Defining Quality Criteria for Nanoplastic Hazard Evaluation: The Case of Polystyrene Nanoplastics and Aquatic Invertebrate Daphnia spp.

Polystyrene nanoparticles are the most investigated type of nanoplastics in environmental hazard studies. It remains unclear whether nanoplastic particles pose a hazard towards aquatic organisms. Thus, it was our aim to investigate whether the existing studies and data provided therein are reliable in terms of data completeness. We used the example of Daphnia spp. studies for the purpose of polystyrene nanoplastic (nanoPS) hazard evaluation. First, a set of quality criteria recently proposed for nanoplastic ecotoxicity studies was applied. These rather general criteria for all types of nanoplastics and different test organisms were then, in the second step, tailored and refined specifically for Daphnia spp. and nanoPS. Finally, a scoring system was established by setting mandatory (high importance) as well as desirable (medium importance) criteria and defining a threshold to pass the evaluation. Among the existing studies on nanoPS ecotoxicity for Daphnia spp. (n = 38), only 18% passed the evaluation for usability in hazard evaluation. The few studies that passed the evaluation did not allow for conclusions on the hazard potential of nanoPS because there was no consensus among the studies. The greatest challenge we identified is in data reporting, as only a few studies presented complete data for hazard evaluation.

Multi-generation exposure to polystyrene nanoplastics showed no major adverse effects in Daphnia magna.

Long-term impacts of plastics exposure to organisms, especially to the smallest plastics fraction, nanoplastics (NPs; ≤1 µm), are yet to be fully understood. The data concerning multiple generations are especially rare - an exposure scenario that is the most relevant from the standpoint of environmental reality aspect. Using Pd-doped 200 nm polystyrene NPs, which allowed for quantification of NPs in trace concentrations, the aim of the study was to evaluate the multigenerational impact of NPs for the freshwater crustacean Daphnia magna. Four consecutive 21-day exposures involving F0-F3 generations of D. magna were conducted according to OECD211. NPs impact (at 0.1 mg/L and 1 mg/L) was assessed in parallel to a comparative particle mesoporous SiO2 of similar size and shape (at 1 mg/L) to deconvolute impacts of variable particle chemistry. D. magna mortality, reproductive endpoints, body length (adults and offspring) and lipid content (offspring) were assessed upon NPs and SiO2 exposures. NPs association with adults and offspring was quantified by ICP-MS through the NPs Pd-dopant. The results showed no NPs impact on D. magna at 0.1 mg/L. At 1 mg NPs/L, the only statistically significant effect on adult organisms was increased fertility in the F3 generation. Conversely, SiO2 induced 80% mortality in F3 adult D. magna and the survived adults were significantly smaller and less fertile than those of other treatments. Both particles induced decreased size and lipid content in F3 offspring. The average NPs body burdens (ng NPs/mg D. magna dwt) for the adult and offspring D. magna were 105 ± 12 and 823 ± 440, respectively at 0.1 mg/L exposure and 503 ± 176 and 621 ± 235, respectively at 1 mg/L exposure. Finally, the results of this study add to the previous findings showing that multi-generation exposure to synthetic nano-sized particles of different chemistries may disturb the energy balance of D. magna.

Hazard of polystyrene micro-and nanospheres to selected aquatic and terrestrial organisms.

Plastics contamination in the environment is a major concern. Risk assessment of micro- and nanoplastics (MPL and NPL) poses significant challenges due to MPL and NPL heterogeneity regarding compositional polymers, particle sizes and morphologies in the environment. Yet, there exists considerable toxicological literature on commercial polystyrene (PS) micro- and nanospheres. Although such particles do not directly represent the environmental MPL and NPL, their toxicity data should be used to advance the hazard assessment of plastics. Here, toxicity data of PS micro- and nanospheres for microorganisms, aquatic and terrestrial invertebrates, fish, and higher plants was collected and analyzed. The evaluation of 294 papers revealed that aquatic invertebrates were the most studied organisms, nanosized PS was studied more often than microsized PS, acute exposures prevailed over chronic exposures, the toxicity of PS suspension additives was rarely addressed, and ∼40 % of data indicated no organismal effects of PS. Toxicity mechanisms were mainly studied in fish and nematode Caenorhabditis elegans, providing guidance for relevant studies in higher organisms. Future studies should focus on environmentally relevant plastics concentrations, wide range of organisms, co-exposures with other pollutants, and method development for plastics identification and quantification to fill the gap of bioaccumulation assessment of plastics.

Effects of environmentally relevant concentrations of microplastics on amphipods.

Lack of microplastics (MP) toxicity studies involving environmentally relevant concentrations and exposure times is concerning. Here we analyzed the potential adverse effects of low density polyethylene (LDPE) MP at environmentally relevant concentration in sub-chronic exposure to two amphipods Gmelinoides fasciatus and Gammarus lacustris, species that naturally compete with each other for their habitats. 14-day exposure to 2 µg/L (8 particles/L corresponding to low exposure) and 2 mg/L (∼8400 particles/L, corresponding to high exposure) of 53-100 µm LDPE MP were used to assess ingestion and egestion of MP, evaluate its effects on amphipod mortality, swimming ability and oxidative stress level. Both amphipod species were effectively ingesting and egesting LDPE MP. On the average, 0.8 and 2.5 MP particles were identified in the intestines of each amphipod exposed to 2 µg/L and 2 mg/L LDPE MP, respectively. Therefore, intestinal MP after 14-day exposure did not fully reflect the differences in LDPE MP exposure concentrations. Increased mortality of both amphipods was observed at 2 mg/L LDPE MP and in case of G. lacustris also at 2 µg/L exposure. The effect of LDPE on swimming activity was observed only in case of G. fasciatus. Oxidative stress marker enzymes SOD, GPx and reduced glutathione GSH varied according to amphipod species and LDPE MP concentration. In general G. lacustris was more sensitive towards LDPE MP induced oxidative stress. Overall, the results suggested that in MP polluted environments, G. lacustris may lose its already naturally low competitiveness and become overcompeted by other more resistant species. The fact that in the sub-chronic foodborne exposure to environmentally relevant and higher LDPE MP concentrations all the observed toxicological endpoints were affected refers to the potential of MP to affect and disrupt aquatic communities in the longer perspective.

Seagrass beds reveal high abundance of microplastic in sediments: A case study in the Baltic Sea.

Microplastic (MPL) contamination in the marine environment is extensively studied yet little is known about the extent of MPL abundance in seagrass beds. The aim of this study was to evaluate MPL accumulation in coastal seagrass (Zostera marina) beds in the Baltic Sea, Estonia. Surface water was sampled by pumping using 40 µm plankton net, and sediments by trowel. MPL was extracted with NaCl, identified by microscopy and ATR-FTIR on selected samples. Surface water in the seagrass beds had 0.04-1.2 (median 0.14) MPL/L, similar to other areas of the Baltic Sea. Sediments had 0-1817 (median 208) MPL/kg (dwt), much higher than previously recorded from adjacent unvegetated and offshore sediments, thereby suggesting a strong ability of the sediments in seagrass beds to retain MPL. Of identified MPL, blue fibres were dominant in both the sampled media. Sediment characterization showed a correlation between MPL counts with poorly sorted sediments.

Techniques Used for Analyzing Microplastics, Antimicrobial Resistance and Microbial Community Composition: A Mini-Review.

Antimicrobial resistance (AMR) is a global health threat. Antibiotics, heavy metals, and microplastics are environmental pollutants that together potentially have a positive synergetic effect on the development, persistence, transport, and ecology of antibiotic resistant bacteria in the environment. To evaluate this, a wide array of experimental methods would be needed to quantify the occurrence of antibiotics, heavy metals, and microplastics as well as associated microbial communities in the natural environment. In this mini-review, we outline the current technologies used to characterize microplastics based ecosystems termed "plastisphere" and their AMR promoting elements (antibiotics, heavy metals, and microbial inhabitants) and highlight emerging technologies that could be useful for systems-level investigations of AMR in the plastisphere.

Long Term Exposure to Virgin and Recycled LDPE Microplastics Induced Minor Effects in the Freshwater and Terrestrial Crustaceans Daphnia magna and Porcellio scaber.

The effects of microplastics (MP) are extensively studied, yet hazard data from long-term exposure studies are scarce. Moreover, for sustainable circular use in the future, knowledge on the biological impact of recycled plastics is essential. The aim of this study was to provide long-term toxicity data of virgin vs recycled (mechanical recycling) low density polyethylene (LDPE) for two commonly used ecotoxicity models, the freshwater crustacean Daphnia magna and the terrestrial crustacean Porcellio scaber. LDPE MP was tested as fragments of 39.8 ± 8.82 µm (virgin) and 205 ± 144 µm (recycled) at chronic exposure levels of 1-100 mg LDPE/L (D. magna) and 0.2-15 g LDPE/kg soil (P. scaber). Mortality, reproduction, body length, total lipid content, feeding and immune response were evaluated. With the exception of very low inconsistent offspring mortality at 10 mg/L and 100 mg/L of recycled LDPE, no MP exposure-related adverse effects were recorded for D. magna. For P. scaber, increased feeding on non-contaminated leaves was observed for virgin LDPE at 5 g/kg and 15 g/kg. In addition, both LDPE induced a slight immune response at 5 g/kg and 15 g/kg with more parameters altered for virgin LDPE. Our results indicated different sublethal responses upon exposure to recycled compared to virgin LDPE MP.

Potential Hazard of Lanthanides and Lanthanide-Based Nanoparticles to Aquatic Ecosystems: Data Gaps, Challenges and Future Research Needs Derived from Bibliometric Analysis.

Lanthanides (Ln), applied mostly in the form of nanoparticles (NPs), are critical to emerging high-tech and green energy industries due to their distinct physicochemical properties. The resulting anthropogenic input of Ln and Ln-based NPs into aquatic environment might create a problem of emerging contaminants. Thus, information on the biological effects of Ln and Ln-based NPs is urgently needed for relevant environmental risk assessment. In this mini-review, we made a bibliometric survey on existing scientific literature with the main aim of identifying the most important data gaps on Ln and Ln-based nanoparticles' toxicity to aquatic biota. We report that the most studied Ln for ecotoxicity are Ce and Ln, whereas practically no information was found for Nd, Tb, Tm, and Yb. We also discuss the challenges of the research on Ln ecotoxicity, such as relevance of nominal versus bioavailable concentrations of Ln, and point out future research needs (long-term toxicity to aquatic biota and toxic effects of Ln to bottom-dwelling species).

Hazard evaluation of polystyrene nanoplastic with nine bioassays did not show particle-specific acute toxicity.

Plastic is a wide-spread pollutant and must be evaluated for potential adverse effects of its breakdown product, microplastic (≤5 mm) along with its subfraction, nanoplastic (1-100 nm). Risk assessment of pollutants cannot be conducted without their toxicity (dose-response) data. In this study, toxicity of polystyrene nanoplastics (PS-NPL) was evaluated using 8 acute and 1 subchronic toxicity assays with 10 organisms of different biological complexity (bacteria, yeast, algae, protozoans, mammalian cells in vitro, crustaceans, midge larvae). Commercial 26 and 100 nm carboxylated PS-NPL spheres were chosen as model and tested in nominal concentrations up to 100 mg/L (1.025·1016 26 nm and 1.83·1014 100 nm particles/L). In most of the assays, both PS-NPL proved non-toxic (L(E)C50 > 100 mg/L) but three tests (V. fischeri, R. subcapitata, D. magna) flagged toxicity in 'as received' 26 nm PS-NPL and D. magna also in 100 nm PS-NPL (EC50 ranging from 13 to 71 mg/L). As, according to manufacturers, both PS-NPL suspensions contained additives (surfactants and biocidal NaN3), the three toxicity tests were repeated also on dialysed PS-NPL and on NaN3. Non-toxicity of dialysed PS-NPL indicated that the toxicity of 'as-received' PS-NPL was not particle-specific but false positive due to water-soluble additives in the PS-NPL preparations. NaN3 was very toxic to D. magna (48 h EC50 = 0.05 ± 0.03 mg NaN3/L), toxic to R. subcapitata (72 h EC50 = 4.97 ± 3.7 mg NaN3/L) and non-toxic to V. fischeri. Toxicity of 'as-received' PS-NPL was not fully explainable by NaN3 but also attributable to other additives in the suspensions. Toxicity research of microplastic using commercial model particles must always consider the potential influence of additives, e.g. test the toxicity of dialysed NPL for comparison. In our study, D. magna, R. subcapitata and V. fischeri were the most sensitive to PS-NPL water-soluble additives and flagged their presence in NPL preparations.

Combined Effects of Test Media and Dietary Algae on the Toxicity of CuO and ZnO Nanoparticles to Freshwater Microcrustaceans Daphnia magna and Heterocypris incongruens: Food for Thought.

The chemical composition of the test medium as well as the presence of algae (microcrustaceans' food) affects the bioavailability and thus the toxicity of metal nanoparticles (NP) to freshwater microcrustaceans. This study evaluated the effect of the addition of algae (Rapidocelis subcapitata at 7.5 × 106 cells/mL) on the toxicity of CuO (primary size 22⁻25 nm) and ZnO NP (10⁻15 nm) to planktic Daphnia magna and benthic Heterocypris incongruens in artificial (mineral) and natural freshwater (lake water). The toxicity of ionic controls, CuSO4 and ZnSO4, was evaluated in parallel. When algae were added and the toxicity was tested in mineral medium, 48 h EC50 of CuO and ZnO NP to D. magna was ~2 mg metal/L and 6-day LC50 of H. incongruens was 1.1 mg metal/L for CuO and 0.36 mg metal/L for ZnO. The addition of algae to D. magna test medium mitigated the toxicity of CuO and ZnO NP 4⁻11-fold when the test was conducted in natural water but not in the artificial freshwater. The addition of algae mitigated the toxicity of CuSO4 (but not ZnSO4) to D. magna at least 3-fold, whatever the test medium. In the 6-day H. incongruens tests (all exposures included algae), only up to 2-fold differences in metal NP and salt toxicity between mineral and natural test media were observed. To add environmental relevance to NP hazard assessment for the freshwater ecosystem, toxicity tests could be conducted in natural water and organisms could be fed during the exposure.

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 (0.05 mg Cu/L and 1 mg Cu/L) than in daphnids exposed to equal or equitoxic concentrations (0.025 mg Cu/L and 0.05 mg 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 24 h 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.

Exposure to sublethal concentrations of Co3O4 and Mn2O3 nanoparticles induced elevated metal body burden in Daphnia magna.

Despite the significant progress made in ecotoxicological research on nanoparticles (NPs), there is still very limited information available regarding the biological effects of certain types of NPs such as Co3O4 and Mn2O3. Only a couple of studies provide data on their impact on aquatic organisms whereas, alarmingly, these NPs have been proposed to have high toxicity potential. In addition, more data are needed to determine whether the adverse effects the metal NPs induce on aquatic organisms are rather due to their chemical or particulate nature. To address these open questions, the (sub)lethal effects of Co and Mn NPs in parallel with the respective soluble metal salts on Daphnia magna were studied. The aims of the current study were to i) assess the acute toxicity of Co3O4 and Mn2O3 NPs (primary size 10-30nm) to D. magna, ii) evaluate whether the acute NP exposure at sublethal concentrations influences D. magna post-exposure feeding behaviour and iii) quantify D. magna metal body burden after exposure and after the post-exposure feeding to estimate the potential of trophic transfer of metals. Flow cytometry and total reflection X-ray fluorescence spectroscopy were applied for feeding and metal body burden evaluations, respectively. CuO NPs (primary size 22-25nm) that are very toxic to D. magna were included in the study as a positive control. Since the release of metal ions is an important possibility for toxicity of metal NPs, soluble Co-, Mn- and Cu-salts were analysed in parallel. The solubilisation of Co3O4 NPs in the OECD202 assay conditions was 0.1% and Mn2O3 NPs 35%. Mn2O3 NPs also produced reactive oxygen species in abiotic conditions. However Co3O4 and Mn2O3 NPs were not acutely toxic to D. magna (48h EC50>100mg metal/L) at OECD202 assay conditions. The 48h EC50 values of soluble Co- and Mn-salts were 3.2mgCo/L and 41mgMn/L, respectively. Post-exposure feeding behaviour after 48h exposure to sublethal concentrations (≤10mg/L) of Co3O4 and Mn2O3 NPs differed from that of the unexposed (control) D. magna only at the highest exposure concentrations but was comparable to the feeding behaviour of the respective metal salt-exposed organisms. Upon 48h exposure, dose-dependent increase of D. magna total metal body burden in case of both the NPs and the soluble salts was observed. After 48h post-exposure feeding with algae C. reinhardtii (depuration): D. magna body burden remained elevated (up to 760-fold compared to the control organism) only in case of the NPs. This may indicate potential for trophic transfer of NPs/heavy metals and thus hazard for freshwater ecosystem.

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 (48 h EC50 1-5.5 µg Ag/L) to D. magna as well as to D. rerio embryos (96 h EC50 8.8-61 µg Ag/L) in both standard media and natural waters with results in good agreement between laboratories. CuO NP toxicity to D. magna differed significantly between the laboratories with 48 h EC50 0.9-11 mg Cu/L in standard media, 5.7-75 mg Cu/L in Lake Greifen and 5.5-26 mg Cu/L in Lake Lucerne. No toxicity of CuO NP to zebrafish embryos was detected up to 100 mg/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.

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

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 100 mg/l. The panel of the bioassays yielded the following toxicity order: Ag > ZnO > CuO > TiO2 > MWCNTs > SiO2 > Au. 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 EC50 = 0.003 mg Ag/l), algae Raphidocelis subcapitata (ZnO and CuO, 72-h EC50 = 0.14 mg Zn/l and 0.7 mg Cu/l, respectively) and murine fibroblasts BALB/3T3 (CuO, 48-h EC50 = 0.7 mg Cu/l). MWCNTs showed toxicity only towards rat alveolar macrophages (EC50 = 15.3 mg/l) assumingly due to high aspect ratio and TiO2 towards R. subcapitata (EC50 = 6.8 mg 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).

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.

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.

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.

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.

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.

Toxicity of nanosized and bulk ZnO, CuO and TiO2 to bacteria Vibrio fischeri and crustaceans Daphnia magna and Thamnocephalus platyurus.

As the production of nanoparticles of ZnO, TiO2 and CuO is increasing, their (eco)toxicity to bacteria Vibrio fischeri and crustaceans Daphnia magna and Thamnocephalus platyurus was studied with a special emphasis on product formulations (nano or bulk oxides) and solubilization of particles. Our innovative approach based on the combination of traditional ecotoxicology methods and metal-specific recombinant biosensors allowed to clearly differentiate the toxic effects of metal oxides per se and solubilized metal ions. Suspensions of nano and bulk TiO2 were not toxic even at 20 g l(-1). All Zn formulations were very toxic: L(E)C50 (mg l(-1)) for bulk ZnO, nanoZnO and ZnSO4.7H2O: 1.8, 1.9, 1.1 (V. fischeri); 8.8, 3.2, 6.1 (D. magna) and 0.24, 0.18, 0.98 (T. platyurus), respectively. The toxicity was due to solubilized Zn ions as proved with recombinant Zn-sensor bacteria. Differently from Zn compounds, Cu compounds had different toxicities: L(E)C50 (mg l(-1)) for bulk CuO, nano CuO and CuSO4: 3811, 79, 1.6 (V. fischeri), 165, 3.2, 0,17 (D. magna) and 95, 2.1, 0.11 (T. platyurus), respectively. Cu-sensor bacteria showed that toxicity to V. fischeri and T. platyurus was largely explained by soluble Cu ions. However, for Daphnia magna, nano and bulk CuO proved less bioavailable than for bacterial Cu-sensor. This is the first evaluation of ZnO, CuO and TiO2 toxicity to V. fischeri and T. platyurus. For nano ZnO and nano CuO this is also a first study for D. magna.