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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

Abstract

Background
The increasing use of nanotechnology means that nanomaterials will enter the environment. Ecotoxicological data are therefore required so that adequate risk assessments can be carried out. In this study, we used a standardized earthworm reproduction test with Eisenia andrei to evaluate three types of TiO2 nanoparticles (NM-101, NM-102, NM-103). The test was performed in natural sandy soil (RefeSol 01A) following Organisation for Economic Co-operation and Development Test Guideline No. 222. The nanoparticles differed in several aspects, such as crystalline structure, size, and the presence or absence of a coating.


Results
Uncoated nanoparticles stimulated earthworm reproduction in a concentration-dependent manner during winter testing, increasing the number of offspring by up to 50% compared to the control. However, there was no stimulation when the same test was performed in the summer. This reflected an underlying circannual rhythm observed in the control soil, characterized by the production of a significantly larger number of juveniles in summer compared with that in winter. The effect of the uncoated TiO2 nanoparticles was to reduce or eliminate the circannual differences by increasing the reproductive rate in winter. Coated TiO2 nanoparticles did not influence earthworm reproduction.


Conclusion
TiO2 appears to affect earthworm reproductive activity by abolishing the circannual rhythm that depresses reproduction in the winter. Further experiments will be necessary to determine (1) the mode of action of the nanoparticles, (2) the important parameters causing the effect (e.g., relevant soil parameters), and (3) the environmental relevance of continuous earthworm reproduction we observed under laboratory conditions.

Main aim of the project was the development of a test strategy on the fate and ecotoxicological effects of synthetic nanomaterials (ENM) in the environment. Furthermore, a tiered risk assessment approach was suggested. For the test strategy development both, conventional and alternative endpoints were considered, and fate and effects were considered separately. First, the literature was reviewed and suitable test methods were identified. Recommendations for the fate and effects testing were provided. The recommendations consider various levels of test complexity as they are to be used in a tiered risk assessment scheme. Furthermore, the proposed test strategy on effects comprises of three sub-steps: Step 1: decision on ENM to be tested; Step 2: testing; Step 3: use of test results.Exemplarily one aspect of the test strategy for the assessment of effects was investigated comprehensively. For the risk assessment (RA) approach, a tiered scheme such as are 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. Besides risk assessment related aspects, the IME-approach also takes into account the basic idea of life-cycle assessment (LCA).

Abstract
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

In view of the advanced development of new specific active pharmaceutical ingredients, the question arises as to whether the established standard procedures for the environmental risk assessment in the context of marketing authorization are still sufficient to adequately cover relevant effects on environmental organisms. The focus of this project is on specific test strategies for substances from the group of oncologicals, cardiologicals and statins, as well as their experimental verification in case studies. Studies with aquatic plants in the Lemna sp. Growth Inhibition Test (⁠OECD⁠ 221), the zebrafish embryo toxicity test (OECD 236) amended with sublethal endpoints and the comet assay with environmentally relevant cell types are discussed as possible adaptations, although not all of them proved to be suitable.

The Water Framework Directive 60/2000/EG (WFD) requires Environmental Quality Standards (EQS) for polluting substances. Member States have to set objectives for individual surface water bodies (Article 4 WFD). The transitional period for Directive 2006/11/EC ended in 2013. Therefore, revision, adoption and implementation of the EQS for the substances on the "Indicative list of main pollutants" (see Annex VIII WFD) is needed to assess the status of the surface waters. In this context, the Federal Environmental Agency (UBA) initiated a project to update the environmental quality standards (EQS) for 10 pollutants (pollutants specific for river basin according to Ordinance on the Protection of Surface Waters (OGewV)) and to compile suggestions for environmental quality standards for 20 new chemicals. The searched data were documented data sheets according to the "Guidance Document No. 27: Technical Guidance for Deriving Environmental Quality Standards" (EC, 2011). The derivation of EQS for the matrices to be considered was performed in accordance with the rules as laid down in the "Guidance Document No. 27". Within this project EQS proposals were derived for the following 30 substances: 2.4-D, arsenic, bezafibrate, carbamazepin, carbendazim, chloroacetic acid, chromium, dibutyltin, dimethoate, dimoxystrobin, epichlorhydrin, erythromycin, flufenacet, flurtamone, imidacloprid, copper, metoprolol, monobutyltin cation, monolinuron, nicosulfuron, omethoate, triphenyl phosphate, phoxim, roxithromycin, sulcotrione, sulfamethoxazole, tetrabutyltin, triclosan, uranium, zinc.

Dpto. Medio Ambiente y Agronomía (INIA) ; 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. ; This project has received funding from the European Union's Horizon 2020 research and innovation programme under BIORIMA grant agreement No 760928 for the testing and under NANORIGO grant agreement No 814530 regarding risk governance. Further, to FCT (Fundação para a Ciência e ...