Cytotoxicity of TiO2 nanoparticles to mussel hemocytes and gill cells in vitro: Influence of synthesis method, crystalline structure, size and additive.

Increasing the production and applications of TiO2 nanoparticles (NPs) has led to grow concerns about the consequences for the environment. In this study, we investigated the effects of a set of TiO2 NPs on the viability of mussel hemocytes and gill cells using neutral red and thiazolyl tetrazolium bromide assays. For this, we compared the cytotoxicity of TiO2 NPs (0.1-100 mg Ti/L) produced by different techniques: rutile NPs (60 nm) produced by milling and containing disodium laureth sulfosuccinate (DSLS), rutile NPs (10, 40 and 60 nm) produced by wet chemistry and anatase/rutile NPs (∼100 nm) produced by plasma synthesis. The commercially available P25 anatase/rutile NPs (10-20 nm) were also tested. Exposures were performed in parallel with their respective bulk forms and the cytotoxicity of the additive DSLS was also tested. Z potential values in distilled water indicated different stabilities depending on the NP type and all NPs tested formed agglomerates/aggregates in cell culture media. In general, TiO2 NPs showed a relatively low and dose-dependent toxicity for both cell models with the two assays tested. NPs produced by milling showed the highest effects, probably due to the toxicity of DSLS. Size-dependent toxicity was found for NPs produced by wet chemistry (10 nm > 40 nm and 60 nm). All TiO2 NPs tested were more toxic than bulk forms excepting for plasma produced ones, which were the least toxic TiO2 tested. The mixture bulk anatase/rutile TiO2 was more toxic than bulk rutile TiO2. In conclusion, the toxicity of TiO2 NPs varied with the mode of synthesis, crystalline structure and size of NPs and can also be influenced by the presence of additives in the suspensions.

Nano-titanium dioxide bioreactivity with human alveolar type-I-like epithelial cells: Investigating crystalline phase as a critical determinant.

There can be significant variability between bioreactivity studies of nanomaterials that are apparently the same, possibly reflecting differences in the models used and differing sources of experimental material. In this study, we have generated two crystal forms of titanium dioxide nanoparticles (nano-TiO2), pure anatase and pure rutile to address the hypothesis that the bioreactivity of these nanoparticles with human alveolar epithelium will depend on their crystal phase. We used a human alveolar type-I-like epithelial cell model (TT1; generated in-house from primary human alveolar epithelial type II cells); these cells cover 95% of the alveolar epithelial surface area and are an important target cell for inhaled nanomaterials. Using literature as a guide, we hypothesised that pure anatase nano-TiO2 would display greater bioreactivity with TT1 cells in comparison to pure rutile nano-TiO2. However, we found the profile and pattern of inflammatory mediator release was similar between these two nano-TiO2 formats, although pure rutile treatment caused a small, but consistently greater, response for IL-6, IL-8 and MCP-1. Interestingly, the temporal induction of oxidative stress (increased reactive oxygen species levels and depleted glutathione) varied markedly between the different nano-TiO2 formats. We have shown that a combination of using nanomaterials synthesised specifically for toxicological study and the use of a highly relevant, reproducible human lung cell model, offers a useful approach to delineating the physicochemical properties of nanomaterials that may be important in their cellular reactivity.

Toxicity and bioaccumulation of sediment-associated silver nanoparticles in the estuarine polychaete, Nereis (Hediste) diversicolor.

In this study, the toxicities of sediment-associated silver added to sediment as commercially available silver nanoparticles (Ag NPs, 20 and 80 nm) and aqueous Ag (AgNO3) to the estuarine polychaete, Nereis (Hediste) diversicolor, were investigated for both individual and subcellular endpoints after 10 d of exposure. Both Ag NP types were characterized in parallel to the toxicity studies and found to be polydispersed and overlapping in size. Burrowing activity decreased (marginally) with increasing Ag concentration and depended on the form of Ag added to sediment. All worms accumulated Ag regardless of the form in which it was added to the sediment, and worm size (expressed as dry weight) was found to significantly affect bioaccumulation such that smaller worms accumulated more Ag per body weight than larger worms. Lysosomal membrane permeability (neutral red retention time, NRRT) and DNA damage (comet assay tail moment and tail DNA intensity %) of Nereis coelomocytes increased in a concentration-dependent manner in all three Ag treatments. Ag NP treatments were more toxic than aqueous Ag for all toxicity endpoints, even though bioaccumulation did not differ significantly among Ag forms. No significant difference in toxicity was observed between the two Ag NP treatments which was attributed to their overlap in particle size.

Bioaccumulation and effects of different-shaped copper oxide nanoparticles in the deposit-feeding snail Potamopyrgus antipodarum.

Copper oxide (CuO) nanoparticles (NPs) are among the most widely used engineered NPs and are thus likely to end up in the environment, predominantly in sediments. Copper oxide NPs have been found to be toxic to a variety of (mainly pelagic) organisms, but to differing degrees. In the present study, the influence of CuO NP shape on bioavailability and toxicity in the sediment-dwelling freshwater gastropod Potamopyrgus antipodarum was examined. In 2 separate studies, snails were exposed to either clean sediment or sediment spiked with either aqueous Cu or CuO NPs of different shapes (rods, spheres, or platelets) at 240 µg Cu/g dry weight of sediment (nominal). In neither of the studies was survival found to be related to Cu form (i.e., free ion vs particle) or shape, whereas snail growth was severely influenced by both form and shape. Reproduction was affected (by CuO NP spheres and aqueous Cu) only when estimated as the total number (live plus dead) of juveniles produced per snail per week. Both the aqueous and particulate forms of Cu were available for uptake by snails when mixed into sediment. However, Cu body burden was not directly related to observed effects. The present study stresses the need for both a better understanding of uptake mechanisms and internal distribution pathways of NPs and an assessment of long-term consequences of NP exposure.

Fate and effects of metal-based nanoparticles in two marine invertebrates, the bivalve mollusc Scrobicularia plana and the annelid polychaete Hediste diversicolor.

The objective of this paper is to synthesize results from seven published research papers employing different experimental approaches to evaluate the fate of metal-based nanoparticles (Ag NPs, Au NPs, CuO NPs, CdS NPs, ZnO NPs) in the marine environment and their effects on two marine endobenthic species, the bivalve Scrobicularia plana and the ragworm Hediste diversicolor. The experiments were carried out under laboratory (microcosms) conditions or under environmentally realistic conditions in outdoor mesocosms. Based on results from these seven papers, we addressed the following research questions: (1) How did the environment into which nanoparticles were released affect their physicochemical properties?, (2) How did the route of exposure (seawater, food, sediment) influence bioaccumulation and effects?, (3) Which biomarkers were the most responsive? and (4) Which tools were the most efficient to evaluate the fate and effects of NPs in the marine environment? The obtained results showed that metal-based NPs in general were highly agglomerated/aggregated in seawater. DGT tools could be used to estimate the bioavailability of metals released from NPs under soluble form in the aquatic environment. Both metal forms (nanoparticulate, soluble) were generally bioaccumulated in both species. Among biochemical tools, GST and CAT were the most sensitive revealing the enhancement of anti-oxidant defenses in both species exposed to sub-lethal concentrations of metal-based NPs. Apoptosis and genotoxicity were frequently observed.

Comparative study using spheres, rods and spindle-shaped nanoplatelets on dispersion stability, dissolution and toxicity of CuO nanomaterials.

Copper oxide nanoparticles with different shapes were used to examine the effect of shape on the various physicochemical properties (reactivity, aggregation, suspension stability) and to examine the behaviour by which CuO nanoparticles exhibit their biological response towards alveolar type-I cells. The different shapes examined in this study include spherical-, rod- and spindle-shaped platelet particles. In vitro dissolution studies (7 days) in 1 mM NaNO3 matrix showed a marked difference in dissolved Cu release between the nanoparticles. However, in serum-free cell-culture media (exposure media to cells), the particles' dissolution was found to be significantly enhanced with close to complete dissolution reported for all particle types. Biological studies showed both shape and size of the CuO nanoparticles tested to have a significant effect on TT-1 cell viability and release of pro-inflammatory cytokines IL-6 and IL-8. This study shows a complex interplay between particulate and dissolved species triggering the biological response. Upon immediate exposure of CuO nanoparticles of different shapes, the particulate form contributes towards the toxicity. However, for any biological response observed over and beyond a period of 24 h, the dissolved fraction becomes significant.

Multi-walled carbon nanotube length as a critical determinant of bioreactivity with primary human pulmonary alveolar cells.

Multiwalled carbon nanotube (MWCNT) length is suggested to critically determine their pulmonary toxicity. This stems from in vitro and in vivo rodent studies and in vitro human studies using cell lines (typically cancerous). There is little data using primary human lung cells. We addressed this knowledge gap, using highly relevant, primary human alveolar cell models exposed to precisely synthesized and thoroughly characterized MWCNTs. In this work, transformed human alveolar type-I-like epithelial cells (TT1), primary human alveolar type-II epithelial cells (ATII) and alveolar macrophages (AM) were treated with increasing concentrations of MWCNTs before measuring cytotoxicity, inflammatory mediator release and MAP kinase signalling. Strikingly, we observed that short MWCNTs (~0.6 µm in length) induced significantly greater responses from the epithelial cells, whilst AM were particularly susceptible to long MWCNTs (~20 µm). These differences in the pattern of mediator release were associated with alternative profiles of JNK, p38 and ERK1/2 MAP kinase signal transduction within each cell type. This study, using highly relevant target human alveolar cells and well defined and characterized MWCNTs, shows marked cellular responses to the MWCNTs that vary according to the target cell type, as well as the aspect ratio of the MWCNT.

Dietary bioavailability of cadmium presented to the gastropod Peringia ulvae as quantum dots and in ionic form.

For quantum dots (QDs) synthesized in solvents that are immiscible in water, dietary, rather than aqueous, exposure is expected to be the primary route of uptake. The estuarine snail Peringia ulvae was presented with mats of simulated detritus spiked with oleic acid capped cadmium sulfide (CdS; 3.1 ± 0.4 nm) or cadmium selenide (CdSe; 4.2 ± 0.8 nm) nanoparticles, synthesized using a microfluidics method, or Cd(2+) (added as Cd[NO3 ]2 ) as a control. A biodynamic modeling approach was used to quantify parameters that describe the dietary accumulation of the Cd forms. Ingestion rates decreased across treatments at higher exposure concentrations, indicating a metal-induced stress response related to Cd dose rather than form. Although Cd was bioavailable from both CdS and CdSe QDs, uptake rate constants from diet were significantly lower than that of Cd(2+) (p < 0.05). After 72 h depuration, however, no loss of Cd was observed from snails that had accumulated Cd from either type of QD. In comparison, snails ingesting Cd(2+) -spiked detritus eliminated 39% of their accumulated body burden per day. The almost identical uptake and efflux rates for Cd in both QDs suggest no effect of the chalcogenide conjugates (S or Se). The findings of the present study indicate that the availability of Cd in the form of nanoparticles and its apparent in vivo persistence will lead to bioaccumulation. The implications of this are discussed.

Bioaccumulation, toxicokinetics, and effects of copper from sediment spiked with aqueous Cu, nano-CuO, or micro-CuO in the deposit-feeding snail, Potamopyrgus antipodarum.

The present study examined the relative importance of copper (aqueous Cu and CuO particles of different sizes) added to sediment to determine the bioaccumulation, toxicokinetics, and effects in the deposit feeder Potamopyrgus antipodarum. In experiment 1, the bioaccumulation of Cu (240 µg Cu/g dry wt of sediment) added as aqueous Cu (CuCl2 ), nano- (6 nm, 100 nm), or micro- (<5 µm) CuO particles in adult snails was measured. In experiment 2, a more comprehensive analysis of the toxicokinetics of Cu (aqueous Cu, 6 nm, or 100 nm) was conducted. In experiment 3, the effects of Cu form (aqueous Cu and 6 nm CuO) on juvenile growth and survival at 0, 30, 60, 120, and 240 µg Cu/g dry weight sediment were assessed. Snails took up less of the 5-µm CuO particles than nano-CuO or aqueous Cu. A substantial fraction of Cu taken up was associated with shell, and this was rapidly lost when snails were transferred to clean sediment. Net uptake rates from sediment amended with 6 nm CuO and aqueous Cu were significantly higher (∼40-50%) than from sediment amended with 100 nm CuO. During 2 wk of depuration, there were no significant differences in depuration rates (kd ) among forms (aqueous Cu: kd = -0.12 wk(-1) ; 6 nm CuO: kd = -0.22 wk(-1) ; 100 nm CuO: kd = -0.2 wk(-1) ). Average juvenile growth was reduced by 0.11 mm (41%) at measured exposure concentrations of 127.2 µg Cu/g dry weight sediment for aqueous Cu and 71.9 µg Cu/g dry weight sediment for 6 nm CuO compared with control; however, differences between forms were not statistically significant. Juvenile snails in the highest exposure concentrations (aqueous Cu and 6-nm CuO groups pooled) reduced their growth by 0.18 mm on average (67%) compared with the control group. Although we observed minor differences in toxicity among Cu forms, effects on juvenile snail growth occurred at bulk sediment concentrations lower than those in the Canadian interim sediment quality guidelines. Characterization of the CuO particles showed that particle size distributions of commercially prepared particles deviated substantially from the manufacturers' specifications and highlighted the importance of fully characterizing particles when using them in toxicity tests.

Multiple cytotoxic and genotoxic effects induced in vitro by differently shaped copper oxide nanomaterials.

In nanotoxicology, the capacity of nanoparticles of the same composition but different shape to induce cytotoxicity and genotoxicity is largely unknown. A series of cytotoxic and genotoxic responses following in vitro exposure to differently shaped CuO nanoparticles (CuO NPs, mass concentrations from 0.1 to 100 µg/ml) were assessed in murine macrophages RAW 264.7 and in peripheral whole blood from healthy volunteers. Cytotoxicity, cytostasis and genotoxicity were evaluated by the colorimetric assay of formazan reduction [3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT)] and by the cytokinesis-block micronucleus cytome (CBMN Cyt) assay. The comet assay was applied for detecting DNA strand breaks and information on oxidative damage to DNA (oxidised purines and pyrimidines). The MTT assay revealed a decrease in cell viability in RAW 264.7 cells and peripheral blood lymphocytes (PBL) with significant dose-effect relationships for the different CuO NP shapes. The comet assay revealed a dose-dependent increase in primary DNA damage, and a significant increase in oxidative damage to DNA was also detectable, as well as increased frequency of micronuclei in binucleated cells, often in a dose-related manner. Proliferative activity, cytotoxicity and apoptotic markers showed a significant trend in the two cell types. Finally, we have differentiated clastogenic events from aneugenic events by fluorescence in situ hybridisation with human and murine pancentromeric probes, revealing for the first time characteristic aneugenic responses related to the shape of CuO NPs and cell type. Independently of size and shape, all CuO NPs revealed a clear-cut cytotoxic and genotoxic potential; this suggests that CuO NPs are good candidates for positive controls in nanotoxicology.

Evaluation of topically applied copper(II) oxide nanoparticle cytotoxicity in human skin organ culture.

The increasing use of nano-sized materials in our environment, and in many consumer products, dictates new safety concerns. In particular, adequate experimental models are needed to evaluate skin toxicity of metal oxide ions, commonly found in cosmetic and dermatologic preparations. We have addressed the biological effects of topically applied copper oxide (CuO) nanoparticles in human skin organ cultures, using light and electron microscopy, and biochemical tests. Nanoparticles were more toxic than micro-sized particles, and their effects were stronger when supplied in growth medium than in topical application. Still topically applied CuO nanoparticles induced inflammatory cytokine secretion and necrosis, especially in epidermis deprived of its protective cornea. Since nanoparticle penetration was not seen, we propose that they may adhere to skin surface, react with the local acidic environment, and generate soluble ions that make their way to inner sites. This work illustrates the abilities of skin organ culture to evaluate the biological effects of topically-applied materials on skin in vitro.

A mesocosm study of fate and effects of CuO nanoparticles on endobenthic species (Scrobicularia plana, Hediste diversicolor).

The fate and effects of CuO nanoparticles (CuO NPs) were examined in endobenthic species (Scrobicularia plana , Hediste diversicolor), under environmentally realistic conditions in outdoor mesocosms (exposure to Cu at 10 µg L(-1) in particulate (CuO NPs) or soluble salt (CuNO(3)) forms) for 21 days. Labile Cu was determined in water and sediment by using diffusive gradient in thin films. No labile Cu being detected from CuO NPs; the observed effects in invertebrates exposed to CuO NPs were mainly attributed to the toxicity of nanoparticulate rather than dissolved Cu toxicity. Bioaccumulation of CuO NPs was observed in both species. Biomarkers were examined at different levels of biological organization: biochemical markers of defense and damage, biomarkers of genotoxicity (comet assay), and behavioral biomarkers (feeding and burrowing). Behavioral biomarkers, antioxidant defenses (catalase, glutathion S-transferase, metallothionein), and genotoxicity are the most sensitive tools to highlight the effect of soluble or nanoparticulate metal forms. Concerning other biomarkers of defense (superoxide dismutase, lactate dehydrogenase, laccase) and damage (thiobarbituric acid reactive substances, acetylcholinesterase, acid phosphatase), no significant effects were detected. This experiment shows the suitability of mesocosms for studying the environmental effects of nanoparticles.

The complexity of nanoparticle dissolution and its importance in nanotoxicological studies.

Dissolution of nanoparticles (NPs) is an important property that alters their abundance and is often a critical step in determining safety of nanoparticles. The dissolution status of the NPs in exposure media (i.e. whether they remain in particulate form or dissolve - and to what extent), strongly affects the uptake pathway, toxicity mechanisms and the environmental compartment in which NPs will have the highest potential impact. A review of available dissolution data on NPs demonstrates there is a range of potential outcomes depending on the NPs and the exposure media. For example two nominally identical nanoparticles, in terms of size and composition, could have totally different dissolution behaviours, subject to different surface modifications. Therefore, it is imperative that toxicological studies are conducted in conjunction with dissolution of NPs to establish the true biological effect of NPs and hence, assist in their regulation.

Respiratory epithelial cytotoxicity and membrane damage (holes) caused by amine-modified nanoparticles.

The respiratory epithelium is a significant target of inhaled, nano-sized particles, the biological reactivity of which will depend on its physicochemical properties. Surface-modified, 50 and 100 nm, polystyrene latex nanoparticles (NPs) were used as model particles to examine the effect of particle size and surface chemistry on transformed human alveolar epithelial type 1-like cells (TT1). Live images of TT1 exposed to amine-modified NPs taken by hopping probe ion conductance microscopy revealed severe damage and holes on cell membranes that were not observed with other types of NPs. This paralleled induction of cell detachment, cytotoxicity and apoptotic (caspase-3/7 and caspase-9) cell death, and increased release of CXCL8 (IL-8). In contrast, unmodified, carboxyl-modified 50 nm NPs and the 100 nm NPs did not cause membrane damage, and were less reactive. Thus, the susceptibility and membrane damage to respiratory epithelium following inhalation of NPs will depend on both surface chemistry (e.g., cationic) and nano-size.

Isotopically modified nanoparticles for enhanced detection in bioaccumulation studies.

This work presents results on synthesis of isotopically enriched (99% (65)Cu) copper oxide nanoparticles and its application in ecotoxicological studies. (65)CuO nanoparticles were synthesized as spheres (7 nm) and rods (7 × 40 nm). Significant differences were observed between the reactivity and dissolution of spherical and rod shaped nanoparticles. The extreme sensitivity of the stable isotope tracing technique developed in this study allowed determining Cu uptake at exposure concentrations equivalent to background Cu concentrations in freshwater systems (0.2-30 µg/L). Without a tracer, detection of newly accumulated Cu was impossible, even at exposure concentrations surpassing some of the most contaminated water systems (>1 mg/L).

Effects of sediment-associated copper to the deposit-feeding snail, Potamopyrgus antipodarum: a comparison of Cu added in aqueous form or as nano- and micro-CuO particles.

Increasing use of engineered nanoparticles (NPs) is likely to result in release of these particles to the aquatic environment where the NPs may eventually accumulate in sediment. However, little is known about the potential ecotoxicity of sediment-associated engineered NPs. We here consider the case of metal oxide NPs using CuO to understand if the effects of NPs differ from micron-sized particles of CuO and aqueous Cu (CuCl2). To address this issue, we compared effects of copper added to the sediment as aqueous Cu, nano- (6 nm) and micro- (<5 µm) CuO particles on the deposit-feeding snail, Potamopyrgus antipodarum. Effects were assessed as mortality, specific growth rate, feeding rate, reproduction, and bioaccumulation after 8 weeks of exposure to nominal concentrations of 0, 30, 60, 120 and 240 µg Cu/g dry weight sediment. The results demonstrate that copper added to sediment as nano-CuO had greater effects on growth, feeding rate, and reproduction of P. antipodarum than copper added as micro-CuO or aqueous Cu. P. antipodarum accumulated more copper in the nano-CuO treatment than in aqueous Cu or micro-CuO treatments, indicating that consideration of metal form may be important when assessing risks of metals to the aquatic environment.

Toxic effects and bioaccumulation of nano-, micron- and ionic-Ag in the polychaete, Nereis diversicolor.

There is increasing concern about the toxicities and potential risks, both still poorly understood, of silver nanoparticles for the aquatic environment after their eventual release via wastewater discharges. In this study, the toxicities of sediment associated nano (<100 nm)-, micron (2-3.5 µm)- and ionic (AgNO(3))-Ag on the sediment-dwelling polychaete, Nereis diversicolor, were compared after 10 days of sediment exposure, using survival, DNA damage (comet assay) and bioaccumulation as endpoints. The nominal concentrations used in all exposure scenarios were 0, 1, 5, 10, 25, and 50 µg Ag/g dry weight (dw) sediment. Our results showed that Ag was able to cause DNA damage in Nereis coelomocytes, and that this effect was both concentration- and Ag form-related. There was significantly greater genotoxicity (higher tail moment and tail DNA intensities) at 25 and 50 µg/g dw in nano- and micron-Ag treatments and at 50 µg/g dw in the ionic-Ag treatment compared to the controls (0µg/g dw). The nano-Ag treatment had the greatest genotoxic effect of the three tested Ag forms, and the ionic-Ag treatment was the least genotoxic. N. diversicolor did accumulate sediment-associated Ag from all three forms. Ag body burdens at the highest exposure concentration were 8.56 ± 6.63, 6.92 ± 5.86 and 9.86 ± 4.94 µg/g dw for worms in nano-, micron- and ionic-Ag treatments, respectively, but there was no significant difference in Ag bioaccumulation among the three treatments.

Behavioural and biochemical responses of two marine invertebrates Scrobicularia plana and Hediste diversicolor to copper oxide nanoparticles.

Engineered nano-sized Cu oxide particles are extensively used in diverse applications. Because aquatic environments are the ultimate "sink" for all contaminants, it is expected that nanoparticles (NP) will follow the same fate. In this study, two marine invertebrates Scrobicularia plana and Hediste diversicolor were chosen as ecotoxicological models. The aim was to evaluate behavioural (burrowing kinetics, feeding rate) and biochemical (biomarkers) responses of S. plana and H. diversicolor exposed in the laboratory to Cu (10 µg L(-1)) added in natural seawater either in the form of engineered nanoparticles (NPs) of CuO or as dissolved Cu in 2% HNO(3). Exposure was characterized by considering (i) the physico-chemical fate of NP (ii) the fraction of labile Cu in experimental media and (iii) Cu bioaccumulation. Results showed high aggregation of CuO NPs in seawater and no additional bioavailable Cu concentrations. Behavioural impairments were observed in S. plana exposed to CuO NPs or soluble Cu whereas in H. diversicolor, only the exposure to soluble Cu led to a burrowing decrease. No obvious neurotoxicity effects were revealed since in both species, no changes in cholinesterasic activity occurred in response to both forms of Cu exposure. Biomarkers of oxidative-stress catalase and glutathione-S-transferase were enhanced in both species whereas superoxide dismutase was increased only in S. plana exposed to CuO NPs. Metallothionein-like protein was increased in bivalves exposed to both forms of Cu. Since, no detectable release of soluble Cu from CuO NPs occurred during the time of experiment, ecotoxicity effects seem to be related to CuO NPs themselves.

Synthesis of isotopically modified ZnO nanoparticles and their potential as nanotoxicity tracers.

Understanding the behavior of engineered nanoparticles in the environment and within organisms is perhaps the biggest obstacle to the safe development of nanotechnologies. Reliable tracing is a particular issue for nanoparticles such as ZnO, because Zn is an essential element and a common pollutant thus present at elevated background concentrations. We synthesized isotopically enriched (89.6%) with a rare isotope of Zn (67Zn) ZnO nanoparticles and measured the uptake of 67Zn by L. stagnalis exposed to diatoms amended with the particles. Stable isotope technique is sufficiently sensitive to determine the uptake of Zn at an exposure equivalent to lower concentration range (<15 µg g(-1)). Without a tracer, detection of newly accumulated Zn is significant at Zn exposure concentration only above 5000 µg g(-1) which represents some of the most contaminated Zn conditions. Only by using a tracer we can study Zn uptake at a range of environmentally realistic exposure conditions.

Characterisation of carbon nanotubes in the context of toxicity studies.

Nanotechnology has the potential to revolutionise our futures, but has also prompted concerns about the possibility that nanomaterials may harm humans or the biosphere. The unique properties of nanoparticles, that give them novel size dependent functionalities, may also have the potential to cause harm. Discrepancies in existing human health and environmental studies have shown the importance of good quality, well-characterized reference nanomaterials for toxicological studies.Here we make a case for the importance of the detailed characterization of nanoparticles, using several methods, particularly to allow the recognition of impurities and the presence of chemically identical but structurally distinct phases. Methods to characterise fully, commercially available multi-wall carbon nanotubes at different scales, are presented.