Differentiating Microplastics from Natural Particles in Aqueous Suspensions Using Flow Cytometry with Machine Learning.

Microplastics (MPs) in natural waters are heterogeneously mixed with other natural particles including algal cells and suspended sediments. An easy-to-use and rapid method for directly measuring and distinguishing MPs from other naturally present colloids in the environment would expedite analytical workflows. Here, we established a database of MP scattering and fluorescence properties, either alone or in mixtures with natural particles, by stain-free flow cytometry. The resulting high-dimensional data were analyzed using machine learning approaches, either unsupervised (e.g., viSNE) or supervised (e.g., random forest algorithms). We assessed our approach in identifying and quantifying model MPs of diverse sizes, morphologies, and polymer compositions in various suspensions including phototrophic microorganisms, suspended biofilms, mineral particles, and sediment. We could precisely quantify MPs in microbial phototrophs and natural sediments with high organic carbon by both machine learning models (identification accuracies over 93%), although it was not possible to distinguish between different MP sizes or polymer compositions. By testing the resulting method in environmental samples through spiking MPs into freshwater samples, we further highlight the applicability of the method to be used as a rapid screening tool for MPs. Collectively, this workflow can be easily applied to a diverse set of samples to assess the presence of MPs in a time-efficient manner.

Herbicide-Induced Shifts in the Periphyton Community Composition Indirectly Affect Feeding Activity and Physiology of the Gastropod Grazer Physella acuta.

Herbicides are well known for unintended effects on freshwater periphyton communities. Large knowledge gaps, however, exist regarding indirect herbicide impacts on primary consumers through changes in the quality of periphyton as a food source (i.e., diet-related effects). To address this gap, the grazer Physella acuta (Gastropoda) was fed for 21 days with periphyton that grew for 15 days in the presence or absence of the herbicide diuron (8 µg/L) to quantify changes in the feeding rate, growth rate, and energy storage (neutral lipid fatty acids; NLFAs) of P. acuta. Periphyton biomass, cell viability, community structure, and FAs served as proxies for food quality that support a mechanistic interpretation of the grazers' responses. Diuron changed the algae periphyton community and fatty acid profiles, indicating alterations in the food quality, which could explain differences in the snails' feeding rate compared to the control. While the snails' growth rate was, despite an effect size of 55%, not statistically significantly changed, NLFA profiles of P. acuta were altered. These results indicate that herbicides can change the food quality of periphyton by shifts in the algae composition, which may affect the physiology of grazers.

Biological components and bioelectronic interfaces of water splitting photoelectrodes for solar hydrogen production.

Artificial photosynthesis (AP) is inspired by photosynthesis in nature. In AP, solar hydrogen can be produced by water splitting in photoelectrochemical cells (PEC). The necessary photoelectrodes are inorganic semiconductors. Light-harvesting proteins and biocatalysts can be coupled with these photoelectrodes and thus form bioelectronic interfaces. We expand this concept toward PEC devices with vital bio-organic components and interfaces, and their integration into the built environment.

Fate and effects of microplastic particles in a periphyton-grazer system.

In the aquatic environment, microplastic particles (MP) can accumulate in microbial communities that cover submerged substrata, i.e. in periphyton. Despite periphyton being the essential food source for grazers in the benthic zones, MP transfer from periphyton to benthic biota and its ecotoxicological consequences are unknown. Therefore, in this study, we investigated the effects of 1) MP on embryonal development of freshwater gastropod Physa acuta embryos, 2) MP on adult Physa acuta individuals through dietary exposure and 3) on the MP surface properties. Embryonal development tests were carried out with spherical polyethylene MP in the size of 1-4 µm (MP). Over a period of 28 days, embryonal development and hatching rate were calculated. In the feeding experiments, periphyton was grown in the presence and absence of MP and was then offered to the adult Physa acuta for 42-152 h. The snails readily ingested and subsequently egested MP, together with the periphyton as shown by MP quantification in periphyton, snail soft body tissue and feces. No selective feeding behavior upon MP exposure was detected. The ingestion of MP had no effect on mortality, feeding and defecation rate. Yet, the reproductive output of snails, measured as the number of egg clutches and numbers of eggs per clutch, decreased after the ingestion of MPs, while the hatching success of snail embryos those parents were exposed remained unaffected. In contrast, hatching rate of snail embryos was significantly reduced upon direct MP exposure. MP optical properties were changed upon the incorporation into the periphyton and the passage through the digestive tract. Our results indicate that MP incorporated in periphyton are bioavailable to aquatic grazers, facilitating the introduction of MP into the food chain and having direct adverse effects on the grazers' reproductive fitness.

Assessing Environmental Risks during the Drug Development Process for Parasitic Vector-Borne Diseases: A Critical Reflection.

Parasitic vector-borne diseases (VBDs) represent nearly 20% of the global burden of infectious diseases. Moreover, the spread of VBDs is enhanced by global travel, urbanization, and climate change. Treatment of VBDs faces challenges due to limitations of existing drugs, as the potential for side effects in nontarget species raises significant environmental concerns. Consequently, considering environmental risks early in drug development processes is critically important. Here, we examine the environmental risk assessment process for veterinary medicinal products in the European Union and identify major gaps in the ecotoxicity data of these drugs. By highlighting the scarcity of ecotoxicological data for commonly used antiparasitic drugs, we stress the urgent need for considering the One Health concept. We advocate for employing predictive tools and nonanimal methodologies such as New Approach Methodologies at early stages of antiparasitic drug research and development. Furthermore, adopting progressive approaches to mitigate ecological risks requires the integration of nonstandard tests that account for real-world complexities and use environmentally relevant exposure scenarios. Such a strategy is vital for a sustainable drug development process as it adheres to the principles of One Health, ultimately contributing to a healthier and more sustainable world.

Linking chemical surface water monitoring and pesticide regulation in selected European countries.

The progress in chemical analytics and understanding of pesticide dynamics in surface waters allows establishing robust data on compounds with frequent exceedances of quality standards. The current chemical, temporal, and spatial coverage of the pesticide monitoring campaigns differs strongly between European countries. A questionnaire revealed differences in monitoring strategies in seven selected European countries; Nordic countries prioritize temporal coverage, while others focus on spatial coverage. Chemical coverage has increased, especially for non-polar classes like synthetic pyrethroids. Sweden combines monitoring data with agricultural practices for derived quantities, while the Netherlands emphasizes spatial coverage to trace contamination sources. None of the EU member states currently has established a process for linking chemical surface water monitoring data with regulatory risk assessment, while Switzerland has recently established a legally defined feedback loop. Due to their design and objectives, most strategies do not capture concentration peaks, especially 2-week composite samples, but also grab samples. Nevertheless, for substances that appear problematic in many data sets, the need for action is evident even without harmonization of monitoring programs. Harmonization would be beneficial, however, for cross-national assessment including risk reduction measures.

Incorporation of sediment- and soil-specific aspects in the Criteria for Reporting and Evaluating Ecotoxicity Data (CRED).

In environmental risk assessment either for registration purposes or for retrospective assessments of monitoring data, the hazard assessment is predominantly based on effect data from ecotoxicity studies. Most regulatory frameworks require studies used for risk assessment to be evaluated for reliability and relevance. Historically, the Klimisch methodology was used in many regulatory procedures where reliability needed to be evaluated. More recently, the Criteria for Reporting and Evaluating Ecotoxicity Data (CRED) have been developed for aquatic ecotoxicity studies, providing more detailed guidance on the evaluation and reporting of not only the reliability but also the relevance of a scientific study. Here, we discuss the application of the CRED methodology for assessing sediment and soil ecotoxicity studies, addressing important sediment- and soil-specific criteria that should be included as part of the CRED evaluation system. We also provide detailed recommendations for the design and reporting of sediment and soil toxicity studies that can be used by scientists and researchers wishing to contribute ecotoxicological data for effect assessments carried out within regulatory frameworks. Integr Environ Assess Manag 2024;00:1-13. © 2024 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

A conceptual framework for landscape-based environmental risk assessment (ERA) of pesticides.

While pesticide use is subject to strict regulatory oversight worldwide, it remains a main concern for environmental protection, including biodiversity conservation. This is partly due to the current regulatory approach that relies on separate assessments for each single pesticide, crop use, and non-target organism group at local scales. Such assessments tend to overlook the combined effects of overall pesticide usage at larger spatial scales. Integrative landscape-based approaches are emerging, enabling the consideration of agricultural management, the environmental characteristics, and the combined effects of pesticides applied in a same or in different crops within an area. These developments offer the opportunity to deliver informative risk predictions relevant for different decision contexts including their connection to larger spatial scales and to combine environmental risks of pesticides, with those from other environmental stressors. We discuss the needs, challenges, opportunities and available tools for implementing landscape-based approaches for prospective and retrospective pesticide Environmental Risk Assessments (ERA). A set of "building blocks" that emerged from the discussions have been integrated into a conceptual framework. The framework includes elements to facilitate its implementation, in particular: flexibility to address the needs of relevant users and stakeholders; means to address the inherent complexity of environmental systems; connections to make use of and integrate data derived from monitoring programs; and options for validation and approaches to facilitate future use in a regulatory context. The conceptual model can be applied to existing ERA methodologies, facilitating its comparability, and highlighting interoperability drivers at landscape level. The benefits of landscape-based pesticide ERA extend beyond regulation. Linking and validating risk predictions with relevant environmental impacts under a solid science-based approach will support the setting of protection goals and the formulation of sustainable agricultural strategies. Moreover, landscape ERA offers a communication tool on realistic pesticide impacts in a multistressors environment for stakeholders and citizens.

In vitro toxicology of ambient particulate matter: correlation of cellular effects with particle size and components.

High concentrations of airborne particulate matter (PM) have been associated with increased rates of morbidity and mortality among exposed populations. Although certain components of PM were suggested to influence these effects, no clear-cut correlation was determined thus far. One of the possible modes of action is the induction of oxidative stress by inhaled PM triggering inflammatory responses. Therefore, the in vitro formation of reactive oxygen species (ROS) in three cell lines in the presence of five subfractions of PM(10), collected in Münster, Germany was investigated. The PM components chloride, nitrate, ammonium, sulfate, 68 chemical elements, and endotoxin were quantified. The highest concentration of endotoxin was found in particles of 0.42-1.2 µm aerodynamic diameters, and therefore probably subject to long-range transport. Intracellular ROS formation in three well established mammalian cell lines (CaCo2, human; MDCK, canine; RAW264.7, mouse) only correlated positively with particle size. The two smallest PM size fractions provoked the highest rise in ROS. However, the latter did not correlate with the concentration of any PM components investigated. The smallest PM size fractions significantly dominated the number of particles. Therefore, the particle number may be most effective in inducing oxidative stress in vitro.

Interference of engineered nanoparticles with in vitro toxicity assays.

Accurate in vitro assessment of nanoparticle cytotoxicity requires a careful selection of the test systems. Due to high adsorption capacity and optical activity, engineered nanoparticles are highly potential in influencing classical cytotoxicity assays. Here, four common in vitro assays for oxidative stress, cell viability, cell death and inflammatory cytokine production (DCF, MTT, LDH and IL-8 ELISA) were assessed for validity using 24 well-characterized engineered nanoparticles. For all nanoparticles, the possible interference with the optical detection methods, the ability to convert the substrates, the influence on enzymatic activity and the potential to bind proinflammatory cytokines were analyzed in detail. Results varied considerably depending on the assay system used. All nanoparticles tested were found to interfere with the optical measurement at concentrations of 50 µg cm⁻² and above when DCF, MTT and LDH assays were performed. Except for Carbon Black, particle interference could be prevented by altering assay protocols and lowering particle concentrations to 10 µg cm⁻². Carbon Black was also found to oxidize H2DCF-DA in a cell-free system, whereas only ZnO nanoparticles significantly decreased LDH activity. A dramatic loss of immunoreactive IL-8 was observed for only one of the three TiO2 particle types tested. Our results demonstrate that engineered nanoparticles interfere with classic cytotoxicity assays in a highly concentration-, particle- and assay-specific manner. These findings strongly suggest that each in vitro test system has to be evaluated for each single nanoparticle type to accurately assess the nanoparticle toxicity.

Cytotoxicity screening of 23 engineered nanomaterials using a test matrix of ten cell lines and three different assays.

BACKGROUND: Engineered nanomaterials display unique properties that may have impact on human health, and thus require a reliable evaluation of their potential toxicity. Here, we performed a standardized in vitro screening of 23 engineered nanomaterials. We thoroughly characterized the physicochemical properties of the nanomaterials and adapted three classical in vitro toxicity assays to eliminate nanomaterial interference. Nanomaterial toxicity was assessed in ten representative cell lines. RESULTS: Six nanomaterials induced oxidative cell stress while only a single nanomaterial reduced cellular metabolic activity and none of the particles affected cell viability. Results from heterogeneous and chemically identical particles suggested that surface chemistry, surface coating and chemical composition are likely determinants of nanomaterial toxicity. Individual cell lines differed significantly in their response, dependent on the particle type and the toxicity endpoint measured. CONCLUSION: In vitro toxicity of the analyzed engineered nanomaterials cannot be attributed to a defined physicochemical property. Therefore, the accurate identification of nanomaterial cytotoxicity requires a matrix based on a set of sensitive cell lines and in vitro assays measuring different cytotoxicity endpoints.

Inhalation of publicly available indoor insecticide spray caused myocardial infarction type II: a case report.

We report on a 70-year-old woman who tried to eliminate ants from her kitchen by applying a publicly available insecticide spray. Immediately afterwards, she felt dyspnoea, superseded by heavy chest pain. High-sensitivity troponin concentration increased from 33 to 149 ng/L (cut-off 50 ng/L). Significant coronary stenosis was excluded by coronary angiography, and the myocardial damage was classified as myocardial infarction type II. After exclusion of other potential mechanisms, we consider a cardiotoxic effect of the insecticide mixture of cypermethrin, tetramethrin, and piperonyl butoxide possible. We conclude that consumer information has to be improved. This concerns sustainable control measures adapted to the target insect species (in this case, the black garden ant Lasius niger), and differentiation between authorized and non-authorized but notified products. The instructions for use should give clear information on vulnerable groups and recommend personal protective equipment. Physicians and authorities should be alert to cardiac side-effects of insecticides.

Characterization of Aquatic Biofilms with Flow Cytometry.

Biofilms are dynamic consortia of microorganism that play a key role in freshwater ecosystems. By changing their community structure, biofilms respond quickly to environmental changes and can be thus used as indicators of water quality. Currently, biofilm assessment is mostly based on integrative and functional endpoints, such as photosynthetic or respiratory activity, which do not provide information on the biofilm community structure. Flow cytometry and computational visualization offer an alternative, sensitive, and easy-to-use method for assessment of the community composition, particularly of the photoautotrophic part of freshwater biofilms. It requires only basic sample preparation, after which the entire sample is run through the flow cytometer. The single-cell optical and fluorescent information is used for computational visualization and biological interpretation. Its main advantages over other methods are the speed of analysis and the high-information-content nature. Flow cytometry provides information on several cellular and biofilm traits in a single measurement: particle size, density, pigment content, abiotic content in the biofilm, and coarse taxonomic information. However, it does not provide information on biofilm composition on the species level. We see high potential in the use of the method for environmental monitoring of aquatic ecosystems and as an initial biofilm evaluation step that informs downstream detailed investigations by complementary and more detailed methods.

Evaluation of Phototrophic Stream Biofilms Under Stress: Comparing Traditional and Novel Ecotoxicological Endpoints After Exposure to Diuron.

Stream biofilms have been shown to be among the most sensitive indicators of environmental stress in aquatic ecosystems and several endpoints have been developed to measure biofilm adverse effects caused by environmental stressors. Here, we compare the effects of long-term exposure of stream biofilms to diuron, a commonly used herbicide, on several traditional ecotoxicological endpoints (biomass growth, photosynthetic efficiency, chlorophyll-a content, and taxonomic composition), with the effects measured by recently developed methods [community structure assessed by flow cytometry (FC-CS) and measurement of extracellular polymeric substances (EPS)]. Biofilms grown from local stream water in recirculating microcosms were exposed to a constant concentration of 20 µg/L diuron over a period of 3 weeks. During the experiment, we observed temporal variation in photosynthetic efficiency, biomass, cell size, presence of decaying cells and in the EPS protein fraction. While biomass growth, photosynthetic efficiency, and chlorophyll-a content were treatment independent, the effects of diuron were detectable with both FC and EPS measurements. This demonstrates that, at least for our experimental setup, a combination of different ecotoxicological endpoints can be important for evaluating biofilm environmental stress and suggests that the more recent ecotoxicological endpoints (FC-CS, EPS protein content and humic substances) can be a useful addition for stream biofilm ecotoxicological assessment.

Carboxylate Functional Groups Mediate Interaction with Silver Nanoparticles in Biofilm Matrix.

Biofilms causing medical conditions or interfering with technical applications can prove undesirably resistant to silver nanoparticle (AgNP)-based antimicrobial treatment, whereas beneficial biofilms may be adversely affected by the released silver nanoparticles. Isolated biofilm matrices can induce reduction of silver ions and stabilization of the formed nanosilver, thus altering the exposure conditions. We thus study the reduction of silver nitrate solution in model experiments under chemically defined conditions as well as in stream biofilms. Formed silver nanoparticles are characterized by state-of-the art methods. We find that isolated biopolymer fractions of biofilm organic matrix are capable of reducing ionic Ag, whereas other isolated fractions are not, meaning that biopolymer fractions contain both reducing agent and nucleation seed sites. In all of the investigated systems, we find that silver nanoparticle-biopolymer interface is dominated by carboxylate functional groups. This suggests that the mechanism of nanoparticle formation is of general nature. Moreover, we find that glucose concentration within the biofilm organic matrix correlates strongly with the nanoparticle formation rate. We propose a simple mechanistic explanation based on earlier literature and the experimental findings. The observed generality of the extracellular polymeric substance/AgNP system could be used to improve the understanding of impact of Ag+ on aqueous ecosystems, and consequently, to develop biofilm-specific medicines and bio-inspired water decontaminants.

Classification and Segmentation of Nanoparticle Diffusion Trajectories in Cellular Micro Environments.

Darkfield and confocal laser scanning microscopy both allow for a simultaneous observation of live cells and single nanoparticles. Accordingly, a characterization of nanoparticle uptake and intracellular mobility appears possible within living cells. Single particle tracking allows to measure the size of a diffusing particle close to a cell. However, within the more complex system of a cell's cytoplasm normal, confined or anomalous diffusion together with directed motion may occur. In this work we present a method to automatically classify and segment single trajectories into their respective motion types. Single trajectories were found to contain more than one motion type. We have trained a random forest with 9 different features. The average error over all motion types for synthetic trajectories was 7.2%. The software was successfully applied to trajectories of positive controls for normal- and constrained diffusion. Trajectories captured by nanoparticle tracking analysis served as positive control for normal diffusion. Nanoparticles inserted into a diblock copolymer membrane was used to generate constrained diffusion. Finally we segmented trajectories of diffusing (nano-)particles in V79 cells captured with both darkfield- and confocal laser scanning microscopy. The software called "TraJClassifier" is freely available as ImageJ/Fiji plugin via https://git.io/v6uz2.

Flow cytometry combined with viSNE for the analysis of microbial biofilms and detection of microplastics.

Biofilms serve essential ecosystem functions and are used in different technical applications. Studies from stream ecology and waste-water treatment have shown that biofilm functionality depends to a great extent on community structure. Here we present a fast and easy-to-use method for individual cell-based analysis of stream biofilms, based on stain-free flow cytometry and visualization of the high-dimensional data by viSNE. The method allows the combined assessment of community structure, decay of phototrophic organisms and presence of abiotic particles. In laboratory experiments, it allows quantification of cellular decay and detection of survival of larger cells after temperature stress, while in the field it enables detection of community structure changes that correlate with known environmental drivers (flow conditions, dissolved organic carbon, calcium) and detection of microplastic contamination. The method can potentially be applied to other biofilm types, for example, for inferring community structure for environmental and industrial research and monitoring.

Mixed messages from benthic microbial communities exposed to nanoparticulate and ionic silver: 3D structure picks up nano-specific effects, while EPS and traditional endpoints indicate a concentration-dependent impact of silver ions.

Silver nanoparticles (AgNP) are currently defined as emerging pollutants in surface water ecosystems. Whether the toxic effects of AgNP towards freshwater organisms are fully explainable by the release of ionic silver (Ag(+)) has not been conclusively elucidated. Long-term effects to benthic microbial communities (periphyton) that provide essential functions in stream ecosystems are unknown. The effects of exposure of periphyton to 2 and 20 µg/L Ag(+) (AgNO3) and AgNP (polyvinylpyrrolidone stabilised) were investigated in artificial indoor streams. The extracellular polymeric substances (EPS) and 3D biofilm structure, biomass, algae species, Ag concentrations in the water phase and bioassociated Ag were analysed. A strong decrease in total Ag was observed within 4 days. Bioassociated Ag was proportional to dissolved Ag indicating a rate limitation by diffusion across the diffusive boundary layer. Two micrograms per liter of AgNO3 or AgNP did not induce significant effects despite detectable bioassociation of Ag. The 20-µg/L AgNO3 affected green algae and diatom communities, biomass and the ratio of polysaccharides to proteins in EPS. The 20-µg/L AgNO3 and AgNP decreased biofilm volume to about 50 %, while the decrease of biomass was lower in 20 µg/L AgNP samples than the 20-µg/L AgNO3 indicating a compaction of the NP-exposed biofilms. Roughness coefficients were lower in 20 µg/L AgNP-treated samples. The more traditional endpoints (biomass and diversity) indicated silver ion concentration-dependent effects, while the newly introduced parameters (3D structure and EPS) indicated both silver ion concentration-dependent effects and effects related to the silver species applied.

Extracellular polymeric substances (EPS) of freshwater biofilms stabilize and modify CeO2 and Ag nanoparticles.

Streams are potential receiving compartments for engineered nanoparticles (NP). In streams, NP may remain dispersed or settle to the benthic compartment. Both dispersed and settling NP can accumulate in benthic biofilms called periphyton that are essential to stream ecosystems. Periphytic organisms excrete extracellular polymeric substances (EPS) that interact with any material reaching the biofilms. To understand the interaction of NP with periphyton it is therefore crucial to study the interaction of NP with EPS. We investigated the influence of EPS on the physicochemical properties of selected NP (CeO2, Ag) under controlled conditions at pH 6, 7.6, 8.6 and light or dark exposure. We extracted EPS from five different periphyton communities, characterized the extracts, and exposed CeO2 and carbonate-stabilized Ag NP (0.5 and 5 mg/L, both 25 nm primary particle size) and AgNO3 to EPS (10 mg/L) over two weeks. We measured NP size distribution, shape, primary particle size, surface plasmon resonance, and dissolution. All EPS extracts were composed of biopolymers, building blocks of humic substances, low molecular weight (Mr) acids, and small amphiphilic or neutral compounds in varying concentrations. CeO2 NP were stabilized by EPS independent of pH and light/dark while dissolution increased over time in the dark at pH 6. EPS induced a size increase in Ag NP in the light with decreasing pH and the formation of metallic Ag NP from AgNO3 at the same conditions via EPS-enhanced photoreduction. NP transformation and formation were slower in the extract with the lowest biopolymer and low Mr acid concentrations. Periphytic EPS in combination with naturally varying pH and light/dark conditions influence the properties of the Ag and CeO2 NP tested and thus the exposure conditions within biofilms. Our results indicate that periphytic organisms may be exposed to a constantly changing mixture of engineered and naturally formed Ag NP and Ag+.