Background Advanced/innovative materials are an undefined group of nano- and micro-particles encompassing diverse material compositions, structures and combinations. Due to their unique properties that enable specific functions during applications, there are concerns about unexpected hazards to humans and the environment. In this study, we provide ecotoxicity data for 36 nano- and microparticles of various inorganic species (single constituents and complex compositions; materials releasing toxic ions and others), morphologies (spheroidal, cubic, flaky, elongated/fibrous) and sizes (10 nm–38 µm). By applying Raphidocelis subcapitata algae growth inhibition and Daphnia magna immobilisation tests according to OECD test guidelines 201 and 202, and extensive material characterisation, we aimed to identify indicators of concern. This would allow better predictions of the hazardous properties of these materials in the future. Results The chemical identity (toxic ion-releasing materials vs. other materials) and agglomeration behaviour, which is affected by size (nm vs. µm) and morphology (fibres vs. others), were obvious drivers of ecotoxicity on R. subcapitata. Differences in morphology had an impact on agglomeration behaviour. Fibres formed agglomerates of varying sizes with entrapped and attached algae. Small compact (e.g. spheroidal) particles attached to algae. A high coverage resulted in high ecotoxicity, while less toxic materials attached to a much lesser extent. No agglomeration of algae and particles was observed for particles with a µm size. Small toxic components of large hybrid materials did not affect ecotoxicity. For D. magna, despite uptake of all materials studied into the gut, the sole indication of toxicity was the release of toxic ions. This is in line with previous observations on nanomaterials. Based on the identified criteria, charts were developed to indicate the expected toxicity of advanced/innovative materials toward algae and daphnia. Conclusion Indicators for the toxicities of advanced materials differ for algae and daphnia. Thus, different materials give rise to concerns for the two aquatic organisms. For D. magna, only the toxic ion-releasing materials are relevant, but for R. subcapitata, more complex interactions between particular materials and cells must be considered. Graphical Abstract

Background The freshwater algae and cyanobacteria growth inhibition test (OECD test guideline 201) is frequently used to assess the ecotoxicity of chemicals or particles. A central issue is the measurement of algal growth by quantifying algal biomass over time. Chlorophyll fluorescence measurements are recommended for the testing of particles. The analysis of in vivo fluorescence is the simplest and fastest approach, but is only suitable if there is no interference with the materials. Therefore, in vitro fluorescence analysis is often preferred. We carried out a comprehensive comparison of chlorophyll fluorescence measurements in vitro and in vivo to evaluate the suitability of rapid in vivo testing for the determination of Raphidocelis subcapitata biomass in the presence of diverse particles. Results For the in vitro measurement, we applied a method that separates particles from chlorophyll using locust bean gum. We tested inorganic and organic particles (including alloys and polymers), ion-releasing and non-releasing materials, and particle sizes in the nanometer to micrometer range with a variety of shapes (spherical, flaky and fibrous). Some of the materials were nontoxic, whereas others showed varying degrees of toxicity (ErC50 = 0.2–100 mg/L in both methods). There were only minor differences between the methods in ErC50 values and the percent inhibition at various test concentrations, but the confidence intervals for the ErC50 values in vivo were narrower and were covered by the range observed in vitro. The in vivo approach showed no limitations, whereas the validity criteria listed in OECD test guideline 201 were not always fulfilled by the in vitro measurements. Conclusion The in vivo approach was a suitable and time-saving method for a wide range of particles, although we cannot completely exclude the possibility that some particles may interfere with fluorescence measurement. To avoid false assessments, pre-tests with simple measurements are therefore recommended. Graphical abstract

Background: Before chemicals, pesticides and biocides are registered and approved, their effects on soil microorganisms must be tested, specifically their impact on nitrogen transformation. Following a request from the European Food Safety Authority (EFSA), the Panel on Plant Protection Products and their Residues provided an opinion document evaluating the science behind the risk assessment of plant protection products in the context of soil-dwelling organisms. The EFSA document concludes that the most relevant community-based microbial test systems should cover the widest possible range of metabolic processes without compromising test sensitivity. The EFSA document refers to the MicroResp test system, stating that although it has not been used to study the effects of pesticides on soil microbial processes, its capability should be investigated in the future. In the scope of harmonization approaches, the recommendations in the EFSA document covering pesticides could also influence the risk assessment and regulation of other kinds of chemicals, including silver nanomaterials. We therefore used the silver nanomaterial NM-300K as a model substance to evaluate the sensitivity of three functional tests covering the activities of different microbial fractions: (1) the potential ammonium oxidation (PAO) test, which considers the first step in nitrification; (2) the Micro-Resp test, which determines respiratory activity by measuring CO 2 evolution; and (3) a colorimetric test system for exoenzyme activity. We also surveyed bacterial 16S rRNA sequence diversity by next-generation sequencing (NGS). Results: There was no major difference in the general sensitivity of the tests, each of which revealed significant effects at silver nanomaterial concentrations of at least 1.67 mg/kg. The PAO test was a robust and sensitive indicator of toxicity, and concentration-effect relationships were calculated for every time interval. The effects on respiration and exoenzyme activities were more variable. Among the three functional tests, the selected exoenzyme activities showed the weakest concentration-effect relationships, although silver concentrations were clearly related to two of the four activities we tested (glucosidase and arylsulfatase). We also observed a relationship between silver concentrations and respiration activity on glucose, cellobiose and alanine substrates. The bacterial orders identified by NGS differed in sensitivity to the silver nanomaterial. We found that the adverse impact on nitrifiers matched the inhibition of PAO activity. EC 50 values calculated for each functional test did not identify a generally superior method. Conclusion: We found that all four test approaches were similar in sensitivity towards the model silver nanomaterial, an ion-releasing substance. We observed advantages and limitations for each test, which must be considered when selecting tests for the registration or approval of substances. It is unclear whether the sensitivity of the tests would be comparable when testing substances that do...

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 (β, 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.