Benjamin J. Shaw

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.

Toxic effects of nanoparticles and nanomaterials: Implications for public health, risk assessment and the public perception of nanotechnology

Abstract Nanomaterials are now being manufactured and used in many products. However, our knowledge of the human health effects and environmental concentrations of engineered nanomaterials or nanoparticles is incomplete. This article gives a toxicologists perspective, outlining possible routes of uptake by humans, environmental concentrations, known or suspected toxic effects, and the practical implication for human health risk assessments and public perception. Humans are already exposed to a range of natural and man-made nanoparticles in the air, and exposure via the food chain, water supply, and medical applications is likely. Toxicology studies on animals, and cells in vitro, raise the possibility of adverse effects on the immune system, oxidative stress related disorders, lung disease and inflammation. However, the doses needed to produce these effects are generally high and it remains to be seen if such exposure is possible via the environment or the work place. Data on exposure is also needed for risk calculations. Current legislation does not specifically address nanoparticles or nanomaterials, and there are concerns about nomenclature, defining nanomaterials as new substance under chemicals regulations such as REACH, and the appropriateness of current test methods.

Histopathological effects of waterborne copper nanoparticles and copper sulphate on the organs of rainbow trout (Oncorhynchus mykiss).

It is unclear whether copper nanoparticles are more toxic than traditional forms of dissolved copper. This study aimed to describe the pathologies in gill, gut, liver, kidney, brain and muscle of juvenile rainbow trout, Oncorhynchus mykiss, exposed in triplicate to either a control (no added Cu), 20 or 100 μg l(-1) of either dissolved Cu (as CuSO(4)) or Cu-NPs (mean primary particle size of 87 ± 27 nm) in a semi-static waterborne exposure regime. Fish were sampled at days 0, 4, and 10 for histology. All treatments caused organ injuries, and the kinds of pathologies observed with Cu-NPs were broadly of the same type as CuSO(4) including: hyperplasia, aneurisms, and necrosis in the secondary lamellae of the gills; swelling of goblet cells, necrosis in the mucosa layer and vacuole formation in the gut; hepatitis-like injury and cells with pyknotic nuclei in the liver; damage to the epithelium of some renal tubules and increased Bowmans space in the kidney. In the brain, some mild changes were observed in the nerve cell bodies in the telencephalon, alteration in the thickness of the mesencephalon layers, and enlargement of blood vessel on the ventral surface of the cerebellum. Changes in the proportional area of muscle fibres were observed in skeletal muscle. Overall the data showed that pathology from CuSO(4) and Cu-NPs were of similar types, but there were some material-type effects in the severity or incidence of injuries with Cu-NPs causing more injury in the intestine, liver and brain than the equivalent concentration of CuSO(4) by the end of the experiment, but in the gill and muscle CuSO(4) caused more pathology.

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.

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

Abstract 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 C60 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 C60 (8.28 ± 0.56 nmol mg−1 protein). No other significant treatment-related differences were observed. Results indicate that dietary exposure to SWCNTs and C60 in rainbow trout did not result in overt toxicity.

Effects of manufactured nanomaterials on fishes: a target organ and body systems physiology approach

Manufactured nanomaterials (NM) are already used in consumer products and exposure modelling predicts releases of ng to low µg l(-1) levels of NMs into surface waters. The exposure of aquatic ecosystems, and therefore fishes, to manufactured NMs is inevitable. This review uses a physiological approach to describe the known effects of NMs on the body systems of fishes and to identify the internal target organs, as well as outline aspects of colloid chemistry relevant to fish biology. The acute toxicity data, suggest that the lethal concentration for many NMs is in the mg l(-1) range, and a number of sublethal effects have been reported at concentrations from c. 100 µg to 1 mg l(-1). Exposure to NMs in the water column can cause respiratory toxicity involving altered ventilation, mucus secretion and gill pathology. This may not lead, however, to overt haematological disturbances in the short term. The internal target organs include the liver, spleen and haematopoietic system, kidney, gut and brain; with toxic effects involving oxidative stress, ionoregulatory disturbances and organ pathologies. Some pathology appears to be novel for NMs, such as vascular injury in the brain of rainbow trout Oncorhynchus mykiss with carbon nanotubes. A lack of analytical methods, however, has prevented the reporting of NM concentrations in fish tissues, and the precise uptake mechanisms across the gill or gut are yet to be elucidated. The few dietary exposure studies conducted show no effects on growth or food intake at 10-100 mg kg(-1) inclusions of NMs in the diet of O. mykiss, but there are biochemical disturbances. Early life stages are sensitive to NMs with reports of lethal toxicity and developmental defects. There are many data gaps, however, including how water quality alters physiological responses, effects on immunity and chronic exposure data at environmentally relevant concentrations. Overall, the data so far suggest that the manufactured NMs are not as toxic as some traditional chemicals (e.g. some dissolved metals) and the innovative, responsible, development of nanotechnology should continue, with potential benefits for aquaculture, fisheries and fish health diagnostics.

Regulatory ecotoxicity testing of nanomaterials – proposed modifications of OECD test guidelines based on laboratory experience with silver and titanium dioxide nanoparticles

Abstract Regulatory ecotoxicity testing of chemicals is of societal importance and a large effort is undertaken at the OECD to ensure that OECD test guidelines (TGs) for nanomaterials (NMs) are available. Significant progress to support the adaptation of selected TGs to NMs was achieved in the context of the project MARINA (http://www.marina-fp7.eu/) funded within the 7th European Framework Program. Eight OECD TGs were adapted based on the testing of at least one ion-releasing NM (Ag) and two inert NMs (TiO2). With the materials applied, two main variants of NMs (ion releasing vs. inert NMs) were addressed. As the modifications of the test guidelines refer to general test topics (e.g. test duration or measuring principle), we assume that the described approaches and modifications will be suitable for the testing of further NMs with other chemical compositions. Firm proposals for modification of protocols with scientific justification(s) are presented for the following tests: growth inhibition using the green algae Raphidocelis subcapitata (formerly: Pseudokirchneriella subcapitata; TG 201), acute toxicity with the crustacean Daphnia magna (TG 202), development toxicity with the fish Danio rerio (TG 210), reproduction of the sediment-living worm Lumbriculus variegatus (TG 225), activity of soil microflora (TGs 216, 217), and reproduction of the invertebrates (Enchytraeus crypticus, Eisenia fetida, TGs 220, 222). Additionally, test descriptions for two further test systems (root elongation of plants in hydroponic culture; test on fish cells) are presented. Ecotoxicological data obtained with the modified test guidelines for TiO2 NMs and Ag NM and detailed method descriptions are available.

A simplified method for determining titanium from TiO2 nanoparticles in fish tissue with a concomitant multi-element analysis.

The reliable detection of nanoparticles (NPs) in fish tissue is required to support ecotoxicological research and food safety investigations. Therefore the current work aimed to develop a simple method to determine Ti from TiO2 NPs in fish tissue whilst simultaneously measuring other elements in the sample. Spike recovery tests showed no differences when digestion was conducted in glass or plastic vials, there was stirring or sonication of the samples, or when sodium dodecyl sulfate was added. However, the addition of 2% Triton X-100 and sonicating and then vortexing of samples immediately prior to analysis did improve recovery (approximately 20% to >90% in trout gill and muscle samples). Method precision and accuracy were good with coefficients of variation <7%. Copper spike recovery results showed that the method is also suitable for multi-element analysis in the same samples. This improved method is simple with high throughput and represents a marked improvement for routine determination Ti from TiO2 NPs in fish tissues.

Toxicity of cerium oxide nanoparticles to the earthworm Eisenia fetida: subtle effects

Environmental context This study investigates the toxicity of cerium oxide nanoparticles to earthworms, key organisms in soil ecosystems. Cerium oxide did not affect survival or reproduction of the earthworms but did exert histological changes. We conclude that current soil guidelines, based simply on metal toxicity, appear to adequately protect against cerium exposure risk, at least for earthworms. Abstract The toxicity of cerium oxide (CeO2) nanoparticles (NPs) in soils is largely unknown. This study aimed to investigate the toxicity of three different CeO2 NPs to the earthworm, Eisenia fetida, for effects on survival (at day 28) and reproduction (at day 56), as well as bioaccumulation and histopathological effects. Eisenia fetida were exposed in standard Lufa 2.2 soil to three CeO2 NPs of different size ranges (5–80nm), one larger particle (300nm) and a cerium salt (ammonium cerium nitrate) over an exposure range from 41–10000mgCekg–1. Survival and reproduction were not affected by the four CeO2 particles, even at the highest exposure concentration tested. Alternatively, 10000mgCekg–1 cerium salt affected survival and reproduction; Median lethal concentration (LC50) and effective concentration (EC50) values were 317.8 and 294.6mgCekg–1. Despite a lack of toxic effect from the different forms of CeO2 particles, there was a dose-dependent increase in cerium in the organisms at all exposure concentrations, and for all material types. Earthworms exposed to CeO2 particles had higher concentrations of total cerium compared to those exposed to ionic cerium, but without exhibiting the same toxic effect. Histological observations in earthworms exposed to the particulate forms of CeO2 did, however, show cuticle loss from the body wall and some loss of gut epithelium integrity. The data suggest that that CeO2 NPs do not affect survival or reproduction in E. fetida over the standard test period. However, there were histological changes that could indicate possible deleterious effects over longer-term exposures.

The acute toxicity of thallium to freshwater organisms: Implications for risk assessment.

The acute toxicity of Tl(I) to the microalga, Pseudokirchneriella subcapitata, the planktonic crustaceans, Daphnia magna and Daphnia pulex, and early-life stage of the zebrafish, Danio rerio, has been studied according to OECD protocols. Toxicological end-point concentrations for the microalga ranged from 17 μg l(-1) for a 72 h EyC25 (yield inhibition) to 80 μg l(-1) for a 72 h ErC50 (growth inhibition). Daphnia were less sensitive to Tl, with 48 h EC50s of about 1000 μg l(-1) and 1200 μg l(-1) for D. magna and D. pulex, respectively; however, end-point concentrations were reduced considerably (to about 510 μg l(-1) and 730 μg l(-1), respectively) when experiments were repeated in dechlorinated Plymouth tap water (rather than OECD medium). The 96 h LC50 for D. rerio was 870 μg l(-1) but a variety of sub-lethal effects, including enlargement of yolk sac and reduction in heart beat rate, were observed when larvae were exposed to lower concentrations. Based on these results, a predicted no effect concentration (PNEC) for Tl in freshwaters of 0.087 μg l(-1) is proposed. The PNEC is an order of magnitude lower than the only (Canadian) water quality guideline for Tl that appears to exist, and is lower than Tl concentrations reported in freshwaters impacted by historical or contemporary metal mining. Our results are also consistent with previous studies that employ different organisms and end-points in that Tl toxicity is dependent on the concentration of K+, the biogeochemical analogue of Tl+. Accordingly, regulation of Tl in the freshwater environment should factor in the relative abundance of K.