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This study aims to assess the effects of Ag particles synthesised by a chemical (Ag 20, 200 nm) and biological method (Ag 23, 27 nm) in aquatic organisms: the bacterium Vibrio fischeri, the alga Desmodesmus subspicatus and the crustacean Daphnia magna. Ag particles exerted toxic effects in all organisms studied with Ag particles 23 nm being the most potent. Although soluble Ag was released in all media, the differences between the tested Ag particles still cannot be explained solely based on soluble Ag. NanoSIMS analysis performed with D. magna showed that apart from their localisation in the gut lumen, Ag 200 nm and Ag NPs 23 nm seemed to pass through the epithelial barrier as well. Ag NPs 23 nm localised in specific areas seemed to be within the ovaries. This study strengthens the argument that size, method of synthesis as well as surface chemistry may affect the uptake and toxic effects of Ag NPs.

This document is the unedited Author’s version of a Submitted Work that was subsequently accepted for publication in Environmental Science and Technology, copyright © American Chemical Society after peer review. To access the final edited and published work see https://pubs.acs.org/doi/10.1021/acs.est.0c03113 In this study, the effects of aged Ag and TiO2 NPs, individually and as a mixture, in wastewater relative to their pristine counterparts on the development of the copepod nauplii (Tisbe battagliai) were investigated. NP behavior in synthetic wastewater and seawater was characterized during aging and exposure. A delayed development and subsequent mortality was observed after 6 days of exposure to aged Ag NPs, with a 2-fold decrease in EC50 (316 μg/L) compared to pristine NPs (EC50 640 μg/L) despite the similar dissolved Ag concentrations measured for aged and pristine Ag NPs (441 μg/L and 378 μg/L, respectively). In co-exposures with TiO2 NPs, higher dissolved Ag levels were measured for aged NPs (238.3 μg/L) relative to pristine NPs (98.57 μg/L). Co-exposure resulted in a slight decrease (15%) in the Ag NP EC50 (270 μg/L) with a 1.9-fold increase in Ag NP retained within the organisms after depuration (2.82% retention) compared to Ag NP single exposures as measured with sp-ICP-MS suggesting that the particles are still bioavailable despite the heteroaggregation observed between Ag, Ti NPs and wastewater components. This study shows that the presence of TiO2 NPs can affect the stability and toxicity of Ag NPs in complex media that cannot be predicted solely based on ionic, total or nanoparticulate concentrations and the need for studying NP interactions in more complex matrices is highlighted.

Due to their small size, nanoplastics (NPLs) possess specific properties which can potentiate their toxicity towards aquatic organisms. As primary producers, microalgae are at the base of aquatic food chains, thus negative impacts of NPLs will likely lead to disturbances in ecosystem productivity. The majority of data available on the toxicity of NPLs is limited to polystyrene and green microalgae, leaving a significant lack of knowledge on impacts of other polymer types across different taxonomic groups. So, the main objective of this study was to evaluate the cell responses of the red microalgae Rhodomonas baltica to plain and carboxylated poly(methyl methacrylate) NPLs (PMMA and PMMA-COOH, 50 nm). Results showed different NPL behaviour in media over time, with PMMA forming micro-scale aggregates and PMMA-COOH maintaining its nominal size range. PMMA caused a higher impact in cellular and physiological parameters than PMMA-COOH, even though a decrease in algal growth was only seen for the later. Overall, PMMA caused a significant decrease in cell viability followed by an increase in cell size and complexity, overproduction of pigments, loss of membrane integrity, hyperpolarization of the mitochondrial membrane, increased production of ROS and LPO, decrease in DNA content and reduced photosynthetic capacity. Conversely, a decrease in algal growth for PMMA-COOH was connected to an impairment in cell cycle and consequent decrease in cell viability, metabolic activity and photosynthetic performance, with negligible effects in ROS formation and pigments content. This study provided a first insight into the mechanistic understanding of the toxic impacts of PMMA and PMMA-COOH NPLs in red microalgae. Results obtained suggest an interaction between both NPLs and R. baltica cell surface that is dependent on particle behaviour and surface chemistry. Future experiments focusing on the in-depth characterization of the mode of action of these particles are recommended.

Calcium oxide (CaO) is being considered as a possible treatment for both the control of echinoderm populations and the treatment against sea lice infestation in Norwegian salmon farms. CaO particles produce an exothermal reaction when in contact with water, which can cause epidermal burns and lesions to certain target organisms leading to death. The aim of the present study was to determine the effects of fine (

Brukte bildekk utgjør et stort avfallsproblem. Dekk skal derfor gjenvinnes i form av ombruk, materialgjenvinning eller energiutnyttelse. Tidlig 2000-tall testet Statens vegvesen bruk av kasserte bildekk som byggemateriale i veibygging. Ved E6 Huggenes (Råde kommune) ble det for eksempel bygd en støyvoll med kasserte bildekk. Denne artikkelen presenterer resultater fra et stor-skala utlekkingsforsøk av denne støyvollen. Forsøket hadde til hensikt å avklare hvorvidt vei- og bildekkrelevante orurensningsstoffer kan lekke ut fra en eldre støyvoll bygd av bildekk, samt vurdere hvorvidt forurensingsnivået i ut lekkingsvannet medfører risiko for skade påvannmiljøet. De fleste forurensningsstoffene i utlekkingsvannet ble målt i lave konsentrasjoner. Unntaket var metallet sink som finnes i betyde lige mengder i dekk. Konsentrasjonen av sink var betydelig høyere enn anbefalte grenseverdi er for når overvann fra veier bør renses. Akutte cellebaserte giftighetstester viste ingen til lav effekt i den organiske fraksjonen av ufortynnet utlekkingsvann. Utlekking av forurensningsstoffer fra Huggenes støyvoll utgjør trolig liten risiko for skader på vannmiljøet.

The use of silver nanomaterials in everyday products, such as cosmetics, textiles, certain types of packaging, etc. is increasing, leading to their release into the environment, including aquatic ecosystems. This last point initiated this investigation on the toxicological effects of Ag nanoparticles (Ag NPs) in the aquatic model organism Danio rerio. For this purpose, zebrafish larvae were exposed to 20nm bare Ag NPs at different concentrations and AgNO3, used as a positive control for Ag+ ions toxicity, at the beginning of their foraging behaviour to determine adverse effects on fitness parameters. We used secondary ion mass spectrometry (SIMS) to determine the localization of Ag and transcriptomics (microarray) to determine the toxicity at the level of gene expression in fish larvae. Exposure to Ag NPs did not result in adverse effects on survival and growth of the fish. However, SIMS analysis showed that Ag NPs mainly concentrate around liver blood vessels and in the interstitial tissue between the intestine and the liver. Gene expression profiles revealed that AgNO3 and Ag NPs impacted common pathways, suggesting similar targets, such as the phototransduction system. However, the Ag NPs showed a broader set of genes impacted following the exposure, including the circadian clock regulation and the photoreception, suggesting specific particle-related effects in addition to those induced by ions.

International audience; Amphipods are one of the most important components of freshwater ecosystems. Among them, gammarids are the most widespread group in Europe and are often used as bioindicators and model organisms in ecotoxicology. However, their use, especially of Gammarus fossarum for the study of the environmental impact of nanoparticles, has been rather limited so far. G. fossarum was selected to assess effects of well-characterized chemically synthesized silver nanoparticles (AgNPs 20nm and 200nm) and "green" laboratory synthetized (from plant leaf extracts) AgNPs (AgNPs 23nm and 27nm). AgNO3 was used as a positive control to compare AgNPs effects and silver ions effects. A multibiomarker approach was used to investigate the sub-lethal effects of AgNPs on physiological and behavioural responses of G. fossarum. Two different experiments were carried out. In a preliminary experiment, two populations of G. fossarum (G.f1 and G.f2) were tested for sensitivity differences and the most sensitive one was exposed, in a final experiment, to sub-lethal concentrations of AgNO3 and the most toxic AgNPs. AgNO3 and AgNPs 23nm led to a significant decrease in survival rates, osmoregulation and locomotor activity. Ag internalisation, performed with Secondary Ion Mass Spectrometry (SIMS), showed the presence of silver in gills of G.f2 exposed to AgNPs 23 and 27nm. This study highlighted the influence of method of synthesis on ion release, uptake and toxic effects of AgNPs on G. fossarum. Osmoregulation appeared to be an effective biomarker indicating the physiological health status of G. fossarum. Locomotor activity, which was the most impacted response, reflects the potential effects of released ions from AgNPs 23nm at the population level as locomotion is necessary for foraging, finding mates and escaping from predators. Therefore, we propose G. fossarum as a suitable model for environmental nanotoxicology, providing information both at individual and population levels.

In this study, a lab-scale wastewater treatment plant (WWTP), simulating biological treatment, received 10 μg/L Ag and 100 μg/L TiO2 nanoparticles (NPs) for 5 weeks. NP partitioning was evaluated by size fractionation (>0.7 μm, 0.1−0.7 μm, 3 kDa-0.1 μm, < 3 kDa) using inductively coupled plasma mass spectrometry (ICP-MS), single particle ICP-MS and transmission electron microscopy. The ecotoxicological effects of the transformed NPs in the effluent were assessed using a battery of marine and freshwater bioassays (algae and crustaceans) and an in vitro gill cell line model (RTgill-W1). TiO2 aggregates were detected in the effluent, whereas Ag NPs (0.1−0.22 μg/L) were associated with S, Cu, Zn. Fractionation showed that >80% of Ag and Ti were associated with the effluent solids. Increased toxicity was observed during weeks 2−3 and the effects were species-dependent; with marine epibenthic copepods and algae being the most sensitive. Increased reactive oxygen species formation was observed in vitro followed by an increase in epithelial permeability. The effluent affected the gill epithelium integrity in vitro and impacted defense pathways (upregulation of multixenobiotic resistance genes). To our knowledge, this is the first study to combine a lab-scale activated sludge WWTP with extensive characterization techniques and ecotoxicological assays to study the effects of transformed NPs in the effluent. Ecotoxicological effects of transformed silver and titanium dioxide nanoparticles in the effluent from a lab-scale wastewater treatment system

Across different species, cellular efflux pumps such as P-glycoprotein (P-gp; also termed multidrug resistance protein 1 [MDR1]) serve as a first line of defense by transporting toxic xenobiotics out of the cell. This mechanism is also active in aquatic organisms such as mussels, fish, and their larvae. Modulation of this resistance mechanism by chemical agents occurring in the environment could result in either higher or lower internal concentrations of toxic or endogenous compounds in cells. The aim of the present study was to explore and quantify the inhibition of the P-gp efflux pumps by several ubiquitous aquatic contaminants. The calcein-acetoxymethyl ester (calcein-AM) assay commonly used in pharmacological research was established with P-gp-overexpressing Madin-Darby canine kidney cells (MDCKII-MDR1) in a 96-well plate, avoiding extra washing, centrifugation, and lysis steps. This calcein-AM-based P-gp cellular efflux pump inhibition assay (CEPIA) was used to study the inhibition by commonly occurring environmental contaminants. Among others, the compounds pentachlorophenol, perfluorooctane sulfonate, and perfluorooctanoate strongly inhibited the P-gp-mediated efflux of calcein-AM while the chloninated alkanes did not seem to interact with the transporter. The fact that common pollutants can be potent modulators of the efflux transporters is a motive to further study whether this increases the toxicity of other contaminants present in the same matrices.

The P-glycoprotein (P-gp, ABCB1) and multidrug resistance associated protein 1 (MRP1), important members of the ABC (ATP-binding cassette) transporters, protect cells and organisms via efflux of xenobiotics and are responsible for the phenomenon of multidrug or multixenobiotic resistance (MXR). In this study we first evaluated, in vitro, the interaction of silver nanoparticles (Ag NPs, 20, 23 and 27 nm), Ag 200 nm particles and Ag ions (AgNO3) with MXR efflux transporters using MDCKII and the P-gp over-expressing MDCKII-MDR1 cells and calcein-AM as a substrate of the transporters. Next the in vivo modulation of MXR activity was studied in Daphnia magna juveniles with the model P-gp and MRP1 inhibitors verapamil-HCl and MK571, respectively. The common environmental contaminants perfluorooctane sulfonate and bisphenol A, previously observed to interfere with the P-gp in vitro, also inhibited the efflux of calcein in vivo. Small-sized Ag NPs (with biomolecules present on the surface) and AgNO3 inhibited the MXR activity in daphnids and MDCKII-MDR1 cells, but abcb1 gene expression remained unchanged. Both Ag NPs and dissolved ions contributed to the effects. This study provides evidence of the interference of Ag NPs and AgNO3 with the MXR activity both in vitro and in D. magna, and should be taken into account when Ag NP toxicity is assessed.

Background The increased incorporation of silver nanoparticles (Ag NPs) into consumer products makes the characterization of potential risk for humans and other organisms essential. The oral route is an important uptake route for NPs, therefore the study of the gastrointestinal tract in respect to NP uptake and toxicity is very timely. The aim of the present study was to evaluate the effects of Ag NPs and ions on a Caco-2/TC7:HT29-MTX intestinal co-culture model with mucus secretion, which constitutes an important protective barrier to exogenous agents in vivo and may strongly influence particle uptake. Methods The presence of the mucus layer was confirmed with staining techniques (alcian blue and toluidine blue). Mono and co-cultures of Caco-2/TC7 and HT29-MTX cells were exposed to Ag NPs (Ag 20 and 200 nm) and AgNO3 and viability (alamar blue), ROS induction (DCFH-DA assay) and IL-8 release (ELISA) were measured. The particle agglomeration in the media was evaluated with DLS and the ion release with ultrafiltration and ICP-MS. The effects of the Ag NPs and AgNO3 on cells in co-culture were studied at a proteome level with two-dimensional difference in gel electrophoresis (2D-DIGE) followed by Matrix Assisted Laser Desorption Ionization - Time Of Flight/ Time Of Flight (MALDI-TOF/TOF) mass spectrometry (MS). Intracellular localization was assessed with NanoSIMS and TEM. Results The presence of mucus layer led to protection against ROS and decrease in IL-8 release. Both Ag 20 and 200 nm NPs were taken up by the cells and Ag NPs 20 nm were mainly localized in organelles with high sulfur content. A dose- and size-dependent increase in IL-8 release was observed with a lack of cytotoxicity and oxidative stress. Sixty one differentially abundant proteins were identified involved in cytoskeleton arrangement and cell cycle, oxidative stress, apoptosis, metabolism/detoxification and stress. Conclusions The presence of mucus layer had an impact on modulating the induced toxicity of NPs. NP-specific effects were observed for uptake, pro-inflammatory response and changes at the proteome level. The low level of overlap between differentially abundant proteins observed in both Ag NPs and AgNO3 treated co-culture suggests size-dependent responses that cannot only be attributed to soluble Ag. Electronic supplementary material The online version of this article (doi:10.1186/s12989-016-0117-9) contains supplementary material, which is available to authorized users.

Mucus layer characterization (Alcian blue staining). Figure S2. Mucus layer characterization (Toluidine blue staining and TEM). Figure S3. Mucus layer characterization (Toluidine blue staining, top view). Figure S4. Cell monolayer integrity evaluation (TEER). Figure S5. Cell-free DCFH-DA assay. Figure S6. TEM images of cells in co-culture exposed to Ag particles. Figure S7. Hierarchical clustering. Table S1. Detailed information on protein identification. Table S2. Cellular Ag content determination. Table S3. KEGG enrichment analysis. (DOCX 1.17 mb)

To understand the potential toxicity of nanoparticles (NPs), it is necessary to evaluate their penetration in biological tissues. For that, cell imaging can be considered as an indispensable diagnosis method. We have used the NanoSIMS50 to detect particles of a few nanometres diameter unambiguously in biological tissues. Animals (Daphnia magna) or cells were exposed to NPs (titaniumdioxide, silver, silicon oxide and copper oxide). Transmission electronic microscopy (TEM) imaging was used as a complementary technique to secondary ion mass spectrometry (SIMS) imaging. The correlation of SIMS and TEM images opens up a new way of studying the toxicological effects of NPs of interest to medicine and pharmacology. Copyright © 2012 John Wiley & Sons, Ltd.

International audience; This paper highlights the use of Al2O3 and SnO2 films as protecting layers to tailor the optical properties and biocompatibility of ZnO nano-films. ZnO, ZnO/Al2O3 and ZnO/SnO2 nano-films have been deposited inside 3-dimensional (3D) structures such as anodic aluminium oxide (AAO) membranes via a gas phase atomic layer deposition (ALD) process. We show that ZnO/Al2O3 multilayers exhibit improved excitonic photoluminescence properties compared to ZnO only, whereas ZnO/SnO2 multilayers quench the near band edge emission signal. In addition, in vitro experiments on Caco-2/TC7 cells and Vibrio fischeri bacteria reveal that ZnO/Al2O3 and ZnO/SnO2 multilayers display enhanced biocompatibility in liquid media compared to ZnO. We attribute the improvement of the biocompatibility to a lower dissolution of the ZnO nano-film when protected by Al2O3 or SnO2.

International audience

Summary Silver nanoparticles (Ag NPs) are among the most promising groups of NPs (particles with all dimensions below 100 nm) for application in numerous consumer products due to their broad spectrum antimicrobial activities. Examples are incorporation in textiles and plastics, personal care products, water filters, food supplements etc. The extensive application and use together with the not yet fully understood properties of Ag NPs as well as the toxicity of Ag itself has raised concerns on potential impact of Ag NPs on human and environmental health. The research conducted within this thesis aimed at the evaluation of potential hazards of Ag NPs and identification of some key factors that determine the toxicity of Ag NPs. A tiered approach was employed using a battery of standard bioassays with model aquatic organisms, followed by the determination of sub-lethal concentrations for mechanistic endpoints, the identification of target tissues and organisms for Ag NP exposure and uptake and the integration of a proteomic tool to identify subtle changes. One of the main uptake routes for Ag NPs is through ingestion making the gastrointestinal epithelium one of the first ports of potential NP uptake and cell-particle interactions. An in vitro co-culture model incorporating a mucus layer mimicking the gastrointestinal epithelium was established for a more realistic evaluation of Ag NP potential toxicity than using intestinal epithelial cells alone. Indeed, the absence of mucus resulted in an overestimation of Ag NP toxicity. To be able to elucidate subtle changes in cellular functions and identification of particle specific effects and NP modes of action, a proteomic approach was employed. Differences and commonalities were observed between the cellular responses induced Ag NPs of different sizes and AgNO3 as a source of free Ag ions. As the Ag NPs are expected to reach the aquatic environment, a combination of adapted standard ecotoxicity assays with organisms of different trophic levels were used to evaluate the toxic effects Ag NPs. Synthetically produced Ag NPs of different sizes (Ag 20 and 200 nm) as well as Ag NPs synthesized by a biological method (using plant leaf extracts of Ocinum sanctum and Azadirachta indica, Ag 23 and 27 nm, respectively) were used as model particles in order to elucidate the relation between size, synthesis method, NP surface properties, ion dissolution and toxicity. Based on earlier indications of interference of another type of NPs with the multi xenobiotic resistance mechanism (MXR), a first line of defense against xenobiotics, the effects of Ag NPs on the MXR mechanism were studied as well. The MXR mechanism is present in all animals, including humans and aquatic organisms. MXR can be compromised by chemical agents that are structurally and chemically unrelated, and interference with MXR could be the basis for enhanced toxicity by contaminant mixtures. A fast in vitro cellular efflux pump inhibition assay (CEPIA) was established evaluating first the effects of contaminants commonly found in the environment. Next, an in vivo CEPIA assay was established using the juvenile D. magna model aquatic organism and the potential of the MXR modulation by Ag NPs and ionic Ag was quantified in vitro and in vivo. This integrated approach revealed that the size and the synthesis method are the factors affecting most the uptake and toxicity in both cells in vitro as well as in vivo in freshwater crustaceans (daphnids) and dissolution in the different media with the biologically synthesized Ag NPs being more potent compared to the conventional Ag NPs. The gastrointestinal tract is expected to be a target site for Ag NPs exposure and a co-culture of Caco-2-TC7 and HT29-MTX cells optimized and employed in the current study represents a more realistic model compared to Caco-2 monocultures. The mucus layer provides an additional protective barrier and its absence can lead to overestimation of effects in in vitro studies. Ag was detected both in the cells in co-culture, in the gut of daphnids’ as well as specific areas, seemingly developing oocytes, indicating a potential translocation of Ag NPs that could have consequences for fecundity. MXR efflux transporters were found to be modulated by Ag at low concentrations (0.18 µg/L), that are slightly lower compared to the predicted environmental concentrations. The extent to which the Ag ions contribute to the effects of Ag NPs depends on the size and surface properties of the Ag NPs. For the conventional, uncoated Ag NPs, the Ag release is minimal and the size is the determining factor while for biologically synthesized particles the biomolecules present due to the synthesis method and Ag release affect most the uptake and effects. The simultaneous presence of Ag ions and NPs releasing ions can lead to an exacerbation of the effects. The proteomic approach was successfully applied and it proved to be a useful technique in discerning subtle cellular changes in response to Ag NP exposure that would otherwise be unnoticed. Ag NPs 20 nm regulated different sets of proteins with a distinct pattern of cellular responses compared to Ag 200 nm and AgNO3, suggesting a different mode of action with effects being particle- and size-dependent. These results obtained during this thesis are promising for future toxicity testing of new materials using invertebrate organisms and more realistic in vitro models leading to more meaningful results and more accurate assessment of Ag NP hazards.

National audience

Across different species, cellular efflux pumps such as P-glycoprotein (P-gp; also termed multidrug resistance protein 1 [MDR1]) serve as a first line of defense by transporting toxic xenobiotics out of the cell. This mechanism is also active in aquatic organisms such as mussels, fish, and their larvae. Modulation of this resistance mechanism by chemical agents occurring in the environment could result in either higher or lower internal concentrations of toxic or endogenous compounds in cells. The aim of the present study was to explore and quantify the inhibition of the P-gp efflux pumps by several ubiquitous aquatic contaminants. The calcein-acetoxymethyl ester (calcein-AM) assay commonly used in pharmacological research was established with P-gp–overexpressing Madin–Darby canine kidney cells (MDCKII–MDR1) in a 96-well plate, avoiding extra washing, centrifugation, and lysis steps. This calcein-AM–based P-gp cellular efflux pump inhibition assay (CEPIA) was used to study the inhibition by commonly occurring environmental contaminants. Among others, the compounds pentachlorophenol, perfluorooctane sulfonate, and perfluorooctanoate strongly inhibited the P-gp–mediated efflux of calcein-AM while the chloninated alkanes did not seem to interact with the transporter. The fact that common pollutants can be potent modulators of the efflux transporters is a motive to further study whether this increases the toxicity of other contaminants present in the same matrices.

Across different species, cellular efflux pumps such as P-glycoprotein (P-gp; also termed multidrug resistance protein 1 [MDR1]) serve as a first line of defense by transporting toxic xenobiotics out of the cell. This mechanism is also active in aquatic organisms such as mussels, fish, and their larvae. Modulation of this resistance mechanism by chemical agents occurring in the environment could result in either higher or lower internal concentrations of toxic or endogenous compounds in cells. The aim of the present study was to explore and quantify the inhibition of the P-gp efflux pumps by several ubiquitous aquatic contaminants. The calcein-acetoxymethyl ester (calcein-AM) assay commonly used in pharmacological research was established with P-gp-overexpressing Madin-Darby canine kidney cells (MDCKII-MDR1) in a 96-well plate, avoiding extra washing, centrifugation, and lysis steps. This calcein-AM-based P-gp cellular efflux pump inhibition assay (CEPIA) was used to study the inhibition by commonly occurring environmental contaminants. Among others, the compounds pentachlorophenol, perfluorooctane sulfonate, and perfluorooctanoate strongly inhibited the P-gp-mediated efflux of calcein-AM while the chloninated alkanes did not seem to interact with the transporter. The fact that common pollutants can be potent modulators of the efflux transporters is a motive to further study whether this increases the toxicity of other contaminants present in the same matrices.

International audience

The rapid expansion of nanotechnology promises to have significant benefits to society, yet there is increasing concern that exposure to nanoparticles (particles typically in vitro and in vivo uptake, biodistribution, and toxicity of AgNPs. Emphasis is placed on the systematization of data over animal and cell models, organs examined, doses applied, the type of particle administration, and the time of examination.