Nanna B. Hartmann

Environmental behavior and ecotoxicity of engineered nanoparticles to algae, plants, and fungi

Developments in nanotechnology are leading to a rapid proliferation of new materials that are likely to become a source of engineered nanoparticles (ENPs) to the environment, where their possible ecotoxicological impacts remain unknown. The surface properties of ENPs are of essential importance for their aggregation behavior, and thus for their mobility in aquatic and terrestrial systems and for their interactions with algae, plants and, fungi. Interactions of ENPs with natural organic matter have to be considered as well, as those will alter the ENPs aggregation behavior in surface waters or in soils. Cells of plants, algae, and fungi possess cell walls that constitute a primary site for interaction and a barrier for the entrance of ENPs. Mechanisms allowing ENPs to pass through cell walls and membranes are as yet poorly understood. Inside cells, ENPs might directly provoke alterations of membranes and other cell structures and molecules, as well as protective mechanisms. Indirect effects of ENPs depend on their chemical and physical properties and may include physical restraints (clogging effects), solubilization of toxic ENP compounds, or production of reactive oxygen species. Many questions regarding the bioavailability of ENPs, their uptake by algae, plants, and fungi and the toxicity mechanisms remain to be elucidated.

Toxicity and bioaccumulation of xenobiotic organic compounds in the presence of aqueous suspensions of aggregates of nano-C60

The potential of C(60)-nanoparticles (Buckminster fullerenes) as contaminant carriers in aqueous systems was studied in a series of toxicity tests with algae (Pseudokirchneriella subcapitata) and crustaceans (Daphnia magna). Four common environmental contaminants (atrazine, methyl parathion, pentachlorophenol (PCP), and phenanthrene) were used as model compounds, representing different physico-chemical properties and toxic modes of action. The aggregates of nano-C(60) formed over 2 months of stirring in water were mixed with model compounds 5 days prior to testing. Uptake and excretion of phenanthrene in 4-days-old D. magna was studied with and without addition of C(60) in aqueous suspensions. It was found that 85% of the added phenanthrene sorbed to C(60)-aggregates >200 nm whereas about 10% sorption was found for atrazine, methyl parathion, and pentachlorophenol. In algal tests, the presence of C(60)-aggregates increased the toxicity of phenanthrene with 60% and decreased toxicity of PCP about 1.9 times. Addition of C(60)-aggregates reduced the toxicity of PCP with 25% in tests with D. magna, whereas a more than 10 times increase in toxicity was observed for phenanthrene when results were expressed as water phase concentrations. Thus, results from both toxicity tests show that phenanthrene sorbed to C(60)-aggregates is available for the organisms. For atrazine and methyl parathion no statistically significant differences in toxicities could be observed in algal and daphnid tests as a result of the presence of C(60)-aggregates. In bioaccumulation studies with phenanthrene in D. magna it was found that the uptake of phenanthrene was faster when C(60) was present in suspension and that a 1.7 times higher steady-state concentration was reached in the animals. However, a very fast clearance took place when animals were transferred to clean water resulting in no accumulation of phenanthrene. This study is the first to demonstrate the influence of C(60)-aggregates on aquatic toxicity and bioaccumulation of other environmentally relevant contaminants. The data provided underline that not only the inherent toxicity of manufactured nanoparticles, but also interactions with other compounds and characterisation of nanoparticles in aqueous suspension are of importance for risk assessment of nanomaterials.

Algal testing of titanium dioxide nanoparticles—Testing considerations, inhibitory effects and modification of cadmium bioavailability

The ecotoxicity of three different sizes of titanium dioxide (TiO(2)) particles (primary particles sizes: 10, 30, and 300nm) to the freshwater green alga Pseudokirchneriella subcapitata was investigated in this study. Algal growth inhibition was found for all three particle types, but the physiological mode of action is not yet clear. It was possible to establish a concentration/dose-response relationship for the three particle sizes. Reproducibility, however, was affected by concentration-dependent aggregation of the nanoparticles, subsequent sedimentation, and possible attachment to vessel surfaces. It is also believed that heteroaggregation, driven by algal exopolymeric exudates, is occurring and could influence the concentration-response relationship. The ecotoxicity of cadmium to algae was investigated both in the presence and absence of 2mg/L TiO(2). The presence of TiO(2) in algal tests reduced the observed toxicity due to decreased bioavailability of cadmium resulting from sorption/complexation of Cd(2+) ions to the TiO(2) surface. However, for the 30nm TiO(2) nanoparticles, the observed growth inhibition was greater than what could be explained by the concentration of dissolved Cd(II) species, indicating a possible carrier effect, or combined toxic effect of TiO(2) nanoparticles and cadmium. These results emphasize the importance of systematic studies of nanoecotoxicological effects of different sizes of nanoparticles and underline the fact that, in addition to particle toxicity, potential interactions with existing environmental contaminants are also of crucial importance in assessing the potential environmental risks of nanoparticles.

ITS-NANO - Prioritising nanosafety research to develop a stakeholder driven intelligent testing strategy

BackgroundTo assess the risk of all nanomaterials (NMs) on a case-by-case basis is challenging in terms of financial, ethical and time resources. Instead a more intelligent approach to knowledge gain and risk assessment is required.MethodsA framework of future research priorities was developed from the accorded opinion of experts covering all major stake holder groups (government, industry, academia, funders and NGOs). It recognises and stresses the major topics of physicochemical characterisation, exposure identification, hazard identification and modelling approaches as key components of the current and future risk assessment of NMs.ResultsThe framework for future research has been developed from the opinions of over 80 stakeholders, that describes the research priorities for effective development of an intelligent testing strategy (ITS) to allow risk evaluation of NMs. In this context, an ITS is a process that allows the risks of NMs to be assessed accurately, effectively and efficiently, thereby reducing the need to test NMs on a case-by-case basis.For each of the major topics of physicochemical characterisation, exposure identification, hazard identification and modelling, key-priority research areas are described via a series of stepping stones, or hexagon diagrams structured into a time perspective. Importantly, this framework is flexible, allowing individual stakeholders to identify where their own activities and expertise are positioned within the prioritisation pathway and furthermore to identify how they can effectively contribute and structure their work accordingly. In other words, the prioritisation hexagon diagrams provide a tool that individual stakeholders can adapt to meet their own particular needs and to deliver an ITS for NMs risk assessment. Such an approach would, over time, reduce the need for testing by increasing the reliability and sophistication of in silico approaches.The manuscript includes an appraisal of how this framework relates to the current risk assessment approaches and how future risk assessment could adapt to accommodate these new approaches. A full report is available in electronic format (pdf) at http://www.nano.hw.ac.uk/research-projects/itsnano.html.ConclusionITS-NANO has delivered a detailed, stakeholder driven and flexible research prioritisation (or strategy) tool, which identifies specific research needs, suggests connections between areas, and frames this in a time-perspective.

Comprehensive in vitro toxicity testing of a panel of representative oxide nanomaterials: First steps towards an intelligent testing strategy

Nanomaterials (NMs) display many unique and useful physico-chemical properties. However, reliable approaches are needed for risk assessment of NMs. The present study was performed in the FP7-MARINA project, with the objective to identify and evaluate in vitro test methods for toxicity assessment in order to facilitate the development of an intelligent testing strategy (ITS). Six representative oxide NMs provided by the EC-JRC Nanomaterials Repository were tested in nine laboratories. The in vitro toxicity of NMs was evaluated in 12 cellular models representing 6 different target organs/systems (immune system, respiratory system, gastrointestinal system, reproductive organs, kidney and embryonic tissues). The toxicity assessment was conducted using 10 different assays for cytotoxicity, embryotoxicity, epithelial integrity, cytokine secretion and oxidative stress. Thorough physico-chemical characterization was performed for all tested NMs. Commercially relevant NMs with different physico-chemical properties were selected: two TiO2 NMs with different surface chemistry – hydrophilic (NM-103) and hydrophobic (NM-104), two forms of ZnO – uncoated (NM-110) and coated with triethoxycapryl silane (NM-111) and two SiO2 NMs produced by two different manufacturing techniques – precipitated (NM-200) and pyrogenic (NM-203). Cell specific toxicity effects of all NMs were observed; macrophages were the most sensitive cell type after short-term exposures (24-72h) (ZnO>SiO2>TiO2). Longer term exposure (7 to 21 days) significantly affected the cell barrier integrity in the presence of ZnO, but not TiO2 and SiO2, while the embryonic stem cell test (EST) classified the TiO2 NMs as potentially ‘weak-embryotoxic’ and ZnO and SiO2 NMs as ‘non-embryotoxic’. A hazard ranking could be established for the representative NMs tested (ZnO NM-110 > ZnO NM-111 > SiO2 NM-203 > SiO2 NM-200 > TiO2 NM-104 > TiO2 NM-103). This ranking was different in the case of embryonic tissues, for which TiO2 displayed higher toxicity compared with ZnO and SiO2. Importantly, the in vitro methodology applied could identify cell- and NM-specific responses, with a low variability observed between different test assays. Overall, this testing approach, based on a battery of cellular systems and test assays, complemented by an exhaustive physico-chemical characterization of NMs, could be deployed for the development of an ITS suitable for risk assessment of NMs. This study also provides a rich source of data for modeling of NM effects.

Adapting OECD Aquatic Toxicity Tests for Use with Manufactured Nanomaterials: Key Issues and Consensus Recommendations.

The unique or enhanced properties of manufactured nanomaterials (MNs) suggest that their use in nanoenabled products will continue to increase. This will result in increased potential for human and environmental exposure to MNs during manufacturing, use, and disposal of nanoenabled products. Scientifically based risk assessment for MNs necessitates the development of reproducible, standardized hazard testing methods such as those provided by the Organisation of Economic Cooperation and Development (OECD). Currently, there is no comprehensive guidance on how best to address testing issues specific to MN particulate, fibrous, or colloidal properties. This paper summarizes the findings from an expert workshop convened to develop a guidance document that addresses the difficulties encountered when testing MNs using OECD aquatic and sediment test guidelines. Critical components were identified by workshop participants that require specific guidance for MN testing: preparation of dispersions, dose metrics, the importance and challenges associated with maintaining and monitoring exposure levels, and the need for reliable methods to quantify MNs in complex media. To facilitate a scientific advance in the consistency of nanoecotoxicology test results, we identify and discuss critical considerations where expert consensus recommendations were and were not achieved and provide specific research recommendations to resolve issues for which consensus was not reached. This process will enable the development of prescriptive testing guidance for MNs. Critically, we highlight the need to quantify and properly interpret and express exposure during the bioassays used to determine hazard values.

The potential of TiO2 nanoparticles as carriers for cadmium uptake in Lumbriculus variegatus and Daphnia magna

The use of engineered nanoparticles (e.g. in industrial applications and consumer products) is increasing. Consequently, these particles will be released into the aquatic environment. Through aggregation/agglomeration and sedimentation, sediments are expected ultimately to be sinks for nanoparticles. Both in the water phase and in the sediments engineered nanoparticles will mix and interact with other environmental pollutants, including metals. In this study the toxicity of cadmium to two freshwater organisms, water column crustacean Daphnia magna and sediment oligochaete Lumbriculus variegatus, was investigated both in the absence and presence of titanium dioxide (TiO(2)) nanoparticles (P25 Evonic Degussa, d: 30 nm). The uptake of cadmium in sub-lethal concentrations was also studied in the absence and presence of 2 mg/L TiO(2) nanoparticles. Formation of larger nanoparticles aggregates/agglomerates was observed and sizes varied depending on media composition (358±13 nm in US EPA moderately hard synthetic freshwater and 1218±7 nm in Elendt M7). TiO(2) nanoparticles are potential carriers for cadmium and it was found that 25% and 6% of the total cadmium mass in the test system for L. variegatus and D. magna tests were associated to suspended TiO(2) particles, respectively. μXRF (micro X-ray fluorescence) analysis confirmed the uptake of TiO(2) in the gut of D. magna. For L. variegatus μXRF analysis indicated attachment of TiO(2) nanoparticles to the organism surface as well as a discrete distribution within the organisms. Though exact localisation in this organism was more difficult to assess, the uptake seems to be within the coelomic cavity. Results show that the overall body burden and toxicity of cadmium to L. variegatus was unchanged by addition of TiO(2) nanoparticles, showing that cadmium adsorption to TiO(2) nanoparticles did not affect overall bioavailability. Despite facilitated uptake of cadmium by TiO(2) nanoparticles in D. magna, resulting in increased total cadmium body burden, no change in toxicity was observed.

Techniques and Protocols for Dispersing Nanoparticle Powders in Aqueous Media—Is there a Rationale for Harmonization?

Selecting appropriate ways of bringing engineered nanoparticles (ENP) into aqueous dispersion is a main obstacle for testing, and thus for understanding and evaluating, their potential adverse effects to the environment and human health. Using different methods to prepare (stock) dispersions of the same ENP may be a source of variation in the toxicity measured. Harmonization and standardization of dispersion methods applied in mammalian and ecotoxicity testing are needed to ensure a comparable data quality and to minimize test artifacts produced by modifications of ENP during the dispersion preparation process. Such harmonization and standardization will also enhance comparability among tests, labs, and studies on different types of ENP. The scope of this review was to critically discuss the essential parameters in dispersion protocols for ENP. The parameters are identified from individual scientific studies and from consensus reached in larger scale research projects and international organizations. A step-wise approach is proposed to develop tailored dispersion protocols for ecotoxicological and mammalian toxicological testing of ENP. The recommendations of this analysis may serve as a guide to researchers, companies, and regulators when selecting, developing, and evaluating the appropriateness of dispersion methods applied in mammalian and ecotoxicity testing. However, additional experimentation is needed to further document the protocol parameters and investigate to what extent different stock dispersion methods affect ecotoxicological and mammalian toxicological responses of ENP.

The challenges of testing metal and metal oxide nanoparticles in algal bioassays: titanium dioxide and gold nanoparticles as case studies

Abstract Aquatic toxicology of engineered nanoparticles is challenged by methodological difficulties stemming partly from highly dynamic and poorly understood behavior of nanoparticles in biological test systems. In this paper scientific and technical challenges of testing not readily soluble nanoparticles in standardised algal growth inhibition tests are highlighted with specific focus on biomass quantification methods. This is illustrated through tests with TiO2 and Au nanoparticles, for which cell-nanoparticle interactions and behavior was studied during incubation. Au NP coating layers changed over time and TiO2 nanoparticle aggregation/agglomeration increased as a function of concentration. Three biomass surrogate measuring techniques were evaluated (coulter counting, cell counting in haemocytometer, and fluorescence of pigment extracts) and out of these the fluorometric methods was found to be most suitable. Background correction was identified as a key issue for biomass quantification, complicated by algae-particle interactions and nanoparticle transformation. Optimisation of the method is needed to reduce further particle interference on measurements.

Aquatic Ecotoxicity Testing of Nanoparticles-The Quest To Disclose Nanoparticle Effects.

Abstract The number of products on the market containing engineered nanoparticles (ENPs) has increased significantly, and concerns have been raised regarding their ecotoxicological effects. Environmental safety assessments as well as relevant and reliable ecotoxicological data are required for the safe and sustainable use of ENPs. Although the number of publications on the ecotoxicological effects and uptake of ENPs is rapidly expanding, the applicability of the reported data for hazard assessment is questionable. A major knowledge gap is whether nanoparticle effects occur when test organisms are exposed to ENPs in aquatic test systems. Filling this gap is not straightforward, because of the broad range of ENPs and the different behavior of ENPs compared to “ordinary” (dissolved) chemicals in the ecotoxicity test systems. The risk of generating false negatives, and false positives, in the currently used tests is high, and in most cases difficult to assess. This Review outlines some of the pitfalls in the aquatic toxicity testing of ENPs which may lead to misinterpretation of test results. Response types are also proposed to reveal potential nanoparticle effects in the aquatic test organisms.