A template wizard for the cocreation of machine-readable data-reporting to harmonize the evaluation of (nano)materials.

Making research data findable, accessible, interoperable and reusable (FAIR) is typically hampered by a lack of skills in technical aspects of data management by data generators and a lack of resources. We developed a Template Wizard for researchers to easily create templates suitable for consistently capturing data and metadata from their experiments. The templates are easy to use and enable the compilation of machine-readable metadata to accompany data generation and align them to existing community standards and databases, such as eNanoMapper, streamlining the adoption of the FAIR principles. These templates are citable objects and are available as online tools. The Template Wizard is designed to be user friendly and facilitates using and reusing existing templates for new projects or project extensions. The wizard is accompanied by an online template validator, which allows self-evaluation of the template (to ensure mapping to the data schema and machine readability of the captured data) and transformation by an open-source parser into machine-readable formats, compliant with the FAIR principles. The templates are based on extensive collective experience in nanosafety data collection and include over 60 harmonized data entry templates for physicochemical characterization and hazard assessment (cell viability, genotoxicity, environmental organism dose-response tests, omics), as well as exposure and release studies. The templates are generalizable across fields and have already been extended and adapted for microplastics and advanced materials research. The harmonized templates improve the reliability of interlaboratory comparisons, data reuse and meta-analyses and can facilitate the safety evaluation and regulation process for (nano) materials.

Toxic Effects of Different Coating-Related Functionalized Nanoparticles on Aquatic Organisms.

The peculiar physico-chemical characteristics of nanomaterials (NMs) and the use of different coatings to improve their expected properties result in a huge amount of nanoforms, which vary in chemical composition, size, shape and surface characteristics. This makes it almost impossible to test all the nanoforms available, and efforts have been made to establish grouping or read-across strategies. The aim of this work was to find a behavior pattern of effect among nanoforms of different metallic core nanoparticles (NPs) (TiO2, CeO2 and Ag NP) with the same coatings (sodium citrate, poly (ethylene glycol), dodecylphosphonic acid or oleylamine). Daphnia magna, rainbow trout and two fish cell lines (PLHC-1 and RTH-149) were exposed to a range of concentrations (up to 100 mg/L) of the uncoated or coated NPs. Ag NPs were the most toxic, followed by CeO2 NPs and finally by TiO2 NPs. The results show that a clear pattern of toxicity in the studied species could not be established related to the coatings. However, it was possible to confirm different inter-species sensitivities. RTH-149 was the most sensitive cell line, and Daphnia magna was more sensitive than fish. Moreover, some differences in coating-core interactions were found between the metal oxide and the metal NPs in Daphnia magna.

Fish acute toxicity of nine nanomaterials: Need of pre-tests to ensure comparability and reuse of data.

Fish acute toxicity tests are commonly used in aquatic environmental risk assessments, being required in different international substances regulations. A general trend in the toxicity testing of nanomaterials (NMs) has been to use standardized aquatic toxicity tests. However, as these tests were primarily developed for soluble chemical, issues regarding particle dissolution, agglomeration or sedimentation during the time of exposure are not considered when reporting the toxicity of NMs. The aim of this study was to characterize the NM behaviour throughout the fish acute test and to provide criteria to assay the toxicity of nine NMs based on TiO2, ZnO, SiO2, BaSO4, bentonite, and carbon nanotubes, on rainbow trout following OECD Test Guideline (TG) nº203. Our results showed the importance of conducting a preliminary test (without fish) when working with NMs. They provide valuable information on, sample monitoring, agglomeration, sedimentation, dissolution, actual concentrations of NMs, needed to design the test. Among the NMs tested, only bentonite nanoparticles were stable during the 96-h pre-test and test in aquarium water. In contrast, the remaining NMs exhibited considerable loss and sedimentation within the first 24 h. The high sedimentation observed for almost all NMs highlights the need of consistently measuring the concentrations throughout the entire duration of the fish acute toxicity test to make reliable concentration-response relationships. Notable differences emerged in LC50 values when using actual concentrations as nominal concentrations overestimated concentrations by up to 85.6%. Among all NMs tested, only ZnO NMs were toxic to rainbow trout. A flow chart was specifically developed for OECD TG 203, aiding users in making informed decisions regarding the selection of test systems and necessary modifications to ensure accurate, reliable, and reusable toxicity data. Our findings might contribute to the harmonization of TG 203 improving result reproducibility and interpretability and supporting the development of read-across and QSAR models.

The Potentiating Effect of Graphene Oxide on the Arylhydrocarbon Receptor (AhR)-Cytochrome P4501A (Cyp1A) System Activated by Benzo(k)fluoranthene (BkF) in Rainbow Trout Cell Line.

The increasing use of graphene oxide (GO) will result in its release into the environment; therefore, it is essential to determine its final fate and possible metabolism by organisms. The objective of this study was to assess the possible role of the aryl hydrocarbon receptor (AhR)-dependent cytochrome P4501A (Cyp1A) detoxification activities on the catabolism of GO. Our hypothesis is that GO cannot initially interact with the AhR, but that after an initial degradation caused by other mechanisms, small fractions of GO could activate the AhR, inducing Cyp1A. The environmental pollutant benzo(k)fluoranthene (BkF) was used for the initial activation of the AhR in the rainbow trout (Oncorhynchus mykiss) cell line RTL-W1. Pre-, co-, and post-exposure experiments with GO were performed and Cyp1A induction was monitored. The strong stimulation of Cyp1A observed in cells after exposure to GO, when BkF levels were not detected in the system, suggests a direct action of GO. The role of the AhR was confirmed by a blockage of the observed effects in co-treatment experiments with αNF (an AhR antagonist). These results suggest a possible role for the AhR and Cyp1A system in the cellular metabolism of GO and that GO could modulate the toxicity of environmental pollutants.

Towards harmonisation of testing of nanomaterials for EU regulatory requirements on chemical safety - A proposal for further actions.

Over the recent years, EU chemicals legislation, guidance and test guidelines have been developed or adapted for nanomaterials to facilitate safe use of nanomaterials. This paper provides an overview of the information requirements across different EU regulatory areas. For each information requirement, a group of 22 experts identified potential needs for further action to accommodate guidance and test guidelines to nanomaterials. Eleven different needs for action were identified, capturing twenty-two information requirements that are specific to nanomaterials and relevant to multiple regulatory areas. These were further reduced to three overarching issues: 1) resolve issues around nanomaterial dispersion stability and dosing in toxicity testing, in particular for human health endpoints, 2) further develop tests or guidance on degradation and transformation of organic nanomaterials or nanomaterials with organic components, and 3) further develop tests and guidance to measure (a)cellular reactivity of nanomaterials. Efforts towards addressing these issues will result in better fit-for-purpose test methods for (EU) regulatory compliance. Moreover, it secures validity of hazard and risk assessments of nanomaterials. The results of the study accentuate the need for a structural process of identification of information needs and knowledge generation, preferably as part of risk governance and closely connected to technological innovation policy.

Bioaccumulation of CuO nanomaterials in rainbow trout: Influence of exposure route and particle shape.

The bioaccumulation potential of spherical and rod-shaped CuO nanomaterials (NMs) was assessed in rainbow trout (Oncorhynchus mykiss) exposed via water and diet following the OECD Test Guideline No. 305. Fish were exposed via diet to both NMs at concentrations of 70 and 500 mg Cu/kg for 15 days, followed by 44 days of depuration. For water-borne exposure, only the rod-shaped CuO NMs were tested at 0.08 and 0.8 mg Cu/L for 28 days, followed by 14 days of depuration. The concentration of Cu was determined in fish whole body to derive biomagnification and bioconcentration factors (BMF and BCF). Different tissues were sampled to investigate the total Cu biodistribution and target organs as well as the particle number-based bioaccumulation of CuO NMs. Estimated BMF and BCF values were below the thresholds of concern. However, shape and route influenced depuration. Following dietary exposure, there was a higher depuration of Cu from fish exposed to the rod-shaped compared to the spherical CuO NMs. A higher depuration was also observed for rod-shaped CuO NMs following the dietary exposure compared the aqueous one. Despite the much higher dietary exposure concentrations of rod-shape CuO NMs, similar Cu body burdens were reached via water. Cu was found in particulate form in different tissues. Although these NMs had a low bioaccumulation potential, differences in distribution and elimination patterns of Cu were observed depending on the exposure route and particle shape. Careful consideration of the most relevant exposure route is needed when designing a bioaccumulation experiment for testing NMs.

Simultaneous Determination of 15 Mycotoxins in Aquaculture Feed by Liquid Chromatography-Tandem Mass Spectrometry.

The use of plant-based fish feed may increase the risk of contamination by mycotoxins. The multiresidue analysis of mycotoxins in fish feed presents many difficulties due to the complexity of the matrix, the different characteristics of the compounds, and their presence in highly different concentration levels. The aim of this study was to develop a selective, sensitive, and efficient analytical method for the simultaneous determination of 15 mycotoxins (regulated and emerging mycotoxins) in aquaculture feed by LC-MS/MS. Sample extraction was performed with ultrasonic assistance, and different cleanup strategies were evaluated. The optimized method was composed by ultrasound-assisted extraction (two cycles, 55 °C, 20 min), followed by cleanup using a Captiva EMR Lipid cartridge. Then, nine commercial samples of aquaculture fish feed were analyzed. Eight of the 15 target mycotoxins were detected in the samples. Results showed that two enniatins (EENB and ENNB1), beauvericin, and fumonisin B2 were detected in all samples. These results show the multi-mycotoxin contamination of fish feed, highlighting the need to improve current knowledge on the occurrence and toxicity of mycotoxins in fish feed, mainly the emerging ones.

Nanopharmaceuticals (Au-NPs) after use: Experiences with a complex higher tier test design simulating environmental fate and effect.

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.

Cytotoxicity of Mycotoxins Frequently Present in Aquafeeds to the Fish Cell Line RTGill-W1.

In the last decades, the aquaculture industry has introduced plant-based ingredients as a source of protein in aquafeeds. This has led to mycotoxin contaminations, representing an ecological, health and economic problem. The aim of this study was to determine in the RTgill-W1 fish cell line the toxicity of fifteen mycotoxins of common occurrence in aquafeeds. To identify the most sensitive endpoint of toxicity, the triple assay was used. It consisted of three assays: alamarBlue, Neutral Red Uptake and CFDA-AM, which revealed the mitochondrial activity, the lysosomal integrity and the plasma membrane integrity, respectively. Most of the assayed mycotoxins were toxic predominantly at lysosomal level (enniatins, beauvericin, zearalenone, ochratoxin A, deoxynivalenol (DON) and its acetylated metabolites 15-O-acetyl-DON and 3-acetyl-DON). Aflatoxins B1 and B2 exerted the greatest effects at mitochondrial level, while fumonisins B1 and B2 and nivalenol were not toxic up to 100 µg/mL. In general, low toxicity was observed at plasma membrane level. The vast majority of the mycotoxins assayed exerted a pronounced acute effect in the fish RTgill-W1 cell line, emphasizing the need for further studies to ascertain the impact of mycotoxin contamination of fish feeds in the aquaculture industry and to establish safe limits in aquafeeds.

Preparation of feed with metal oxide nanoparticles for nanomaterial dietary exposure to fish and use in OECD TG 305.

The first step of nanomaterial accumulation in the aquatic environment is the uptake of particulate material. For substances with very low water solubility, exposure via water may be of limited relevance in comparison to the dietary route. The OECD Test Guideline 305 for bioaccumulation testing in fish using dietary exposure recommends to add substances to fish food following methodologies normally used in aquaculture (e.g. with a corn or fish oil vehicle). The feasibility of using such an approach for the testing of manufactured nanomaterials (MNs), due to their unique physical characteristics and solubility, needs to be investigated. In this study an easy, cost-effective method to prepare metal oxide nanoparticle (NP) spiked feed to give the required dietary exposure concentration to fish is described. Metal oxide NP (CeO2,TiO2 and ZnO) dispersions were prepared in oil (sunflower or olive oil) and used to soak fish feed pellets. NP surface deposition and homogeneity of distribution were analysed and confirmed. Discrepancies between nominal and measured concentrations highlighted the need to measure the achieved concentration in MN-spiked feed. The present method provides stable concentrations for bioaccumulation testing of MNs in fish through the dietary route. A method for•Fish feed preparation using nanomaterial-oil suspensions.•Homogenous spiking of nanomaterials on feed.•Nanomaterials stably maintained on feed immersed in water until eaten by fish.

The protective effect of stilbenes resveratrol and pterostilbene individually and combined with mycotoxin citrinin in human adenocarcinoma HT-29 cell line in vitro.

This study was focused to determine an individual and combined effect of mycotoxin citrinin (CIT) and two compounds of the stilbene family- resveratrol (RES) and his dimethyl ether analogue pterostilbene (PTE) which have many health benefits. As a model the human adenocarcinoma cell line HT-29 was used which may exhibits the properties of small intestine cells. Viability, plasma membrane integrity, lysosomal functionality, intracellular production of superoxide anions and superoxide dismutase activity were examined. The results indicate that concentrations of 50 and 100 µg/mL of the tested compounds were cytotoxic in mostly monitored parameters and probably caused apoptosis. HT-29 cells were more sensitive to PTE than to RES with a higher antioxidant effect of PTE than RES, which may be caused by its chemical structure. Both stilbenes at medium doses act as effective superoxide anions scavengers leading to reduction of oxidative stress and consequent cell damage. The nontoxic concentration of RES (25 µg/mL) protects the HT-29 cell line faced to the toxicity of CIT at 25 µg/mL by increasing viability of cells and by reducing the superoxide production induced by CIT concentrations of 12.5 µg/mL and 25 µg/mL.

Effect of Gamma-Radiation on Zearalenone-Degradation, Cytotoxicity and Estrogenicity.

Zearalenone (ZEA) is produced in cereals by different species of Fusarium, being a non-steroidal estrogenic mycotoxin. Despite having a low acute toxicity, ZEA strongly interferes with estrogen receptors. Gamma-radiation has been investigated to eliminate mycotoxins from food and feed, showing promising results. The present study aims to investigate the gamma-radiation effect on ZEA at different moisture conditions and to evaluate the cytotoxicity and estrogenicity of the irradiated ZEA. Different concentrations of dehydrated ZEA and aqueous solutions of ZEA were exposed to gamma-radiation doses ranging from 0.4 to 8.6 kGy and the mycotoxin concentration determined after exposure by high performance liquid chromatography (HPLC) with fluorescence detection. Following this, the cytotoxicity of irradiated samples was assessed in HepG2 cells, by measuring alterations of metabolic activity, plasma membrane integrity and lysosomal function, and their estrogenicity by measuring luciferase activity in HeLa 9903 cells. Gamma-radiation was found to be effective in reducing ZEA, with significant increases in degradation with increased moisture content. Furthermore, a reduction of cytotoxicity with irradiation was observed. ZEA estrogenicity was also increasingly reduced with increasing radiation doses, but mainly in aqueous solutions. These results suggest reduction of ZEA levels and of its toxicity in food and feed commodities may be achieved by irradiation.

Comparing in vivo data and in silico predictions for acute effects assessment of biocidal active substances and metabolites for aquatic organisms.

The purpose of this study was to determine the acute toxicity in aquatic organisms of one biocidal active substance and six metabolites derived from biocidal active substances and to assess the suitability of available QSAR models to predict the obtained values. We have reported the acute toxicity in sewage treatment plant (STP) microorganisms, in the freshwater microalgae Pseudokirchneriella subcapitata and in Daphnia magna following OECD test methods. We have also identified in silico models for acute toxicity of these trophic levels currently available in widely recognized platforms such as VEGA and the OECD QSAR ToolBox. A total of six, four and two models have been selected for Daphnia, algae and microorganisms, respectively. Finally, we have compared the in silico and in vivo data for the seven compounds plus two previously assayed biocidal substances. None of the compounds tested were toxic for Daphnia and STP microorganisms. For microalgae, CGA71019 (1,2,4 triazole) presented an ErC50 value of 38.3 mg/L. The selected in silico models have provided lower EC50 values and are therefore more conservative. Models from the OECD QSAR ToolBox predicted values for 7 out of 9 and for 4 out of 9 chemicals for Daphnia and P. subcapitata, respectively. No predictive models were identified in such platform for STP microorganism's acute effects. In terms of models's specificity, biocide-specific models, developed from curated datasets integrated by biocidal active substances and implemented in VEGA, perform better in the case of microalgae but for Daphnia an alternative, non biocide-specific has revealed a better performance. For STP microorganisms only biocide-specific models have been identified.

Acute toxic effects caused by the co-exposure of nanoparticles of ZnO and Cu in rainbow trout.

The toxic effects produced by the co-exposure to low- and non-toxic concentrations of zinc oxide (ZnONPs) and copper nanoparticles (CuNPs) was assessed in rainbow trout following the OECD Test Guideline 203. Four groups of trouts were exposed for 96 h to a range of concentrations (0.0425-0.34 mg/L) of CuNPs (50 nm) in combination with a fixed non-toxic concentration (1.25 mg/L) of ZnONPs (25 nm) determined from an independent concentration-response study. One additional group was exposed to the highest concentration of CuNPs alone. Behaviour and mortality were observed during the experiment. After 96 h exposure, accumulated levels of Cu and Zn in the fish were measured by ICP-MS and ICP-OES, respectively. The induction of oxidative stress in liver and gills was evaluated by the glutathione-S-transferase (GST) activity and the reduced glutathione (GSH) / oxidized glutathione (GSSG) ratio. The ethoxyresorufin-O-deethylase (EROD) activity was also assessed. The results showed that CuNPs at the highest tested concentration do not cause acute toxicity, whereas exposure to all mixtures caused mortality, which was inversely proportional to the concentration of CuNPs (from 28% to 86% survival). Accumulated levels of Cu and Zn in the fish increased with the increasing concentrations of CuNPs, suggesting that the presence of CuNPs favours the entry of Zn. In general, the GST activity increased significantly in the gills of co-exposed groups, whereas the GSH/GSSG ratio was altered in the liver. The EROD activity was not modified. In conclusion, the co-exposure to these NPs potentiates their toxicity, observing an alteration of the GST activity and GSH/GSSG ratio in gill and liver, which was more pronounced at the lowest concentration of CuNPs. The lower toxic effect observed with the highest concentrations of CuNPs coincides with a greater internalization of Zn.

Acute hazard of biocides for the aquatic environmental compartment from a life-cycle perspective.

One of the aims of the European project LIFE-COMBASE is to build a computational tool to predict the acute toxicity for aquatic organisms of biocidal active substances and its environmental degradation products. A database was implemented compiling toxicity data for these substances in organisms of the freshwater/marine and sewage treatment plant compartments. The goal of this study is to analyze the compiled data to identify the possible hazard of these compounds for the aquatic compartments. Several official and scientific databases were consulted. Data from 196 biocidal substances and 206 environmental metabolites were collected for the taxonomic groups, including fish, invertebrates, algae and sewage treatment plant (STP) microorganisms. Substances were categorized for their toxicity in four groups, considering values of L(E)C50, according to EU Regulation (EC) No 1272/2008. >50% of the parent were located in category 1 (L(E)C50 ≤ 1 mg/L) for fish, invertebrates and algae, indicating a high toxicity for the freshwater/marine compartments. However >60% were not toxic for STP microorganisms. Metabolites were mainly less toxic than the parent compounds, but 22-36% presented the same toxicity and ~6% were more toxic. No toxicological information was found for ~50% of the metabolites for fish, invertebrates and algae, reaching the 96% for the microorganisms. In addition, information on toxicity to the STP microorganisms was only found for 40% of the parent compounds. The high percentage of toxic metabolites and the scarcity of data for these compounds indicate the need to further study their impact in the aquatic compartments.

Gamma irradiation effects on ochratoxin A: Degradation, cytotoxicity and application in food.

Ochratoxin A (OTA) is one of the main mycotoxins that can be found in food. The use of gamma radiation is a technique for preserving food that may exert some effects on mycotoxins. OTA was irradiated in its dry form, in aqueous and in methanolic solutions, and in wheat flour, grape juice and wine. Additionally, the toxicity of OTA irradiated in water was tested. In aqueous solutions, more than 90% of the OTA was degraded by γ-radiation doses ≥2.5kGy, and a 2-fold reduction in OTA cytotoxicity was observed. In food matrices, the elimination of OTA by γ-radiation was found more difficult, as radiation doses of 30kGy eliminate at most 24% of the OTA. Higher moisture content of food matrices did not substantially increase OTA elimination. It is concluded that OTA is very sensitive to irradiation in water solutions but resistant in its dry form and in food matrices.

Negligible cytotoxicity induced by different titanium dioxide nanoparticles in fish cell lines.

Titanium dioxide nanoparticles (TiO2-NPs) have a wide number of applications in cosmetic, solar and paint industries due to their photocatalyst and ultraviolet blocking properties. The continuous increase in the production of TiO2-NPs enhances the risk for this manufactured nanomaterial to enter water bodies through treated effluents or agricultural amendments. TiO2-NPs have shown very low toxicity in a number of aquatic organisms. However, there are no conclusive data about their deleterious effects and on their possible mechanisms of toxic action. At this level, in vitro cell culture systems are a useful tool to gain insight about processes underlying the toxicity of a wide variety of substances, including nanomaterials. Differences in the physiology of different taxa make advisable the use of cells coming from the taxon of interest, but collecting data from a variety of cellular types allows a better understanding of the studied processes. Taking all this into account, the aim of the present study was to assess the toxicity of three types of TiO2-NP, rutile hydrophobic (NM-103), rutile hydrophilic (NM-104) and rutile-anatase (NM-105), obtained from the EU Joint Research Centre (JRC) repository, using various fish cell lines (RTG-2, PLHC-1, RTH-149, RTL-W1) and rainbow trout primary hepatocytes. For comparative purposes, the effect of different dispersion protocols, end-point assays and extended exposure time was studied in a fish cell line (RTG-2) and in the rat hepatoma cell line (H4IIE). TiO2-NPs dispersions showed a variable degree of aggregation in cell culture media. Disruption of mitochondrial metabolic activity, plasma membrane integrity and lysosome function was not detected in any cell line after exposure to TiO2-NPs at any time and concentration ranges tested. These results are indicative of a low toxicity of the TiO2-NPs tested and show the usefulness of fish cells maintained in vitro as high throughput screening methods that can facilitate further testing in the framework of integrated testing strategies.

Androgens and androgenic activity in broiler manure assessed by means of chemical analyses and in vitro bioassays.

The use of manure as an agricultural amendment is increasing the release of steroid hormones into the environment. Most research in this field has focused on estrogenic phenomena, with less attention paid to androgenic substances. The present study assessed androgenic activity in broiler manure using in vitro approaches based on cells stably transfected with androgen receptor. Leaching experiments were also performed to observe whether endocrine disruptors present in manure pass through a soil column and potentially reach groundwater. In parallel, an analytical chemistry method was used to determine the contribution of the most important natural androgens to androgenicity. Samplings were performed at 4 farms in 2 seasons. All but 2 samples showed androgen activity. In leakage experiments, however, no androgenic activity was detectable in leachates or in soils after leaching. According to the analytical results, androgenicity can be attributed mainly (but not completely) to androstenedione, and dihydrotestosterone. Similarly to the bioassays, chemical analysis did not reveal the presence of any androgen in leachates or soils. These results point to a rapid degradation of the substances responsible for androgenic activity in soils under the experimental conditions of the present study. However, the long-term effects associated with the constant and intensive application of manure to agricultural land require further attention. Environ Toxicol Chem 2017;36:1746-1754. © 2016 SETAC.

Mechanisms underlying the enhancement of toxicity caused by the coincubation of zinc oxide and copper nanoparticles in a fish hepatoma cell line.

Ecosystems are exposed to a wide variety of individual substances, including at the nano-scale; and the potential adverse effects of their interactions are an increasing concern. The purpose of the present study was to determine whether zinc oxide nanoparticles (ZnONPs) at a no-observed-effect concentration modulate the cytotoxicity of copper nanoparticles (CuNPs) in the fish hepatoma cell line PLHC-1 after 48 h of exposure and the contribution of the released ions to these effects. Cells were exposed to 50-nm CuNPs (0.39-25.0 µg/mL), alone or in combination with ZnONPs (25 nm or 100 nm), at 6.25 µg/mL. Cells were exposed to suspensions of NPs or to their supernatants, as well as to their combinations. The effects on cell viability were assessed through cytotoxicity assays. Changes in cell morphology and metal internalization were also evaluated. The cytotoxicity exerted by CuNPs was enhanced in the presence of nontoxic concentrations of ZnONPs. On the contrary, Zn ions protected the cell line from the CuNP toxicity, this effect being related to an increase in the intracellular levels of Zn. This increase of metal was not observed in cells exposed to both ZnONPs and CuNPs, even when they were visualized inside the cell. The results indicated that the internalization of ZnONPs, but not the Zn ions, was responsible for the enhanced toxicity of the CuNPs. Environ Toxicol Chem 2016;35:2562-2570. © 2016 SETAC.

Comparative Cytotoxicity Study of Silver Nanoparticles (AgNPs) in a Variety of Rainbow Trout Cell Lines (RTL-W1, RTH-149, RTG-2) and Primary Hepatocytes.

Among all classes of nanomaterials, silver nanoparticles (AgNPs) have potentially an important ecotoxicological impact, especially in freshwater environments. Fish are particularly susceptible to the toxic effects of silver ions and, with knowledge gaps regarding the contribution of dissolution and unique particle effects to AgNP toxicity, they represent a group of vulnerable organisms. Using cell lines (RTL-W1, RTH-149, RTG-2) and primary hepatocytes of rainbow trout (Oncorhynchus mykiss) as in vitro test systems, we assessed the cytotoxicity of the representative AgNP, NM-300K, and AgNO3 as an Ag+ ion source. Lack of AgNP interference with the cytotoxicity assays (AlamarBlue, CFDA-AM, NRU assay) and their simultaneous application point to the compatibility and usefulness of such a battery of assays. The RTH-149 and RTL-W1 liver cell lines exhibited similar sensitivity as primary hepatocytes towards AgNP toxicity. Leibovitz's L-15 culture medium composition (high amino acid content) had an important influence on the behaviour and toxicity of AgNPs towards the RTL-W1 cell line. The obtained results demonstrate that, with careful consideration, such an in vitro approach can provide valuable toxicological data to be used in an integrated testing strategy for NM-300K risk assessment.