Short-term test for the toxicogenomic assessment of ecotoxic modes of action in Myriophyllum spicatum.

In environmental risk assessment of substances, the 14-day growth inhibition test following OECD test guideline 239 is employed to assess toxicity in the macrophyte Myriophyllum spicatum. Currently, this test evaluates physiological parameters and does not allow the identification of the mode of action (MoA) by which adverse effects are induced. However, for an improved ecotoxicity assessment of substances, knowledge about their ecotoxic MoA in non-target organisms is required. It has previously been suggested that the identification of gene expression changes can contribute to MoA identification. Therefore, we developed a shortened three-day assay for M. spicatum including the transcriptomic assessment of global gene expression changes and applied this assay to two model substances, the herbicide and photosynthesis inhibitor bentazone and the pharmaceutical and HMG-CoA reductase inhibitor atorvastatin. Due to the lack of a reference genome for M. spicatum we performed a de novo transcriptome assembly followed by a functional annotation to use the toxicogenomic results for MoA discrimination. The gene expression changes induced by low effect concentrations of these substances were used to identify differentially expressed genes (DEGs) and impaired biological functions for the respective MoA. We observed both concentration-dependent numbers and differentiated patterns of DEGs for both substances. While bentazone impaired genes involved in the response to reactive oxygen species as well as light response, and also genes involved in developmental processes, atorvastatin exposure led to a differential regulation of genes related to brassinosteroid response as well as potential metabolic shifts between the mevalonate and methyl erythritol 4-phosphate pathway. Based on these responses, we identified biomarker candidates for the assessment of MoA in M. spicatum. Utilizing the shortened assay developed in this study, the investigation of the identified biomarker candidates may contribute to the development of future MoA-specific screening approaches in the ecotoxicological hazard prediction using aquatic non-standard model organisms.

Palladium-doped and undoped polystyrene nanoplastics in a chronic toxicity test for higher plants: Impact on soil, plants and ammonium oxidizing bacteria.

There is a lack of knowledge about the fate and impact of microplastics (MPs) and nanoplastics (NPs), as well as their potential uptake and impact on plants and microorganisms. The predicted environmental concentrations (PEC) of frequent polymers in soils are low, and therefore, difficult to detect with the available techniques, which explains the knowledge gaps. Therefore, model particles (polystyrene particles (PS-P), 343 nm) and palladium (Pd) nanoparticle-doped polystyrene particles (PS-Pd-PS-P, 442 nm) were synthesized, characterized, and subsequently applied to agricultural soils (Cambisol, Podzol, PS target contents: 25 mg kg-1, 75 mg kg-1, 225 mg kg-1). A combination of different techniques, such as inductively coupled plasma-mass spectrometry (ICP-MS), pyrolysis-gas chromatography-mass spectrometry (Pyr-GC-MS), dynamic light scattering (DLS), and scanning electron microscopy (SEM), were used to characterize the particles in the dispersions, soils and plants. The spiked soils were applied to a chronical plant toxicity test with oat (Avena sativa). The applied particle contents could be recovered from both soils by ICP-MS (Pd, 89% - 99%), and Pyr-GC-MS (PS, 73% - 120%). Moreover, non-aggregated particles in soils and on oat roots were visualized through SEM. The ratio obtained for the Pd contents in oat roots to that in the Cambisol (2.2-2.7) and the Podzol (2.3-2.6) implied that particles accumulated on the root surface or in the roots. No Pd was detected in the oat shoots, which indicated that no translocation occurred from the roots to the shoots. Despite particle accumulation at or in the roots, no clear effects on plant growth were observed. Furthermore, the soil microorganisms (Podzol) and the soil water repellency (Cambisol, Podzol) showed no clear monotone concentration-response relationship after exposure to PS-P and PS-Pd-PS-P. The findings are complex and illustrate the urgent need for further sophisticated experimental studies to elucidate the impacts of NPs on physicochemical soil function, plants, and soil organisms. The model PS-P doped with Pd nanoparticles significantly enhanced the development and validation of methods for investigating MPs and NPs in environmental matrices, highlighting their considerable potential for further studies.

Short-Term Test for Toxicogenomic Analysis of Ecotoxic Modes of Action in Lemna minor.

In the environmental risk assessment of substances, toxicity to aquatic plants is evaluated using, among other methods, the 7 dayLemna sp. growth inhibition test following the OECD TG 221. So far, the test is not applicable for short-term screening of toxicity, nor does it allow evaluation of toxic modes of action (MoA). The latter is also complicated by the lack of knowledge of gene functions in the test species. Using ecotoxicogenomics, we developed a time-shortened 3 day assay inLemna minor which allows discrimination of ecotoxic MoA. By examining the changes in gene expression induced by low effect concentrations of the pharmaceutical atorvastatin and the herbicide bentazon at the transcriptome and proteome levels, we were able to identify candidate biomarkers for the respective MoA. We developed a homology-based functional annotation pipeline for the reference genome ofL. minor, which allowed overrepresentation analysis of the gene ontologies affected by both test compounds. Genes affected by atorvastatin mainly influenced lipid synthesis and metabolism, whereas the bentazon-responsive genes were mainly involved in light response. Our approach is therefore less time-consuming but sensitive and allows assessment of MoA in L. minor. Using this shortened assay, investigation of expression changes of the identified candidate biomarkers may allow the development of MoA-specific screening approaches in the future.

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.

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.

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.

Silver nanoparticles in sewage sludge: Bioavailability of sulfidized silver to the terrestrial isopod Porcellio scaber.

Silver nanoparticles (AgNPs) are efficiently converted during the wastewater-treatment process into sparingly soluble Ag sulfides (Ag2 S). In several countries, sewage sludge is used as a fertilizer in agriculture. The bioavailability of sulfidized Ag to the terrestrial isopod Porcellio scaber was investigated. Sewage sludge containing transformed AgNPs was obtained from a laboratory-scale sewage-treatment plant operated according to Organisation for Economic Co-operation and Development (OECD) guideline 303a. The results of transmission electron microscopy with energy dispersive X-ray of sludge samples suggest that AgNPs were completely transformed to Ag2 S. Adult isopods were exposed to OECD 207 soil substrate amended with the AgNP spiked sludge for 14 d (uptake phase) followed by an elimination phase in unspiked soil of equal duration. Most of the Ag measured in P. scaber at the end of the uptake phase was found in the hindgut (71%), indicating that only a minor part of the estimated Ag content was actually assimilated by the isopods with 16.3 and 12.7% found in the carcass and hepatopancreas, respectively. As a result of this, the Ag content of the animals dropped following transition to unspiked sludge within 2 d to one-third of the previously measured Ag concentration and remained stable at this level until the end of the elimination period. The present study shows that Ag2 S in sewage sludge is bioavailable to the terrestrial isopod P. scaber. Environ Toxicol Chem 2018;37:1606-1613. © 2018 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals, Inc. on behalf of SETAC.

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.

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.

Approach on environmental risk assessment of nanosilver released from textiles.

Based on the increased utilization of nanosilver (silver nanomaterials=AgNM) as antibacterial agent, there is the strong need to assess the potential environmental implication associated with its new application areas. In this study an exemplary environmental risk assessment (ERA) of AgNM applied in textiles was performed. Environmental exposure scenarios (via municipal sewage treatment plant (STP)) with wastewater supply from domestic homes) were developed for three different types of textiles equipped with AgNM. Based on these scenarios predicted environmental concentrations (PECs) were deduced for STPs and for the environmental compartments surface water, sediment as well as soil. These PECs were related to PNECs (predicted no effect concentrations). PNECs were deduced from results of ecotoxicity tests of a selected AgNM (NM-300K). Data on ecotoxicology were derived from various tests with activated sludge, cyanobacteria, algae, daphnids, fish, duckweed, macrophytes, chironomids, earthworms, terrestrial plants as well as soil microorganisms. Emission data for the AgNM NM-300K from textiles were derived from washing experiments. The performed ERA was based on the specifications defined in the ECHA Guidances on information requirements and chemical safety assessment. Based on the chosen scenarios and preconditions, no environmental risk of the AgNM NM-300K released from textiles was detected. Under conservative assumptions a risk quotient for surface water close to 1 indicated that the aquatic compartment may be affected by an increased emission of AgNM to the environment due to the high sensitivity of aquatic organisms to silver. Based on the successful retention of AgNM in the sewage sludge and the still ongoing continual application of sewage sludge on farmland it is recommended to introduce a threshold for total silver content in sewage sludge into the respective regulations. Regarding potential risk mitigation measures, it is emphasized to preferably directly introduce AgNM into the textile fiber since this will strongly minimize the release of AgNM during washing. If this is not possible due to technical limitations or other reasons, the introduction of a threshold level controlling the release of AgNM from textiles is suggested. It has to be noted that this study is a case study which is only valid for the investigated NM-300K and its potential application in textiles.

Test strategy for assessing the risks of nanomaterials in the environment considering general regulatory procedures.

BACKGROUND: Engineered nanomaterials (ENMs) are marketed as a substance or mixtures and are additionally used due to their active agent properties in products such as pesticides or biocides, for which specific regulations apply. Currently, there are no specific testing strategies for environmental fate and effects of ENMs within the different regulations. An environmental test and risk assessment strategy for ENMs have been developed considering the general principles of chemical assessment. RESULTS: The test strategy has been developed based on the knowledge of national and international discussions. It also takes into account the conclusions made by the OECD WPMN which held an expert meeting in January 2013. For the test strategy development, both conventional and alternative endpoints were discussed and environmental fate and effects were addressed separately. CONCLUSION: A tiered scheme as commonly used in the context of precautionary environmental risk assessment was suggested including the use of mathematical models and trigger values to either stop the procedure or proceed to the next tier. There are still several gaps which have to be filled, especially with respect to fate, to develop the test strategy further. The test strategy features a general approach. It is not specified to fulfil the information requirements of certain legislation (e.g. plant protection act, biocide regulation, REACH). However, the adaption of single elements of the strategy to the specific needs of certain legislation will provide a valuable contribution in relation to the testing of nanomaterials.

Influence of soil properties on the effect of silver nanomaterials on microbial activity in five soils.

We investigated the effects of silver nanomaterials (AgNMs) on five well-characterized soils with distinct physicochemical properties using two standardized test systems. The carbon transformation test (OECD217) showed minimal sensitivity whereas the ammonia oxidizing bacteria test (ISO 15685) showed extreme sensitivity over 28 days of exposure. AgNM toxicity was compared with the physicochemical properties of the soils, revealing that toxicity declined with increasing clay content and increasing pH. AgNM toxicity did not appear to be affected by the organic carbon content of the soil. Our results showed that AgNM toxicity cannot be attributed to any single soil property but depends on the same parameters that determine the toxicity of conventional chemicals. Recommendations in the test guidelines for soil ecotoxicity studies are therefore applicable to AgNMs as well as conventional chemicals.

The toxicity of silver nanoparticles to zebrafish embryos increases through sewage treatment processes.

Silver nanoparticles (AgNPs) are widely believed to be retained in the sewage sludge during sewage treatment. The AgNPs and their derivatives, however, re-enter the environment with the sludge and via the effluent. AgNP were shown to occur in surface water, while evidence of a potential toxicity of AgNPs in aquatic organisms is growing. This study aims to examine the toxicity of AgNPs to the embryos of the aquatic vertebrate model zebrafish (Danio rerio) before and after sewage treatment plants (STPs) processes. Embryos were treated with AgNP (particle size: >90 % <20 nm) and AgNO3 in ISO water for 48 h and consequently displayed effects such as delayed development, tail malformations and edema. For AgNP, the embryos were smaller than the controls with conspicuously smaller yolk sacs. The corresponding EC50 values of 48 hours post fertilization (hpf) were determined as 73 µg/l for AgNO3 and 1.1 mg/l for AgNP. Whole-mount immunostainings of primary and secondary motor neurons also revealed secondary neurotoxic effects. A TEM analysis confirmed uptake of the AgNPs, and the distribution within the embryo suggested absorption across the skin. Embryos were also exposed (for 48 h) to effluents of AgNP-spiked model STP with AgNP influent concentrations of 4 and 16 mg/l. These embryos exhibited the same malformations than for AgNO3 and AgNPs, but the embryo toxicity of the sewage treatment effluent was higher (EC50 = 142 µg/l; 48 hpf). On the other hand, control STP effluent spiked with AgNPs afterwards was less toxic (EC50 = 2.9 mg/l; 48 hpf) than AgNPs in ISO water. This observation of an increased fish embryo toxicity of STP effluents with increasing AgNP influent concentrations identifies the accumulation of AgNP in the STP as a potential source of effluent toxicity.

Effects of silver nanoparticles and silver nitrate in the earthworm reproduction test.

The widespread use of silver nanoparticles (Ag-NPs), for example, in textiles and cleaning products, means that they are likely to reach the environment via biosolids or the effluent from wastewater treatment plants. The aim of the present study was to determine the ecotoxicity of Ag-NPs in the earthworm reproduction test using Eisenia andrei. In addition to the usual endpoints, the authors also investigated the uptake and accumulation of Ag by adult earthworms and the concentration of free Ag(+) in soil pore water. Silver nanoparticles and Ag nitrate showed similar toxicities in the earthworm reproduction test. The uptake of Ag from Ag-NPs in the earthworm was slightly higher than the uptake of Ag from Ag nitrate. Spiked soils showed a concentration-dependent effect on reproduction, but there was no concentration-dependent increase in the amount of Ag in earthworm tissues. The authors noted a concentration-dependent increase in the levels of free Ag(+) in the soil pore water regardless of the Ag source. The number of juveniles is a more suitable endpoint than biomass or mortality. The uptake of Ag does not appear to inhibit reproduction. Instead, inhibition seems to reflect Ag(+) released into the soil pore water, which affects cocoons and juveniles in the soil. Analysis of transformed Ag-NPs after purification in wastewater treatment plants would provide additional information.