Deborah Berhanu

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.

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.

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.

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

Abstract 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.

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.

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.

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.

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

Abstract 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.

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.