Kaja Kasemets

Toxicity of nanoparticles of CuO, ZnO and TiO2 to microalgae Pseudokirchneriella subcapitata

Toxicities of ZnO, TiO2 and CuO nanoparticles to Pseudokirchneriella subcapitata were determined using OECD 201 algal growth inhibition test taking in account potential shading of light. The results showed that the shading effect by nanoparticles was negligible. ZnO nanoparticles were most toxic followed by nano CuO and nano TiO2. The toxicities of bulk and nano ZnO particles were both similar to that of ZnSO4 (72 h EC50 approximately 0.04 mg Zn/l). Thus, in this low concentration range the toxicity was attributed solely to solubilized Zn2+ ions. Bulk TiO2 (EC50=35.9 mg Ti/l) and bulk CuO (EC50=11.55 mg Cu/l) were less toxic than their nano formulations (EC50=5.83 mg Ti/l and 0.71 mg Cu/l). NOEC (no-observed-effect-concentrations) that may be used for risk assessment purposes for bulk and nano ZnO did not differ (approximately 0.02 mg Zn/l). NOEC for nano CuO was 0.42 mg Cu/l and for bulk CuO 8.03 mg Cu/l. For nano TiO2 the NOEC was 0.98 mg Ti/l and for bulk TiO2 10.1 mg Ti/l. Nano TiO2 formed characteristic aggregates entrapping algal cells that may contribute to the toxic effect of nano TiO2 to algae. At 72 h EC50 values of nano CuO and CuO, 25% of copper from nano CuO was bioavailable and only 0.18% of copper from bulk CuO. Thus, according to recombinant bacterial and yeast Cu-sensors, copper from nano CuO was 141-fold more bioavailable than from bulk CuO. Also, toxic effects of Cu oxides to algae were due to bioavailable copper ions. To our knowledge, this is one of the first systematic studies on effects of metal oxide nanoparticles on algal growth and the first describing toxic effects of nano CuO towards algae.

Toxicity of nanoparticles of ZnO, CuO and TiO2 to yeast Saccharomyces cerevisiae

The aim of this study was to evaluate the toxic effect of nanosized ZnO, CuO and TiO(2) to Saccharomyces cerevisiae - a widely used unicellular eukaryotic model organisms in molecular and cell biology. The effect of metal oxide nanoparticles, their bulk forms and respective ionic forms were compared. The bioavailable Zn(2+) and Cu(2+) ions in the growth medium were quantified by recombinant microbial sensors. Nano and bulk TiO(2) were not toxic even at 20000 mg/l. Both, nano and bulk ZnO were of comparable toxicity (8-h EC(50) 121-134 mg ZnO/l and 24-h EC(50) 131-158 mg/l). The toxicity was explained by soluble Zn-ions as proved by the microbial sensor. However, nano CuO was about 60-fold more toxic than bulk CuO: 8-h EC(50) were 20.7 and 1297 mg CuO/l and 24-h EC(50) were 13.4 and 873 mg/l, respectively. The increase in toxicity of both CuO formulations at 24th hour of growth was due to the increased dissolution of copper ions from CuO over time. Comparison of EC(50) values of nano CuO, bulk CuO and Cu(2+) with bioavailable copper concentrations in the growth medium showed that the solubilized Cu-ions explained only about 50% of the toxicity of both, nano and bulk CuO. To our knowledge, this is the first study that evaluates the toxicity of ZnO, CuO and TiO(2) nanoparticles to S.cerevisiae.

Toxicity of ZnO and CuO nanoparticles to ciliated protozoa Tetrahymena thermophila

The toxic effects of nanoparticles (NPs) of ZnO and CuO to particle-ingesting model organism protozoa Tetrahymena thermophila were evaluated. Nano-ZnO was remarkably more toxic than nano-CuO (EC(50) values approximately 5mg metal/l versus 128mg metal/l). Toxic effect of CuO depended on particle size: nano-CuO was about 10-20 times more toxic than bulk CuO. However, when calculated on basis of bioavailable copper (quantified using recombinant Cu-sensor bacteria) the 4-h EC(50) values of nano- and bulk formulations were comparable (2.7 and 1.9mg bioavailable Cu/l, respectively), and statistically different from the EC(50) value of Cu(2+) (1.1mg/l). Differently from CuO particles, bulk and nanosized ZnO as well as Zn(2+) were of similar toxicity (4-h EC(50) values 3.7 and 3.9mg bioavailable Zn/l, respectively, and 4.9mg Zn(2+)/l). Thus, the toxic effect of both, CuO and ZnO (nano)particles to protozoa was caused by their solubilised fraction. The toxic effects of the copper compounds were not dependent on exposure time (4 and 24h), whereas the toxicity of zinc compounds was about 1.5 times lower after 24h of exposure than after 4h, probably due to adaptation. In summary, we recommend T. thermophila as a simple eukaryotic particle-ingesting model organism for the toxicity screening of NPs. For the high throughput testing we suggest to use the 4-h assay on microplates using ATP and/or propidium iodide for the evaluation of cell viability.

Size-Dependent Toxicity of Silver Nanoparticles to Bacteria, Yeast, Algae, Crustaceans and Mammalian Cells In Vitro

The concept of nanotechnologies is based on size-dependent properties of particles in the 1–100 nm range. However, the relation between the particle size and biological effects is still unclear. The aim of the current paper was to generate and analyse a homogenous set of experimental toxicity data on Ag nanoparticles (Ag NPs) of similar coating (citrate) but of 5 different primary sizes (10, 20, 40, 60 and 80 nm) to different types of organisms/cells commonly used in toxicity assays: bacterial, yeast and algal cells, crustaceans and mammalian cells in vitro. When possible, the assays were conducted in ultrapure water to minimise the effect of medium components on silver speciation. The toxic effects of NPs to different organisms varied about two orders of magnitude, being the lowest (∼0.1 mg Ag/L) for crustaceans and algae and the highest (∼26 mg Ag/L) for mammalian cells. To quantify the role of Ag ions in the toxicity of Ag NPs, we normalized the EC50 values to Ag ions that dissolved from the NPs. The analysis showed that the toxicity of 20–80 nm Ag NPs could fully be explained by released Ag ions whereas 10 nm Ag NPs proved more toxic than predicted. Using E. coli Ag-biosensor, we demonstrated that 10 nm Ag NPs were more bioavailable to E. coli than silver salt (AgNO3). Thus, one may infer that 10 nm Ag NPs had more efficient cell-particle contact resulting in higher intracellular bioavailability of silver than in case of bigger NPs. Although the latter conclusion is initially based on one test organism, it may lead to an explanation for “size-dependent“ biological effects of silver NPs. This study, for the first time, investigated the size-dependent toxic effects of a well-characterized library of Ag NPs to several microbial species, protozoans, algae, crustaceans and mammalian cells in vitro.

Biotests and Biosensors for Ecotoxicology of Metal Oxide Nanoparticles: A Minireview

Nanotechnologies have become a significant priority worldwide. Several manufactured nanoparticles - particles with one dimension less than 100 nm - are increasingly used in consumer products. At nanosize range, the properties of materials differ substantially from bulk materials of the same composition, mostly due to the increased specific surface area and reactivity, which may lead to increased bioavailability and toxicity. Thus, for the assessment of sustainability of nanotechnologies, hazards of manufactured nanoparticles have to be studied. Despite all the above mentioned, the data on the potential environmental effects of nanoparticles are rare. This mini-review is summarizing the emerging information on different aspects of ecotoxicological hazard of metal oxide nanoparticles, focusing on TiO2, ZnO and CuO. Various biotests that have been successfully used for evaluation of ecotoxic properties of pollutants to invertebrates, algae and bacteria and now increasingly applied for evaluation of hazard of nanoparticles at different levels of the aquatic food-web are discussed. Knowing the benefits and potential drawbacks of these systems, a suite of tests for evaluation of environmental hazard of nanoparticles is proposed. Special attention is paid to the influence of particle solubility and to recombinant metal-sensing bacteria as powerful tools for quantification of metal bioavailability. Using recombinant metal-specific bacterial biosensors and multitrophic ecotoxicity assays in tandem will create new scientific knowledge on the respective role of ionic species and of particles in toxicity of metal oxide nanoparticles.

Mechanisms of toxic action of Ag, ZnO and CuO nanoparticles to selected ecotoxicological test organisms and mammalian cells in vitro: A comparative review

Abstract Silver, ZnO and CuO nanoparticles (NPs) are increasingly used as biocides. There is however increasing evidence of their threat to “non-target” organisms. In such a context, the understanding of the toxicity mechanisms is crucial for both the design of more efficient nano-antimicrobials, i.e. for “toxic by design” and at the same time for the design of nanomaterials that are biologically and/or environmentally benign throughout their life-cycle (safe by design). This review provides a comprehensive and critical literature overview on Ag, ZnO and CuO NPs’ toxicity mechanisms on the basis of various environmentally relevant test species and mammalian cells in vitro. In addition, factors modifying the toxic effect of nanoparticles, e.g. impact of the test media, are discussed. Literature analysis revealed three major phenomena driving the toxicity of these nanoparticles: (i) dissolution of nanoparticles, (ii) organism-dependent cellular uptake of NPs and (iii) induction of oxidative stress and consequent cellular damages. The emerging information on quantitative structure–activity relationship modeling of nanomaterials’ toxic effects and the challenges of extrapolation of laboratory results to the environment are also addressed.

High throughput kinetic Vibrio fischeri bioluminescence inhibition assay for study of toxic effects of nanoparticles.

Despite of the growing production and use of nanoparticles (NPs) in various applications, current regulations, including EC new chemical policy REACH, fail to address the environmental, health, and safety risks posed by NPs. This paper shows that kinetic Vibrio fischeri luminescence inhibition test--Flash Assay--that up to now was mainly used for toxicity analysis of solid and colored environmental samples (e.g. sediments, soil suspensions), is a powerful tool for screening the toxic properties of NPs. To demonstrate that Flash Assay (initially designed for a tube luminometer) can also be adapted to a microplate format for high throughput toxicity screening of NPs, altogether 11 chemicals were comparatively analyzed. The studied chemicals included bulk and nanosized CuO and ZnO, polyethylenimine (PEI) and polyamidoamine dendrimer generations 2 and 5 (PAMAM G2 and G5). The results showed that EC50 values of 30-min Flash Assay in tube and microplate formats were practically similar and correlated very well (log-logR2=0.98), classifying all analyzed chemicals, except nano CuO (that was more toxic in cuvette format), analogously when compared to the risk phrases of the EC Directive 93/67/EEC for ranking toxicity of chemicals for aquatic organisms. The 30-min EC50 values of nanoscale organic cationic polymers (PEI and dendrimers) ranged from 215 to 775 mg/l. Thirty-minute EC50 values of metal oxides varied largely, ranging from approximately 4 mg/l (bulk and nano ZnO) to approximately 100 mg/l (nano CuO) and approximately 4000 mg/l (bulk CuO). Thus, considering an excellent correlation between both formats, 96-well microplate Flash Assay can be successfully used for high throughput evaluation of harmful properties of chemicals (including organic and inorganic NPs) to bacteria.

Changes in the Daphnia magna midgut upon ingestion of copper oxide nanoparticles: a transmission electron microscopy study.

This work is a follow-up of our previous paper (Heinlaan et al., 2008. Chemosphere 71, 1308-1316) where we showed about 50-fold higher acute toxicity of CuO nanoparticles (NPs) compared to bulk CuO to water flea Daphnia magna. In the current work transmission electron microscopy (TEM) was used to determine potential time-dependent changes in D. magna midgut epithelium ultrastructure upon exposure to CuO NPs compared to bulk CuO at their 48 h EC(50) levels: 4.0 and 175 mg CuO/L, respectively. Special attention was on potential internalization of CuO NPs by midgut epithelial cells. Ingestion of both CuO formulations by daphnids was evident already after 10 min of exposure. In the midgut lumen CuO NPs were dispersed whereas bulk CuO was clumped. By the 48th hour of exposure to CuO NPs (but not to equitoxic concentrations of bulk CuO) the following ultrastructural changes in midgut epithelium of daphnids were observed: protrusion of epithelial cells into the midgut lumen, presence of CuO NPs in circular structures analogous to membrane vesicles from holocrine secretion in the midgut lumen. Implicit internalization of CuO NPs via D. magna midgut epithelial cells was not evident however CuO NPs were no longer contained within the peritrophic membrane but located between the midgut epithelium microvilli. Interestingly, upon exposure to CuO NPs bacterial colonization of the midgut occurred. Ultrastructural changes in the midgut of D. magna upon exposure to CuO NPs but not to bulk CuO refer to its nanosize-related adverse effects. Time-dependent solubilisation of CuO NPs and bulk CuO in the test medium was quantified by recombinant Cu-sensor bacteria: by the 48th hour of exposure to bulk CuO, the concentration of solubilised copper ions was 0.05 ± 0.01 mg Cu/L that was comparable to the acute EC(50) value of Cu-ions to D. magna (48 h CuSO(4) EC(50) = 0.07 ± 0.01 mg Cu/L). However, in case of CuO NPs, the solubilised Cu-ions 0.01 ± 0.001 mg Cu/L, explained only part of the toxicity.

Biotests and biosensors in ecotoxicological risk assessment of field soils polluted with zinc, lead, and cadmium.

The combined chemical and ecotoxicological hazard evaluation study was conducted on 60 smelter-influenced soils containing 1 to 13, 50 to 653, and 100 to 1,198 mg/kg of Cd, Pb, and Zn, respectively. For these soils (liquid-to-soil ratio = 10), water extractability of Zn, Cd, and Pb was less than 0.19% (median values). Acetic acid (0.11 M) extracted 23, 9.7, and 0.7% of Cd, Zn, and Pb, respectively. Although heavy metal concentrations in the studied soils were high, the toxic effects of water extracts were observed only in few samples and in few biotests (algae Selenastrum capricornutum and metal detector assay). For most of the aquatic test organisms (e.g., crustaceans, photobacteria), the bioavailable concentrations of metals in soil-water extracts were either subtoxic, or the adverse effects were compensated by soil nutrients, etc. However, analysis of the soils with recombinant Cd sensor Bacillus subtilis (pTOO24) showed that about 65% of these apparently subtoxic samples contained bioavailable Cd when analyzed in the suspension assay (detection limit 1.5 mg Cd/kg soil), indicating the desorption of Cd induced by direct contact of bacteria with soil particles. The median bioavailable fraction of Cd (1%) was 23-fold lower than the fraction extracted by acetic acid. The Pb-Cd sensor Staphylococcus aureus (pT0024) detected bioavailable Pb only in the suspensions of five of the most lead-polluted soils (>417 mg Pb/kg): the median bioavailability of Pb was 0.42%. Consequently, the hazard assessment relying on total metal levels in soils should be revised by critical comparison with data obtained from bioassays. Development and use of biosensors (excellent tools for mechanistic studies and signaling hazard already at subtoxic level) should be encouraged.

Exposure to CuO Nanoparticles Changes the Fatty Acid Composition of Protozoa Tetrahymena thermophila

In the current study, the toxicity mechanism of nanosized CuO (nCuO) to the freshwater ciliated protozoa Tetrahymena thermophila was studied. Changes in fatty acid profile, lipid peroxidation metabolites and reactive oxygen species (ROS) were measured. Bulk CuO and CuSO(4) served as controls for size and solubility and 3,5-dichorophenol (3,5-DCP) as a control for a chemical known to directly affect the membrane composition. Exposure to all copper compounds induced the generation of ROS, whereas nCuO was most potent. The latter effect was not solely explained by solubilized Cu-ions and was apparently particle-related. 24 h exposure of protozoa to 80 mg/L of nCuO (EC50) significantly decreased the proportion of two major unsaturated fatty acids (UFA) (C18:3 cis-6,9,12, C18:2 cis-9,12), while it increased the relative amount of two saturated fatty acids (SFA) (C18:0, C16:0). Analogous effect was not observed when protozoa were exposed to equitoxic suspensions of bulk CuO, Cu-ions or 3,5-DCP. As changes in the UFA:SFA upon exposure of protozoa to nCuO were not detected at 2 h exposure and no simultaneous dose- or time-dependent lipid peroxidation occurred, it is likely that one of the adaptation mechanisms of protozoa to nCuO was lowering membrane fluidity by the inhibition of de novo synthesis of fatty acid desaturases. This is the first study of the effects of nanoparticles on the membrane fatty acid composition.