(2) NPs), or composite of several metals. The physiological effects and toxicity of trace metals in the traditional dissolved form are relatively well known and the overall aim of this review was to use our existing conceptual framework of metal toxicity in fish to compare and contrast the effects of nanometals. Conceptually, there are some fundamental differences that relate to bioavailability and uptake. The chemistry and behaviour of nanometals involves dynamic aspects of aggregation theory, rather than the equilibrium models traditionally used for free metal ions. Some NPs, such as Cu-NPs, may also release free metal ions from the surface of the particle. Biological uptake of NPs is not likely via ion transporters, but endocytosis is a possible uptake mechanism. The body distribution, metabolism, and excretion of nanometals is poorly understood and hampered by a lack of methods for measuring NPs in tissues. Although data sets are still limited, emerging studies on the acute toxicity of nanometals have so far shown that these materials can be lethal to fish in the mg-µgl(-1) range, depending on the type of material. Evidence suggests that some nanometals can be more acutely toxic to some fish than dissolved forms. For example, juvenile zebrafish have a 48-h LC(50) of about 0.71 and 1.78mgl(-1) for nano- and dissolved forms of Cu respectively. The acute toxicity of metal NPs is not always explained, or only partly explained, by the presence of free metal ions; suggesting that other novel mechanisms may be involved in bioavailability. Evidence suggests that nanometals can cause a range of sublethal effects in fish including respiratory toxicity, disturbances to trace elements in tissues, inhibition of Na(+)K(+)-ATPase, and oxidative stress. Organ pathologies from nanometals can be found in a range of organs including the gill, liver, intestine, and brain. These sublethal effects suggest some common features in the sublethal responses to nanometals compared to metal salts. Effects on early life stages of fish are also emerging, with reports of nanometals crossing the chorion (e.g., Ag-NPs), and suggestions that the nano-forms of some metals (Cu-NPs and ZnO NPs) may be more toxic to embryos or juveniles, than the equivalent metal salt. It remains possible that nanometals could interfere with, and/or stimulate stress responses in fish; but data has yet to be collected on this aspect. We conclude that nanometals do have adverse physiological effects on fish, and the hazard for some metal NPs will be different to the traditional dissolved forms of metals.

Dietary toxicity of single-walled carbon nanotubes and fullerenes (C60) in rainbow trout (Oncorhynchus mykiss).

The objective of this investigation was to compare the toxicity of two manufactured carbon nanomaterials (CNs) to determine if shape influenced toxicity. Juvenile rainbow trout Oncorhynchus mykiss were fed a control diet (no CN addition), or a diet supplemented with 500 mg single-walled carbon nanotubes (SWCNT) kg(-1) or 500 mg C(60) kg(-1) for six weeks. Fish growth, haematology, tissue ion concentrations, histopathology, osmoregulation, and biochemistry were evaluated. At week 4, but not on weeks 2 and 6, significant elevation in brain TBARS (an indication of lipid peroxidation) was observed in fish exposed to SWCNTs (16.2 ± 1.38 nmol mg(-1) protein) compared to the control (9.11 ± 0.81 nmol mg(-1) protein) and fish exposed to C(60) (8.28 ± 0.56 nmol mg(-1) protein). No other significant treatment-related differences were observed. Results indicate that dietary exposure to SWCNTs and C(60) in rainbow trout did not result in overt toxicity.

Dietary exposure to titanium dioxide nanoparticles in rainbow trout, (Oncorhynchus mykiss): no effect on growth, but subtle biochemical disturbances in the brain.

Our laboratory recently reported gut pathology following incidental ingestion of titanium dioxide nanoparticles (TiO(2) NPs) during aqueous exposures in trout, but there are almost no data on dietary exposure to TiO(2) NPs in fish. The aim of this experiment was to observe the sub-lethal effects of dietary exposure to TiO(2) NPs in juvenile rainbow trout (Oncorhynchus mykiss). Stock solutions of dispersed TiO(2) NPs were prepared by sonication without the use of solvents and applied to a commercial trout diet. Fish were exposed in triplicate to either, control (no added TiO(2)), 10, or 100 mg kg(-1) TiO(2) NPs diets for 8 weeks followed by a 2 week recovery period where all fish were fed the control diet. TiO(2) NPs had no impact on growth or nutritional performance, and no major disturbances were observed in red or white blood cell counts, haematocrits, whole blood haemoglobin, or plasma Na(+). Ti accumulation occurred in the gill, gut, liver, brain and spleen during dietary TiO(2) exposure. Notably, some of these organs, especially the brain, did not clear Ti after exposure. The brain also showed disturbances to Cu and Zn levels (statistically significant at weeks 4 and 6; ANOVA or Kruskal-Wallis, P < 0.05) and a 50% inhibition of Na(+)K(+)-ATPase activity during TiO(2) NP exposure. Na(+)K(+)-ATPase activity was unaffected in the gills and intestine. Total glutathione in the gills, intestine, liver and brain were not affected by dietary TiO(2) NPs, but thiobarbituric acid reactive substances (TBARS) showed up to 50% decreases in the gill and intestine. We conclude that TiO(2) NPs behave like other toxic dietary metals where growth rate and haematology can be protected during sub-lethal exposures, but in the case of TiO(2) NPs this may be at the expense of critical organs such as the brain and the spleen.

Toxicity of titanium dioxide nanoparticles to rainbow trout (Oncorhynchus mykiss): gill injury, oxidative stress, and other physiological effects.

Mammalian and in vitro studies have raised concerns about the toxicity of titanium dioxide nanoparticles (TiO2 NPs), but there are very limited data on ecotoxicity to aquatic life. This paper is an observational study where we aim to describe the toxicity of TiO2 NPs to the main body systems of rainbow trout. Stock solutions of dispersed TiO2 NPs were prepared by sonication without using solvents. A semi-static test system was used to expose rainbow trout to either a freshwater control, 0.1, 0.5, or 1.0 mg l(-1) TiO2 NPs for up to 14 days. Exposure to TiO2 NPs caused some gill pathologies including oedema and thickening of the lamellae. No major haematological or blood disturbances were observed in terms of red and white blood cell counts, haematocrit values, whole blood haemoglobin, and plasma Na+ or K+ concentrations. Tissue metal levels (Na+, K+, Ca2+ and Mn) were generally unaffected. However, some exposure concentration-dependent changes in tissue Cu and Zn levels were observed, especially in the brain. Exposure to TiO2 NPs caused statistically significant decreases in Na+K+-ATPase activity (ANOVA, P<0.05) in the gills and intestine, and a trend of decreasing enzyme activity in the brain (the latter was not statistically significant). Thiobarbituric acid reactive substances (TBARS) showed exposure concentration-dependent and statistically significant (ANOVA or Kruskal-Wallis test, P<0.05) increases (two-fold or more) in the gill, intestine and brain, but not the liver during exposure to TiO2 NPs compared to controls. TiO2 NP exposure caused statistically significant (ANOVA, P<0.05) increases in the total glutathione levels in the gills, but depletion of hepatic glutathione compared to controls. Total glutathione levels in the brain and intestine were unaffected. Liver cells exposed to TiO2 NPs showed minor fatty change and lipidosis, and some hepatocytes showed condensed nuclear bodies (apoptotic bodies). Fish probably ingested water containing TiO2 NPs during exposure (stress-induced drinking) which may have resulted in some areas of erosion on the intestinal epithelium. Overall we conclude that titanium dioxide nanoparticles are not a major ionoregulatory toxicant, or haemolytic, at the concentration and exposure times used. Respiratory distress is a concern and sub-lethal toxicity involves oxidative stress, organ pathologies, and the induction of anti-oxidant defences, such as glutathione.

Toxicity of single walled carbon nanotubes to rainbow trout, (Oncorhynchus mykiss): respiratory toxicity, organ pathologies, and other physiological effects.

Mammalian studies have raised concerns about the toxicity of carbon nanotubes (CNTs), but there is very limited data on ecotoxicity to aquatic life. We describe the first detailed report on the toxicity of single walled carbon nanotubes (SWCNT) to rainbow trout, using a body systems approach. Stock solutions of dispersed SWCNT were prepared using a combination of solvent (sodium dodecyl sulphate, SDS) and sonication. A semi-static test system was used to expose rainbow trout to either a freshwater control, solvent control, 0.1, 0.25 or 0.5 mgl(-1) SWCNT for up to 10 days. SWCNT exposure caused a dose-dependent rise in ventilation rate, gill pathologies (oedema, altered mucocytes, hyperplasia), and mucus secretion with SWCNT precipitation on the gill mucus. No major haematological or blood disturbances were observed in terms of red and white blood cell counts, haematocrits, whole blood haemoglobin, and plasma Na(+) or K(+). Tissue metal levels (Na(+), K(+), Ca(2+), Cu, Zn and Co) were generally unaffected. However some dose-dependent changes in brain and gill Zn or Cu were observed (but not tissue Ca(2+)), that were also partly attributed to the solvent. SWCNT exposure caused statistically significant increases in Na(+)K(+)-ATPase activity in the gills and intestine, but not in the brain. Thiobarbituric acid reactive substances (TBARS) showed dose-dependent and statistically significant decreases especially in the gill, brain and liver during SWCNT exposure compared to controls. SWCNT exposure caused statistically significant increases in the total glutathione levels in the gills (28%) and livers (18%), compared to the solvent control. Total glutathione in the brain and intestine remained stable in all treatments. Pathologies in the brain included possible aneurisms or swellings on the ventral surface of the cerebellum. Liver cells exposed to SWCNT showed condensed nuclear bodies (apoptotic bodies) and cells in abnormal nuclear division. Overt fatty change or wide spread lipidosis was absent in the liver. Fish ingested water containing SWCNT during exposure (presumably stress-induced drinking) which resulted in precipitated SWCNT in the gut lumen and intestinal pathology. Aggressive behaviour and fin nipping caused some mortalities at the end of the experiment, which may be associated with the gill irritation and brain injury, although the solvent may also partly contributed to aggression. Overall we conclude that SWCNTs are a respiratory toxicant in trout, the fish are able to manage oxidative stress and osmoregulatory disturbances, but other cellular pathologies raise concerns about cell cycle defects, neurotoxicity, and as yet unidentified blood borne factors that possibly mediate systemic pathologies.