Margit Heinlaan

Ecotoxicity of nanoparticles of CuO and ZnO in natural water

The acute toxicity of CuO and ZnO nanoparticles in artificial freshwater (AFW) and in natural waters to crustaceans Daphnia magna and Thamnocephalus platyurus and protozoan Tetrahymena thermophila was compared. The L(E)C(50) values of nanoCuO for both crustaceans in natural water ranged from 90 to 224 mg Cu/l and were about 10-fold lower than L(E)C(50) values of bulk CuO. In all test media, the L(E)C(50) values for both bulk and nanoZnO (1.1-16 mg Zn/l) were considerably lower than those of nanoCuO. The natural waters remarkably (up to 140-fold) decreased the toxicity of nanoCuO (but not that of nanoZnO) to crustaceans depending mainly on the concentration of dissolved organic carbon (DOC). The toxicity of both nanoCuO and nanoZnO was mostly due to the solubilised ions as determined by specific metal-sensing bacteria.

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.

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.

Toxicity of 11 Metal Oxide Nanoparticles to Three Mammalian Cell Types In Vitro.

The knowledge on potential harmful effects of metallic nanomaterials lags behind their increased use in consumer products and therefore, the safety data on various nanomaterials applicable for risk assessment are urgently needed. In this study, 11 metal oxide nanoparticles (MeOx NPs) prepared using flame pyrolysis method were analyzed for their toxicity against human alveolar epithelial cells A549, human epithelial colorectal cells Caco2 and murine fibroblast cell line Balb/c 3T3. The cell lines were exposed for 24 h to suspensions of 3-100 μg/mL MeOx NPs and cellular viability was evaluated using. Neutral Red Uptake (NRU) assay. In parallel to NPs, toxicity of soluble salts of respective metals was analyzed, to reveal the possible cellular effects of metal ions shedding from the NPs. The potency of MeOx to produce reactive oxygen species was evaluated in the cell-free assay. The used three cell lines showed comparable toxicity responses to NPs and their metal ion counterparts in the current test setting. Six MeOx NPs (Al2O3, Fe3O4, MgO, SiO2, TiO2, WO3) did not show toxic effects below 100 µg/mL. For five MeOx NPs, the averaged 24 h IC50 values for the three mammalian cell lines were 16.4 µg/mL for CuO, 22.4 µg/mL for ZnO, 57.3 µg/mL for Sb2O3, 132.3 µg/mL for Mn3O4 and 129 µg/mL for Co3O4. Comparison of the dissolution level of MeOx and the toxicity of soluble salts allowed to conclude that the toxicity of CuO, ZnO and Sb2O3 NPs was driven by release of metal ions. The toxic effects of Mn3O4 and Co3O4 could be attributed to the ROS-inducing ability of these NPs. All the NPs were internalized by the cells according to light microscopy studies but also proven by TEM, and internalization of Co3O4 NPs seemed to be most prominent in this aspect. In conclusion, this work provides valuable toxicological data for a library of 11 MeOx NPs. Combining the knowledge on toxic or non-toxic nature of nanomaterials may be used for safe-by-design approach.

An interlaboratory comparison of nanosilver characterisation and hazard identification: harmonising techniques for high quality data

Within the FP7 EU project NanoValid a consortium of six partners jointly investigated the hazard of silver nanoparticles (AgNPs) paying special attention to methodical aspects that are important for providing high-quality ecotoxicity data. Laboratories were supplied with the same original stock dispersion of AgNPs. All partners applied a harmonised procedure for storage and preparation of toxicity test suspensions. Altogether ten different toxicity assays with a range of environmentally relevant test species from different trophic levels were conducted in parallel to AgNP characterisation in the respective test media. The paper presents a comprehensive dataset of toxicity values and AgNP characteristics like hydrodynamic sizes of AgNP agglomerates and the share (%) of Ag(+)-species (the concentration of Ag(+)-species in relation to the total measured concentration of Ag). The studied AgNP preparation (20.4±6.8 nm primary size, mean total Ag concentration 41.14 mg/L, 46-68% of soluble Ag(+)-species in stock, 123.8±12.2 nm mean z-average value in dH2O) showed extreme toxicity to crustaceans Daphnia magna, algae Pseudokirchneriella subcapitata and zebrafish Danio rerio embryos (EC50<0.01 mg total Ag/L), was very toxic in the in vitro assay with rainbow trout Oncorhynchus mykiss gut cells (EC50: 0.01-1 mg total Ag/L); toxic to bacteria Vibrio fischeri, protozoa Tetrahymena thermophila (EC50: 1-10 mg total Ag/L) and harmful to marine crustaceans Artemia franciscana (EC50: 10-100 mg total Ag/L). Along with AgNPs, also the toxicity of AgNO3 was analyzed. The toxicity data revealed the same hazard ranking for AgNPs and AgNO3 (i.e. the EC50 values were in the same order of magnitude) proving the importance of soluble Ag(+)-species analysis for predicting the hazard of AgNPs. The study clearly points to the need for harmonised procedures for the characterisation of NMs. Harmonised procedures should consider: (i) measuring the AgNP properties like hydrodynamic size and metal ions species in each toxicity test medium at a range of concentrations, and (ii) including soluble metal salt control both in toxicity testing as well as in Ag(+)-species measurements. The present study is among the first nanomaterial interlaboratory comparison studies with the aim to improve the hazard identification testing protocols.

Multilaboratory evaluation of 15 bioassays for (eco)toxicity screening and hazard ranking of engineered nanomaterials: FP7 project NANOVALID

Abstract Within EU FP7 project NANOVALID, the (eco)toxicity of 7 well-characterized engineered nanomaterials (NMs) was evaluated by 15 bioassays in 4 laboratories. The highest tested nominal concentration of NMs was 100u2009mg/l. The panel of the bioassays yielded the following toxicity order: Agu2009>u2009ZnOu2009>u2009CuOu2009>u2009TiO2u2009>u2009MWCNTsu2009>u2009SiO2u2009>u2009Au. Ag, ZnO and CuO proved very toxic in the majority of assays, assumingly due to dissolution. The latter was supported by the parallel analysis of the toxicity of respective soluble metal salts. The most sensitive tests/species were Daphnia magna (towards Ag NMs, 24-h EC50u2009=u20090.003u2009mg Ag/l), algae Raphidocelis subcapitata (ZnO and CuO, 72-h EC50u2009=u20090.14u2009mg Zn/l and 0.7u2009mg Cu/l, respectively) and murine fibroblasts BALB/3T3 (CuO, 48-h EC50u2009=u20090.7u2009mg Cu/l). MWCNTs showed toxicity only towards rat alveolar macrophages (EC50u2009=u200915.3u2009mg/l) assumingly due to high aspect ratio and TiO2 towards R. subcapitata (EC50u2009=u20096.8u2009mg Ti/l) due to agglomeration of TiO2 and entrapment of algal cells. Finally, we constructed a decision tree to select the bioassays for hazard ranking of NMs. For NM testing, we recommend a multitrophic suite of 4 in vitro (eco)toxicity assays: 48-h D. magna immobilization (OECD202), 72-h R. subcapitata growth inhibition (OECD201), 30-min Vibrio fischeri bioluminescence inhibition (ISO2010) and 48-h murine fibroblast BALB/3T3 neutral red uptake in vitro (OECD129) representing crustaceans, algae, bacteria and mammalian cells, respectively. Notably, our results showed that these assays, standardized for toxicity evaluation of “regular” chemicals, proved efficient also for shortlisting of hazardous NMs. Additional assays are recommended for immunotoxicity evaluation of high aspect ratio NMs (such as MWCNTs).

Natural water as the test medium for Ag and CuO nanoparticle hazard evaluation: An interlaboratory case study

Engineered nanoparticles (NPs) have realistic potential of reaching natural waterbodies and of exerting toxicity to freshwater organisms. The toxicity may be influenced by the composition of natural waters as crucial NP properties are influenced by water constituents. To tackle this issue, a case study was set up in the framework of EU FP7 NanoValid project, performing an interlaboratory hazard evaluation of NPs in natural freshwater. Ag and CuO NPs were selected as model NPs because of their potentially high toxicity in the freshwater. Daphnia magna (OECD202) and Danio rerio embryo (OECD236) assays were used to evaluate NP toxicity in natural water, sampled from Lake Greifen and Lake Lucerne (Switzerland). Dissolution of the NPs was evaluated by ultrafiltration, ultracentrifugation and metal specific sensor bacteria. Ag NP size was stable in natural water while CuO NPs agglomerated and settled rapidly. Ag NP suspensions contained a large fraction of Ag(+) ions and CuO NP suspensions had low concentration of Cu(2+) ions. Ag NPs were very toxic (48xa0h EC50 1-5.5xa0μg Ag/L) to D.xa0magna as well as to D.xa0rerio embryos (96xa0h EC50 8.8-61xa0μg Ag/L) in both standard media and natural waters with results in good agreement between laboratories. CuO NP toxicity to D.xa0magna differed significantly between the laboratories with 48xa0h EC50 0.9-11xa0mg Cu/L in standard media, 5.7-75xa0mg Cu/L in Lake Greifen and 5.5-26xa0mg Cu/L in Lake Lucerne. No toxicity of CuO NP to zebrafish embryos was detected up to 100xa0mg/L independent of the medium used. The results show that Ag and CuO NP toxicity may be higher in natural water than in the standard media due to differences in composition. NP environmental hazard evaluation can and should be carried out in natural water to obtain more realistic estimates on the toxicity.

Evaluation of the effect of test medium on total Cu body burden of nano CuO-exposed Daphnia magna: A TXRF spectroscopy study

Toxicity of Cu and Cu-based nanoparticles (NPs) to aquatic biota is usually mitigated in natural freshwater compared to organics-free artificial freshwater. The main aim of this study was to evaluate whether mitigated toxicity is accompanied by lower total copper body burden in the freshwater crustacean Daphnia magna and whether CuO NPs are more hazardous in this aspect than soluble Cu salts. Total copper body burden in different media (OECD202 artificial freshwater and two natural freshwaters) was measured by a relatively novel technique - total reflection X-ray fluorescence (TXRF) spectroscopy - which proved suitable for the analysis of individual juvenile daphnids. Mean copper body burden was 2.8-42 times higher in daphnids exposed to CuO NPs (0.05xa0mg Cu/L and 1xa0mg Cu/L) than in daphnids exposed to equal or equitoxic concentrations (0.025xa0mg Cu/L and 0.05xa0mg Cu/L) of CuSO4. Using natural freshwater instead of artificial one resulted in increased copper burden after exposure to CuO NPs but not after exposure to Cu salt. After 24xa0h post-exposure depuration in the presence of algae Raphidocelis subcapitata, total copper body burden in daphnids exposed to CuO NPs sharply decreased while in daphnids exposed to Cu salt it did not. Despite the CuO NP toxicity mitigating effect of natural freshwater, total copper body burden of aquatic crustaceans in natural waterbodies may be greater than could be predicted based on the results obtained using artificial freshwater as the test medium.