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Environmental stressors are assessed through methods that quantify their impacts on a wide range of metrics including species density, growth rates, reproduction, behaviour and physiology, as on host-pathogen interactions and immunocompetence. Environmental stress may induce additional sublethal effects, like mutations and epigenetic signatures affecting offspring via germline mediated transgenerational inheritance, shaping phenotypic plasticity, increasing disease susceptibility, tissue pathologies, changes in social behaviour and biological invasions. The growing diversity of pollutants released into aquatic environments requires the development of a reliable, standardised and 3R (replacement, reduction and refinement of animals in research) compliant in vitro toolbox. The tools have to be in line with REACH regulation 1907/2006/EC, aiming to improve strategies for potential ecotoxicological risks assessment and monitoring of chemicals threatening human health and aquatic environments. Aquatic invertebrates' adult stem cells (ASCs) are numerous and can be pluripotent, as illustrated by high regeneration ability documented in many of these taxa. This is of further importance as in many aquatic invertebrate taxa, ASCs are able to differentiate into germ cells. Here we propose that ASCs from key aquatic invertebrates may be harnessed for applicable and standardised new tests in ecotoxicology. As part of this approach, a battery of modern techniques and endpoints are proposed to be tested for their ability to correctly identify environmental stresses posed by emerging contaminants in aquatic environments. Consequently, we briefly describe the current status of the available toxicity testing and biota-based monitoring strategies in aquatic environmental ecotoxicology and highlight some of the associated open issues such as replicability, consistency and reliability in the outcomes, for understanding and assessing the impacts of various chemicals on organisms and on the entire aquatic environment. Following this, we describe the benefits of aquatic invertebrate ASC-based tools for better addressing ecotoxicological questions, along with the current obstacles and possible overhaul approaches., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Colloidal silver products are sold for a wide range of disinfectant and health applications. This has increased the potential for human exposure to silver nanoparticles (AgNPs) and ions (Ag + ), for which oral ingestion is considered to be a major route of exposure. Our objective was to evaluate and compare the toxicity of two commercially available colloidal silver products on two human intestinal epithelial models under realistic exposure conditions. Mesosilver™ and AgC were characterized and a concentration range between 0.1 and 12 μg/mL chosen. Caco-2 cells vs. co-culture of Caco-2 and mucus-secreting HT29-MTX cells (90/10) were used. Repeated exposure was carried out to determine cell viability over 18 days of cell differentiation in 24-well plates. Selected concentrations (0.1, 1, and 3 μg/mL) were tested on cells cultured in E-plates and Transwells with the same repeated exposure regimen, to determine cell impedance, and cell viability and trans-epithelial electrical resistance (TEER), respectively. Silver uptake, intracellular localisation, and translocation were determined by CytoViva™, HIM-SIMS, and ICP-MS. Genotoxicity was determined on acutely-exposed proliferating Caco-2 cells by γH2AX immunofluorescence staining. Repeated exposure of a given concentration of AgC, which is composed solely of ionic silver, generally exerted more toxic effects on Caco-2 cells than Mesosilver™, which contains a mix of AgNPs and ionic silver. Due to its patchy structure, the presence of mucus in the Caco-2/HT29-MTX co-culture only slightly mitigated the deleterious effects on cell viability. Increased genotoxicity was observed for AgC on proliferating Caco-2 cells. Silver uptake, intracellular localisation, and translocation were similar. In conclusion, Mesosilver™ and AgC colloidal silver products show different levels of gut toxicity due to the forms of distinct silver (AgNPs and/or Ag + ) contained within. This study highlights the applicability of high-resolution (chemical) imaging to detect and localize silver and provides insights into its uptake mechanisms, intracellular fate and cellular effects., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Organic-inorganic perovskite solar cells (PSCs) are promising candidates as photovoltaic cells. Recently, they have attracted significant attention due to certified power conversion efficiencies exceeding 23%, low-cost engineering, and superior electrical/optical characteristics. These PSCs extensively utilize a perovskite-structured composite with a hybrid of Pb-based nanomaterials. Operation of them may cause the release of Pb-based nanoparticles. However, limited information is available regarding the potential toxicity of Pb-based PSCs on various organisms. This study conducted a battery of in vitro and in vivo toxicity bioassays for three quintessential Pb-based PSCs (CH 3 NH 3 PbI 3 , NHCHNH 3 PbBr 3 , and CH 3 NH 3 PbBr 3 ) using progressively more complex forms of life. For all species tested, the three different perovskites had comparable toxicities. The viability of Caco-2/TC7 cells was lower than that of A549 cells in response to Pb-based PSC exposure. Concentration-dependent toxicity was observed for the bioluminescent bacterium Vibrio fischeri, for soil bacterial communities, and for the nematode Caenorhabditis elegans. Neither of the tested Pb-based PSCs particles had apparent toxicity to Pseudomonas putida. Among all tested organisms, V. fischeri showed the highest sensitivity with EC 50 values (30 min of exposure) ranging from 1.45 to 2.91 mg L -1 . Therefore, this study recommends that V. fischeri should be preferably utilized to assess. PSC toxicity due to its increased sensitivity, low costs, and relatively high throughput in a 96-well format, compared with the other tested organisms. These results highlight that the developed assay can easily predict the toxic potency of PSCs. Consequently, this approach has the potential to promote the implementation of the 3Rs (Replacement, Reduction, and Refinement) principle in toxicology and decrease the dependence on animal testing when determining the safety of novel PSCs., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The specific and unique properties of silver nanoparticles (AgNPs) and gold nanoparticles (AuNPs), make them of high interest for different scientific and industrial applications. Their increasing use will inevitably lead to their release in the environment and aquatic ecosystems where they may represent a threat to aquatic organisms. Being a widespread and important component of the aquatic macroinvertebrate assemblage, amphipods and more specifically Gammarus fossarum will certainly be exposed to AgNPs and AuNPs. For these reasons, G. fossarum was selected as model organism for this study. The aim of the present work was the evaluation of the influence of both size (20, 40 and 80 nm) and surface coating (citrate CIT, polyethylene glycol PEG) on the acute toxicity of AgNPs and AuNPs on G. fossarum. We investigated the effects of AgNPs and AuNPs on the uptake by G. fossarum, NP tissue distribution and the expression of stress related genes by the use of ICP-MS, NanoSIMS50, Cytoviva®, and Rt-qPCR, respectively. Ag and Au bioaccumulation revealed a significant surface-coating dependence, with CIT-AgNPs and CIT-AuNPs showing the higher bio-accumulation potential in G. fossarum as compared to PEG-NPs. Opposite to that, no size-dependent effects on the bioaccumulation potential was observed. SIMS imaging and CytoViva® revealed an influence of the type of metal on the tissue distribution after uptake, with AgNPs detected in the cuticle and the gills of G. fossarum, while AuNPs were detected in the gut area. Furthermore, AgNPs were found to up-regulate CuZnSOD gene expression while AuNPs led to its down-regulation. Modulation of SOD may indicate generation of reactive species of oxygen and a possible activation of antioxidant defence in order to prevent and defend the organism from oxidative stress. However, further investigations are still needed to better define the mechanisms underlying the observed AgNPs and AuNPs effects., (Copyright © 2018 Elsevier B.V. All rights reserved.)

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 AgNO 3 , 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 AgNO 3 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., (Copyright © 2017 Elsevier B.V. All rights reserved.)

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., (Copyright © 2016 Elsevier B.V. All rights reserved.)

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., (Copyright © 2016 Elsevier B.V. All rights reserved.)