Reduced bioavailability of Au and isotopically enriched 109Ag nanoparticles transformed through a pilot wastewater treatment plant in Hyallela azteca under environmentally relevant exposure scenarios.

Wastewater Treatment Plants (WWTP) are a major repository and entrance path of nanoparticles (NP) in the environment and hence play a major role in the final fate and toxicity to biota of NP in the aquatic environment. Studies on silver nanoparticles (AgNP) transport via the WWTP system and uptake by aquatic biota have so far been carried out using unrealistically high AgNP concentrations, unlikely to be encountered in the aquatic environment. The use of high AgNP concentrations is necessitated by both the low sensitivity of the detection methods used and the need to distinguish background Ag from spiked AgNP. In this study, isotopically enriched 109AgNP were synthesized to overcome these shortcomings and characterized by a broad range of methods including transmission electron microscopy, dynamic and electrophoretic light scattering. 109AgNP and gold NP (AuNP) were spiked to a pilot wastewater treatment plant fed with municipal wastewater for up to 21 days. AuNP were used as chemically less reactive tracer. The uptake of the pristine and transformed NP present in the effluent was assessed using the benthic amphipod Hyalella azteca in fresh- and brackish water exposures at environmentally relevant concentrations of 30 to 500 ng Au/L and 39 to 260 ng Ag/L. The unique isotopic signature of the 109AgNP allowed to detect the material at environmentally relevant concentrations in the presence of a much higher natural Ag background. The results show that the transformations reduce the NP uptake at environmentally relevant exposure concentrations. For 109Ag, lower accumulation factors (AF) were obtained after exposure to transformed NP (250-350) compared to the AF values obtained for pristine 109AgNP (750-840). The reduced AF values observed for H. azteca exposed to effluent from the AuNP-spiked WWTP indicate that biological transformation processes (e.g. eco-corona formation) seem to be involved in addition to chemical transformation.

Wastewater-Aged Silver Nanoparticles in Single and Combined Exposures with Titanium Dioxide Affect the Early Development of the Marine Copepod Tisbe battagliai.

In this study, the effects of aged Ag and TiO2 nanoparticles (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 were observed after 6 days of exposure to aged Ag NPs, with a twofold 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 and 378 µg/L, respectively). In coexposures with TiO2 NPs, higher dissolved Ag levels were measured for aged NPs (238.3 µg/L) relative to pristine NPs (98.57 µg/L). Coexposure resulted in a slight decrease (15%) in the Ag NP EC50 (270 µg/L) with a 1.9-fold increase in the 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.

Characterization of cell responses in Rhodomonas baltica exposed to PMMA nanoplastics.

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.

Determining the risk of calcium oxide (CaO) particle exposure to marine organisms.

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 (<0.8 mm) and coarse (<2.5 mm) CaO particles to a range of marine species from different taxonomic groups: two echinoderms (Asterias ruben and Strongylocentrotus droebachiensis); two crustaceans (Carcinus maenas and Tisbe battagliai); two molluscs (Mytilus edulis and Hinia reticulata); a polychaete (Nereis pelagica); a fish (Cyclopterus sp.); and seaweed germlings (Fucus vesiculosus). Overall, the fine CaO particles were more toxic to the selected marine species than the coarse particles. Coarse CaO particle effects were only observed in four of the nine species tested (A. rubens, S. droebachiensis, N. pelagica, T. battagliai) with similar LC50 values between 207 and 268 g/m2. For the fine CaO particles, the lowest LC50 was for the epibenthic copepod (T. battagliai) at 3.14 g/m2, followed by the sea urchin (20.1 g/m2), starfish (22.2 g/m2), ragworm (29.6 g/m2), and netted dog whelk (41.9 g/m2). Lump sucker fish exhibited significant mortalities only at the highest fine CaO concentration tested (320 g/m2) and recorded an LC50 of 226 g/m2. The toxicity data were used to generate species sensitivity distributions (SSDs) for both fine and coarse CaO particles. The hazard concentrations for 5% of the species (HC5) calculated from the SSDs, based on NOEC values, for the coarse and fine particles were 35.5 and 1.5 g/m2 respectively. Using a recommended assessment factor of 5, the Predicted No Effect Concentration (PNEC) was calculated as 7.1 and 0.3 g/m2 for coarse and fine CaO particles respectively.

Ecotoxicological Effects of Transformed Silver and Titanium Dioxide Nanoparticles in the Effluent from a Lab-Scale Wastewater Treatment System.

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.

Fate and effects of silver nanoparticles on early life-stage development of zebrafish (Danio rerio) in comparison to silver nitrate.

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.

Inhibition of multixenobiotic resistance transporters (MXR) by silver nanoparticles and ions in vitro and in Daphnia magna.

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 27nm), Ag 200nm 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.

Gammarus fossarum (Crustacea, Amphipoda) as a model organism to study the effects of silver nanoparticles.

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.

Effects of silver nanoparticles and ions on a co-culture model for the gastrointestinal epithelium.

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.

P-gp efflux pump inhibition potential of common environmental contaminants determined in vitro.

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.

Ag nanoparticles: size- and surface-dependent effects on model aquatic organisms and uptake evaluation with NanoSIMS.

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.