(Re)Conceptualizing decision-making tools in a risk governance framework for emerging technologies-the case of nanomaterials.

The utility of decision-making tools for the risk governance of nanotechnology is at the core of this paper. Those working in nanotechnology risk management have been prolific in creating such tools, many derived from European FP7 and H2020-funded projects. What is less clear is how such tools might assist the overarching ambition of creating a fair system of risk governance. In this paper, we reflect upon the role that tools might and should play in any system of risk governance. With many tools designed for the risk governance of this emerging technology falling into disuse, this paper provides an overview of extant tools and addresses their potential shortcomings. We also posit the need for a data readiness tool. With the EUs NMP13 family of research consortia about to report to the Commission on ways forward in terms of risk governance of this domain, this is a timely intervention on an important element of any risk governance system.

Analytical and toxicological aspects of nanomaterials in different product groups: Challenges and opportunities.

The widespread integration of engineered nanomaterials into consumer and industrial products creates new challenges and requires innovative approaches in terms of design, testing, reliability, and safety of nanotechnology. The aim of this review article is to give an overview of different product groups in which nanomaterials are present and outline their safety aspects for consumers. Here, release of nanomaterials and related analytical challenges and solutions as well as toxicological considerations, such as dose-metrics, are discussed. Additionally, the utilization of engineered nanomaterials as pharmaceuticals or nutraceuticals to deliver and release cargo molecules is covered. Furthermore, critical pathways for human exposure to nanomaterials, namely inhalation and ingestion, are discussed in the context of risk assessment. Analysis of NMs in food, innovative medicine or food contact materials is discussed. Specific focus is on the presence and release of nanomaterials, including whether nanomaterials can migrate from polymer nanocomposites used in food contact materials. With regard to the toxicology and toxicokinetics of nanomaterials, aspects of dose metrics of inhalation toxicity as well as ingestion toxicology and comparison between in vitro and in vivo conclusions are considered. The definition of dose descriptors to be applied in toxicological testing is emphasized. In relation to potential exposure from different products, opportunities arising from the use of advanced analytical techniques in more unique scenarios such as release of nanomaterials from medical devices such as orthopedic implants are addressed. Alongside higher product performance and complexity, further challenges regarding material characterization and safety, as well as acceptance by the general public are expected.

Leaching of Titanium Dioxide Nanomaterials from Agricultural Soil Amended with Sewage Sludge Incineration Ash: Comparison of a Pilot Scale Simulation with Standard Laboratory Column Elution Experiments.

Nanoscale titanium dioxide (nTiO2 (Hombikat UV 100 WP)) was applied to sewage sludge that was incinerated in a large-scale waste treatment plant. The incineration ash produced was applied to soil as fertilizer at a realistic rate of 5% and investigated in pilot plant simulations regarding its leaching behavior for nTiO2. In parallel, the applied soil material was subject to standard column leaching (DIN 19528) in order to test the suitability of the standard to predict the leaching of nanoscale contaminants from treated soil material. Relative to the reference material (similar composition but without nTiO2 application before incineration) the test material had a total TiO2 concentration, increased by a factor of two or 3.8 g/kg, respectively. In contrast, the TiO2 concentration in the respective leachates of the simulation experiment differed by a factor of around 25 (maximum 91.24 mg), indicating that the added nTiO2 might be significantly mobilisable. Nanoparticle specific analysis of the leachates (spICP-MS) confirmed this finding. In the standard column elution experiment the released amount of TiO2 in the percolates between test and reference material differed by a factor of 4 to 6. This was also confirmed for the nTiO2 concentrations in the percolates. Results demonstrate that the standard column leaching, developed and validated for leaching prediction of dissolved contaminants, might be also capable to indicate increased mobility of nTiO2 in soil materials. However, experiments with further soils are needed to verify those findings.

Ecotoxicity and fate of silver nanomaterial in an outdoor lysimeter study after twofold application by sewage sludge.

The increasing use of antibacterial silver nanomaterials (AgNM) in consumer products leads to their release into sewers. High amounts of AgNM become retained in sewage sludge, which causes their accumulation in agricultural soils when sewage sludge is applied as fertilizer. This increase in AgNM arouses concerns about toxicity to soil organisms and transfer within trophic levels. Long-term field studies simulating the sewage sludge pathway to soils are sparse, and the effects of a second sewage sludge application are unknown. In this perennial field lysimeter study, a twofold application of AgNM (NM-300K, 2 + 3 mg AgNM/kg dry matter soil (DMS)) and a onefold application of silver nitrate (AgNO3, 2 mg Ag/kg DMS) by sewage sludge to the uppermost 20 cm of the soil (Cambisol) were applied. The response of microorganisms to the applications was determined by measuring the inhibition of ammonium-oxidizing bacteria (AOB). Silver concentration in soil, leachates, and crops were measured after acid digestion by inductively coupled plasma mass spectrometry (ICP-MS). Almost no vertical Ag translocation to deeper soil layers and negligible Ag release to leachates suggest that soil is a large sink for AgNM and AgNO3. For AgNM, an increase in toxicity to AOB was shown after the second sewage sludge application. The application of AgNO3 resulted in long-term toxicity comparable to the toxicity of AgNM. Low root uptake from both AgNM- and AgNO3-spiked lysimeters to crops indicates their incomplete immobilization, which is why food chain uptake cannot completely be excluded. However, the root-shoot barrier for wheat (9.8 → 0.1 mg/kg) and skin body barrier for sugar beets (1.0 → 0.2 mg/kg) will further reduce the accumulation within trophic levels. Moreover, the applied AgNM concentration was above the predicted environmental concentration, which is why the root uptake might be negligible in agricultural practice.

Nanopharmaceuticals (Au-NPs) after use: Experiences with a complex higher tier test design simulating environmental fate and effect.

The current environmental hazard assessment is based on the testing of the pristine substance. However, it cannot be excluded that (nano)pharmaceuticals are excreted into sewage during the use phase followed by entry into wastewater treatment plants (WWTPs). Sorption to sewage sludge or release via effluent can result in modified ecotoxicological effects which possibly can only be detected with a modified test approach. The objective of our study was to investigate a realistic exposure scenario for metallic nanoparticles (NPs) in pharmaceutical products, excreted into effluent, and released into the environment after treatment in WWTPs. The test approach was illustrated by using gold (Au) NPs. Effluent from model WWTPs were investigated in aquatic tests (Daphnia magna, fish cell lines). Sewage sludge was used as a sole food source (Eisenia fetida) or mixed with soil and used as test medium (soil microorganisms, Folsomia candida, Enchytraeus crypticus). To cover the aspect of regulation, the test systems described in OECD-test guidelines (OECD TG 201, 211, 220, 232, 249, 317) were applied. Modifications and additional test approaches were included to meet the needs arising out of the testing of nanomaterials and of the exposure scenarios. The results were assessed regarding the suitability of the test design and the toxicity of Au-NPs. Except for activated sludge as a sole food source for E.fetida, the selected test approach is suitable for the testing of nanomaterials. Additional information can be gained when compared to the common testing of the pristine nanomaterials in the standardized test systems. Effects of Au-NPs were observed in concentrations exceeding the modeled environmental.

Development of an Alternative Test System for Chronic Testing of Lotic Macroinvertebrate Species: A Case Study with the Insecticide Imidacloprid.

There are currently few suitable test systems for the chronic toxicity testing of aquatic macroinvertebrates under stream conditions. Therefore, a new test system mimicking running water conditions was developed for testing with lotic insects. This system uses small test cages, with 10 of these suspended inside each 25-L container and rotating at 0.1 m/s, to create a water flow for the individual organism inside each cage. To test the performance of the new exposure system, chronic effects (21 d) of the neonicotinoid imidacloprid were investigated with field-collected larvae of the stonefly Protonemura sp. Endpoints were survival, growth, and/or emergence (depending on the developmental stage of the larvae at the start of the exposure). Two experiments conducted 1 yr apart showed good reproducibility: growth 10% effect concentration (EC10) values were 15.3 and 18.5 µg/L and no-observed-effect concentration (NOEC) values were 30.3 and 21.5 µg/L. A third experiment, performed with further-developed larval instars, showed a significant effect of imidacloprid on emergence (with EC10 of 5.97 µg/L and NOEC of 2.89 µg/L) and a significant effect on survival (with median lethal concentration of 44.7 µg/L). The results of the present study show that the newly developed test system provides a suitable approach for toxicity testing with stonefly larvae and potentially for other lotic macroinvertebrate species. Environ Toxicol Chem 2021;40:2229-2239. © 2021 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Microbial Population Dynamics in Model Sewage Treatment Plants and the Fate and Effect of Gold Nanoparticles.

Adequate functioning of a sewage treatment plant (STP) is essential to protect the downstream aquatic environment (ECHA 2017), and information on the degradability of chemicals and their toxicity to activated sludge microorganisms is required. An environmental realistic higher tier test is a STP simulation test as described in OECD 303A (2001) which for nanoparticles can also be used to study their sorption behavior to activated sludge. However, information is limited on the influence of synthetic sewage on the microbial community of the activated sludge. A modified community can result in modifications of the sludge floccules affecting the sorption behavior. The main objective of our study was to show whether a representative microbial diversity remains under standardized test conditions as described in OECD 303A (2001) using synthetic sewage as influent. Furthermore, we investigated whether just considering the functional properties of a STP (elimination of dissolved organic carbon; nitrification), is sufficient for an assessment of gold nanoparticles (AuNPs) or whether the influence on microbial diversity also needs to be considered. AuNPs were used as a case study due to their rising medical applications and therefore increasing probability to reach the sewer and STP. The results can provide significant input for the interpretation of results from the regulatory point of view. To deliver these objectives, the general changes of the microbial population in activated sludge and its influence on the degradation activity (dissolved organic carbon (DOC) and inorganic nitrogen) using freshly collected sludge from the municipal STP in an artificial test system as a model STP in accordance with OECD 303A (2001) were assessed. Additionally, we evaluated the potential impact of AuNPs and its dispersant on the microbial composition and the overall impact on the function of the STP in terms of DOC degradation and nitrogen removal to observe if an assessment based on functional properties is sufficient. The bacteria composition in our study, evaluated at a class level, revealed commonly described environmental bacteria. Proteobacteria (ß, α, δ) accounted for more than 50% but also nitrifying bacteria as Nitrospira were present. Our results show that mainly within the first 7 days of an acclimatization phase by addition of synthetic sewage, the bacterial community changed. Even though AuNPs can have antibacterial properties, no adverse effects on the function and structure of the microorganisms in the STP could be detected at concentrations of increased modeled PEC values by a factor of about 10,000. Complementary to other metallic nanomaterials, gold nanomaterials also sorb to a large extent to the activated sludge. If activated sludge is used as fertilizer on agricultural land, gold nanoparticles can be introduced into soils. In this case, the effect on soil (micro)organisms must be investigated more closely, also taking into account the structural diversity.

Attachment Efficiency of Nanomaterials to Algae as an Important Criterion for Ecotoxicity and Grouping.

Engineered nanomaterials (ENMs) based on CeO2 and TiO2 differ in their effects on the unicellular green alga Raphidocelis subcapitata but these effects do not reflect the physicochemical parameters that characterize such materials in water and other test media. To determine whether interactions with algae can predict the ecotoxicity of ENMs, we studied the attachment of model compounds (three subtypes of CeO2 and five subtypes of TiO2) to algal cells by light microscopy and electron microscopy. We correlated our observations with EC50 values determined in growth inhibition assays carried out according to the Organisation for Economic Co-operation and Development (OECD) test guideline 201. Light microscopy revealed distinct patterns of ENM attachment to algal cells according to the type of compound, with stronger interactions leading to greater toxicity. This was confirmed by electron microscopy, which allowed the quantitative assessment of particle attachment. Our results indicate that algal extracellular polymeric substances play an important role in the attachment of ENMs, influencing the formation of agglomerates. The attachment parameters in short-term tests predicted the toxicity of CeO2 and TiO2 ENMs and can be considered as a valuable tool for the identification of sets of similar nanoforms as requested by the European Chemicals Agency in the context of grouping and read-across.

The nanoGRAVUR framework to group (nano)materials for their occupational, consumer, environmental risks based on a harmonized set of material properties, applied to 34 case studies.

The project nanoGRAVUR (BMBF, 2015-2018) developed a framework for grouping of nanomaterials. Different groups may result for each of the three distinct perspectives of occupational, consumer and environmental safety. The properties, methods and descriptors are harmonised between the three perspectives and are based on: Tier 1 intrinsic physico-chemical properties (what they are) or GHS classification of the non-nano-form (human tox, ecotox, physical hazards); Tier 2 extrinsic physico-chemical properties, release from nano-enabled products, in vitro assays with cells (where they go; what they do); Tier 3 case-specific tests, potentially in vivo studies to substantiate the similarity within groups or application-specific exposure testing. Amongst all properties, dissolution and transformation are least modulated by different nanoforms within one substance, whereas dustiness, dispersion stability, abiotic and especially in vitro surface reactivity vary more often between different nanoforms. The methods developed or selected by nanoGRAVUR fill several gaps highlighted in the ProSafe reviews, and are useful to implement (i) the concept of nanoforms of the European Chemicals Agency (ECHA) and (ii) the concept of discrete forms of the United States Environmental Protection Agency (EPA). One cannot assess the significance of a dissimilarity, if the dynamic range of that property is unknown. Benchmark materials span dynamic ranges that enable us to establish bands, often with order-of-magnitude ranges. In 34 case studies we observed high biological similarity within each substance when we compared different (nano)forms of SiO2, BaSO4, kaolin, CeO2, ZnO, organic pigments, especially when we compared forms that are all untreated on the surface. In contrast, different Fe2O3 or TiO2 (nano)forms differ more significantly. The same nanoforms were also integrated in nano-enabled products (NEPs) for automotive coatings, clinker-reduced cements, cosmetic sunscreen, and lightweight polymers.

Long-term effects of three different silver sulfide nanomaterials, silver nitrate and bulk silver sulfide on soil microorganisms and plants.

Silver nanomaterials (AgNMs) are released into sewers and consequently find their way to sewage treatment plants (STPs). The AgNMs are transformed en route, mainly into silver sulfide (Ag2S), which is only sparingly soluble in water and therefore potentially less harmful than the original AgNMs. Here we investigated the toxicity and fate of different sulfidized AgNMs using an exposure scenario involving the application of five different test materials (NM-300K, AgNO3, Ag2S NM-300K, Ag2S NM and bulk Ag2S) into a simulated STP for 10 days. The sewage sludge from each treatment was either dewatered or anaerobically digested for 35 days and then mixed into soil. We then assessed the effect on soil microorganisms over the next 180 days. After 60 days, a subsample of each test soil was used to assess chronic toxicity in oat plants (Avena sativa L) and a potential uptake into the plants. The effect of each AgNM on the most sensitive test organism was also tested without the application of sewage sludge. Although Ag sulfidized species are considered poorly soluble and barely bioavailable, we observed toxic effects on soil microorganisms. Furthermore, whether or not the AgNM was sulfidized before or during the passage through the STP, comparable effects were observed on ammonium oxidizing bacteria after sewage sludge application and incubation for 180 days. We observed the uptake of Ag into oat roots following the application of all test substances, confirming their bioavailability. The oat shoots generally containing less Ag than the roots.

Silver nanoparticles in sewage treatment plant effluents: chronic effects and accumulation of silver in the freshwater amphipod Hyalella azteca.

BACKGROUND: Increasing amounts of engineered nanoparticles (NPs) in wastewater can reach the aquatic environment by passing through the sewage treatment plant (STP). NPs can induce ecotoxicological effects due to their specific chemical properties. However, their bioavailability and toxicity are potentially influenced by transformation processes caused by substances present in the STP, e.g., humic acids or sulfides. Due to the lack of a test system allowing to test NPs under realistic environmental conditions, we coupled two existing test systems, the activated sludge simulation test (OECD TG 303A 2001) and the chronic exposure test with the freshwater amphipod Hyalella azteca (Environment Canada 2013), to gain a test scenario that allows to consider the altered behavior and fate of NPs induced by the STP process. This should improve the environmental realism of the chronic exposure test with Hyalella. In the first study, we tested the STP effluent containing AgNPs. In the second and third study, tap water and control STP effluent were spiked with AgNPs and used as test media. RESULTS: The chronic exposure studies with the freshwater amphipod H. azteca showed that the investigated AgNPs lose most of their toxicity while passing through the STP. Over all studies with total Ag concentrations ranging from 0.85 to 68.70 µg/L, significant effects of the AgNPs were only observed in the survival of test animals exposed to tap water containing the highest Ag concentration (62.59 µg/L). Accumulation of silver in the body of test animals was clearly dependent on the pretreatment of the AgNPs. Silver ions (Ag+) released from AgNPs are supposed to be the major pathway leading to body burden following exposure to test media containing AgNPs. CONCLUSION: The coupled test system is suitable for testing substances that can reach the environment via the STP effluent. The investigated AgNPs lose most of their toxicity while passing through the STP. Accumulation of silver in the animals exposed to the different treatments was apparent, whereby silver ions (Ag+) released from AgNPs were supposed to be the major pathway leading to body burden.

Environmental Impacts by Fragments Released from Nanoenabled Products: A Multiassay, Multimaterial Exploration by the SUN Approach.

Nanoenabled products (NEPs) have numerous outdoor uses in construction, transportation or consumer scenarios, and there is evidence that their fragments are released in the environment at low rates. We hypothesized that the lower surface availability of NEPs fragment reduced their environmental effects with respect to pristine nanomaterials. This hypothesis was explored by testing fragments generated by intentional micronisation ("the SUN approach"; Nowack et al. Meeting the Needs for Released Nanomaterials Required for Further Testing: The SUN Approach. Environmental Science & Technology, 2016 (50), 2747). The NEPs were composed of four matrices (epoxy, polyolefin, polyoxymethylene, and cement) with up to 5% content of three nanomaterials (carbon nanotubes, iron oxide, and organic pigment). Regardless of the type of nanomaterial or matrix used, it was observed that nanomaterials were only partially exposed at the NEP fragment surface, indicating that mostly the intrinsic and extrinsic properties of the matrix drove the NEP fragment toxicity. Ecotoxicity in multiple assays was done covering relevant media from terrestrial to aquatic, including sewage treatment plant (biological activity), soil worms (Enchytraeus crypticus), and fish (zebrafish embryo and larvae and trout cell lines). We designed the studies to explore the possible modulation of ecotoxicity by nanomaterial additives in plastics/polymer/cement, finding none. The results support NEPs grouping by the matrix material regarding ecotoxicological effect during the use phase. Furthermore, control results on nanomaterial-free polymer fragments representing microplastic had no significant adverse effects up to the highest concentration tested.

Nanomaterials: certain aspects of application, risk assessment and risk communication.

Development and market introduction of new nanomaterials trigger the need for an adequate risk assessment of such products alongside suitable risk communication measures. Current application of classical and new nanomaterials is analyzed in context of regulatory requirements and standardization for chemicals, food and consumer products. The challenges of nanomaterial characterization as the main bottleneck of risk assessment and regulation are presented. In some areas, e.g., quantification of nanomaterials within complex matrices, the establishment and adaptation of analytical techniques such as laser ablation inductively coupled plasma mass spectrometry and others are potentially suited to meet the requirements. As an example, we here provide an approach for the reliable characterization of human exposure to nanomaterials resulting from food packaging. Furthermore, results of nanomaterial toxicity and ecotoxicity testing are discussed, with concluding key criteria such as solubility and fiber rigidity as important parameters to be considered in material development and regulation. Although an analysis of the public opinion has revealed a distinguished rating depending on the particular field of application, a rather positive perception of nanotechnology could be ascertained for the German public in general. An improvement of material characterization in both toxicological testing as well as end-product control was concluded as being the main obstacle to ensure not only safe use of materials, but also wide acceptance of this and any novel technology in the general public.

Environmental Risk Assessment Strategy for Nanomaterials.

An Environmental Risk Assessment (ERA) for nanomaterials (NMs) is outlined in this paper. Contrary to other recent papers on the subject, the main data requirements, models and advancement within each of the four risk assessment domains are described, i.e., in the: (i) materials, (ii) release, fate and exposure, (iii) hazard and (iv) risk characterisation domains. The material, which is obviously the foundation for any risk assessment, should be described according to the legislatively required characterisation data. Characterisation data will also be used at various levels within the ERA, e.g., exposure modelling. The release, fate and exposure data and models cover the input for environmental distribution models in order to identify the potential (PES) and relevant exposure scenarios (RES) and, subsequently, the possible release routes, both with regard to which compartment(s) NMs are distributed in line with the factors determining the fate within environmental compartment. The initial outcome in the risk characterisation will be a generic Predicted Environmental Concentration (PEC), but a refined PEC can be obtained by applying specific exposure models for relevant media. The hazard information covers a variety of representative, relevant and reliable organisms and/or functions, relevant for the RES and enabling a hazard characterisation. The initial outcome will be hazard characterisation in test systems allowing estimating a Predicted No-Effect concentration (PNEC), either based on uncertainty factors or on a NM adapted version of the Species Sensitivity Distributions approach. The risk characterisation will either be based on a deterministic risk ratio approach (i.e., PEC/PNEC) or an overlay of probability distributions, i.e., exposure and hazard distributions, using the nano relevant models.

Long-term effects of sulfidized silver nanoparticles in sewage sludge on soil microflora.

The use of silver nanoparticles (AgNPs) in consumer products such as textiles leads to their discharge into wastewater and consequently to a transfer of the AgNPs to soil ecosystems via biosolids used as fertilizer. In urban wastewater systems (e.g., sewer, wastewater treatment plant [WWTP], anaerobic digesters) AgNPs are efficiently converted into sparingly soluble silver sulfides (Ag2 S), mitigating the toxicity of the AgNPs. However, long-term studies on the bioavailability and effects of sulfidized AgNPs on soil microorganisms are lacking. Thus we investigated the bioavailability and long-term effects of AgNPs (spiked in a laboratory WWTP) on soil microorganisms. Before mixing the biosolids into soil, the sludges were either anaerobically digested or directly dewatered. The effects on the ammonium oxidation process were investigated over 140 d. Transmission electron microscopy (TEM) suggested an almost complete sulfidation of the AgNPs analyzed in all biosolid samples and in soil, with Ag2 S predominantly detected in long-term incubation experiments. However, despite the sulfidation of the AgNPs, soil ammonium oxidation was significantly inhibited, and the degree of inhibition was independent of the sludge treatment. The results revealed that AgNPs sulfidized under environmentally relevant conditions were still bioavailable to soil microorganisms. Consequently, Ag2 S may exhibit toxic effects over the long term rather than the short term. Environ Toxicol Chem 2017;36:3305-3313. © 2017 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals, Inc. on behalf of SETAC.

Ecotoxicity and fate of a silver nanomaterial in an outdoor lysimeter study.

Sewage sludge is repeatedly applied as fertilizer on farmland due to its high nutrient content. This may lead to a significant increase of silver nanomaterials (AgNM) in soil over years. Therefore, our aim was to investigate the ecotoxicity and fate of AgNM under environmentally relevant conditions in outdoor lysimeters over 25 months. Two AgNM concentrations (1.7 and 8.0 mg/kg dry matter soil) were applied via sewage sludge into soil. In subsamples of the soil, incubated under laboratory conditions for 180 days, the comparability of outdoor and laboratory results regarding ecotoxicity was determined. The results from our long term lysimeter experiments show no detectable horizontal displacement in combination with very low remobilization to the percolate water. Thus, indicate that the sludge applied AgNM remains nearly immobile in the pathway between soils and leachate. However, Ag uptake to the roots of wheat and canola suggests that the chemical conditions in the rhizosphere induce AgNM remobilization from the incorporated sewage sludge even after two harvesting cycles. At the higher AgNM concentration a steady inhibition of the soil microflora was observed over 25 month in the lysimeter study, while there was no effect at the lower AgNM concentration. The results of the laboratory experiment reflect the findings of the lysimeter study and indicate that a risk assessment for AgNM based on data from laboratory tests is acceptable.

Regulatory ecotoxicity testing of nanomaterials - proposed modifications of OECD test guidelines based on laboratory experience with silver and titanium dioxide nanoparticles.

Regulatory ecotoxicity testing of chemicals is of societal importance and a large effort is undertaken at the OECD to ensure that OECD test guidelines (TGs) for nanomaterials (NMs) are available. Significant progress to support the adaptation of selected TGs to NMs was achieved in the context of the project MARINA ( http://www.marina-fp7.eu/ ) funded within the 7th European Framework Program. Eight OECD TGs were adapted based on the testing of at least one ion-releasing NM (Ag) and two inert NMs (TiO2). With the materials applied, two main variants of NMs (ion releasing vs. inert NMs) were addressed. As the modifications of the test guidelines refer to general test topics (e.g. test duration or measuring principle), we assume that the described approaches and modifications will be suitable for the testing of further NMs with other chemical compositions. Firm proposals for modification of protocols with scientific justification(s) are presented for the following tests: growth inhibition using the green algae Raphidocelis subcapitata (formerly: Pseudokirchneriella subcapitata; TG 201), acute toxicity with the crustacean Daphnia magna (TG 202), development toxicity with the fish Danio rerio (TG 210), reproduction of the sediment-living worm Lumbriculus variegatus (TG 225), activity of soil microflora (TGs 216, 217), and reproduction of the invertebrates (Enchytraeus crypticus, Eisenia fetida, TGs 220, 222). Additionally, test descriptions for two further test systems (root elongation of plants in hydroponic culture; test on fish cells) are presented. Ecotoxicological data obtained with the modified test guidelines for TiO2 NMs and Ag NM and detailed method descriptions are available.

Considerations of Environmentally Relevant Test Conditions for Improved Evaluation of Ecological Hazards of Engineered Nanomaterials.

Engineered nanomaterials (ENMs) are increasingly entering the environment with uncertain consequences including potential ecological effects. Various research communities view differently whether ecotoxicological testing of ENMs should be conducted using environmentally relevant concentrations-where observing outcomes is difficult-versus higher ENM doses, where responses are observable. What exposure conditions are typically used in assessing ENM hazards to populations? What conditions are used to test ecosystem-scale hazards? What is known regarding actual ENMs in the environment, via measurements or modeling simulations? How should exposure conditions, ENM transformation, dose, and body burden be used in interpreting biological and computational findings for assessing risks? These questions were addressed in the context of this critical review. As a result, three main recommendations emerged. First, researchers should improve ecotoxicology of ENMs by choosing test end points, duration, and study conditions-including ENM test concentrations-that align with realistic exposure scenarios. Second, testing should proceed via tiers with iterative feedback that informs experiments at other levels of biological organization. Finally, environmental realism in ENM hazard assessments should involve greater coordination among ENM quantitative analysts, exposure modelers, and ecotoxicologists, across government, industry, and academia.

Single versus repeated applications of CuO and Ag nanomaterials and their effect on soil microflora.

Nanomaterials enter the terrestrial environment via the repeated application of sludge to soils over many years. The goal of this investigation was to compare the effects of CuO and Ag nanomaterials on soil microorganisms after a single application and after repeated applications ultimately resulting in the same test concentrations. The effect on soil microorganisms was determined using the ammonium oxidation (ISO 15685), enzymatic activity patterns (ISO 22939) and MicroResp™ tests on days 28, 56 and 84. The comparability of single and repeated applications of ion-releasing nanomaterials depended on the test endpoint and duration. No significant differences between single and repeated applications were observed when testing nitrifying microorganisms and exoenzymes, but differences were observed in the substrate-induced respiration test. The three test systems used together provide more comprehensive information about the impact of different nanomaterials on the soil microflora and its diversity.

The MARINA Risk Assessment Strategy: A Flexible Strategy for Efficient Information Collection and Risk Assessment of Nanomaterials.

An engineered nanomaterial (ENM) may actually consist of a population of primary particles, aggregates and agglomerates of various sizes. Furthermore, their physico-chemical characteristics may change during the various life-cycle stages. It will probably not be feasible to test all varieties of all ENMs for possible health and environmental risks. There is therefore a need to further develop the approaches for risk assessment of ENMs. Within the EU FP7 project Managing Risks of Nanoparticles (MARINA) a two-phase risk assessment strategy has been developed. In Phase 1 (Problem framing) a base set of information is considered, relevant exposure scenarios (RESs) are identified and the scope for Phase 2 (Risk assessment) is established. The relevance of an RES is indicated by information on exposure, fate/kinetics and/or hazard; these three domains are included as separate pillars that contain specific tools. Phase 2 consists of an iterative process of risk characterization, identification of data needs and integrated collection and evaluation of data on the three domains, until sufficient information is obtained to conclude on possible risks in a RES. Only data are generated that are considered to be needed for the purpose of risk assessment. A fourth pillar, risk characterization, is defined and it contains risk assessment tools. This strategy describes a flexible and efficient approach for data collection and risk assessment which is essential to ensure safety of ENMs. Further developments are needed to provide guidance and make the MARINA Risk Assessment Strategy operational. Case studies will be needed to refine the strategy.