The overall aim of this work package is to identify the main body systems and modes of toxic effect of ENMs. WP7 will also provide in vivo data on translocation mechanisms and the uptake rates of ENMs for WP9. The work in this work package is carefully coordinated toxic effect of ENMs across a range of organisms from microbes to mammals.
This will provide in vivo- level data for the hazard classification of the ENMs by mode of toxic action on different body systems (e.g. respiratory toxicant, liver toxicity etc.) and by overall magnitude of toxicity (e.g. low toxicity, toxic, very toxic).
This traditional hazard classification approach is intended to provide both the regulatory agencies and industry with information in a format they can use now in their current procedures, and will therefore have high immediate impact.
In parallel, tissue samples from the experiments will be used for state-of- the-art molecular biology in WP10 and WP11 to develop a new systems biology predictive model that will be incorporated into the “ENM safety classifier” in WP12.
Outcomes of the workpackage
The key role of WP7 in the NANOSOLUTIONS project was to provide in vivo data on the exposure of a wide range of organisms to ENMs. In addition, the work package also aimed to generate tissue samples that would be utilized for molecular biology (global RNA sequencing and proteomics) by other WPs in the project. All of the data has subsequently been used to build and calibrate the Nanosafety Classifier tool. The specific tasks in WP7 included; (i) In vivo exposures of test organisms to ENMs (task 7.1), (ii) mechanistic investigations on ex-vivo samples to determine mode of action and details of the biological effects (task 7.2), and (iii) accumulation and uptake kinetics studies to understand the bioaccumulation risk from ENMs (task 7.3). All the work has been achieved and the in vivo data has been made available to the EU Nanosafety data hub. Experiments were conducted on several model organisms. These were the soil bacterium Escherichia coli K-12 strain, earthworms (Eisenia fetida), a nematode worm that lives in the soil (Caenorhabditis elegans), the freshwater invertebrate (Daphnia magna), the edible marine mussel (Mytilus edulis), freshwater-adapted rainbow trout (Oncorhynchus mykiss), zebrafishes (Danio rerio), and mice.
One of the central scientific hypothesis in NANOSOLUTIONS was that the surface coating rather than the core chemistry should impart the biological effect. The experiments demonstrated some differential effects of the coatings. However, the effects were not consistent across materials or by model organisms. Furthermore, traditional toxicological end points (survival, growth, reproduction) and biochemical assays did not reveal a consistent ranking of the materials by coatings. In the zebrafish studies, it was possible to cluster data by material-type and possibly by coating using proteomics, provided the data were carefully sorted for the life stage. Regardless of the coating, overall the CuO, Ag NPs, and CdTe QD materials were the most toxic, while nanodiamonds and the TiO2 materials had limited toxicity across all the model organisms tested. The toxic effects of MWCNTs were generally moderate, between these extremes. The observations were generally consistent with ranking toxicity by the chemistry of the core. The toxic mechanisms observed in the experiments included known modes of toxicity including oxidative stress, ionoregulatory toxicity, genotoxicity, inflammation, immune effects, and bioenergetics effects on growth and/or reproduction. There was no evidence of a single nano-specific or novel toxic mechanism.
Bioaccumulation and uptake kinetic studies were also conducted. The effort focused on the organisms that were large enough to dissect in order to obtain tissues for internal uptake measurements, or where a whole body burden could be measured. The studies especially focused on the CuO materials in earthworms, Daphnia magna, marine mussels and in zebrafish, as these materials were previously shown to be toxic to these organisms and the total Cu could be readily detected. Some data on TiO2 in marine mussels was also obtained. Overall, the net Cu accumulation (uptake) from the CuO material exposures was broadly the same magnitude as that of the metal salt, CuSO4, although the latter was a little faster and achieved at lower doses in the fish and earthworm studies.
In earthworms the CuO materials were cleared, as measured by decreasing body burdens over time in clean media. The apparent nano bioaccumulation factors (nBAF) were <1 indicating that the materials are not bioaccumulative in earthworms. This may be expected for an essential metal with a well-documented homeostatic mechanism in animal cells. In earthworms, there were no clear differences in the accumulation rates for different coatings; except for the CuO-PEG, which was slower. However, the accumulated Cu from the CuO-PEG exposures also had slow clearance from the body, and so the bioaccumulation risk was heading towards the hazard threshold with a nBAF of 0.8. In the zebrafish studies, concentration-dependent accumulation was seen in all treatments for the ENMs, but there was an important life-stage effect; with greater apparent uptake in the embryos compared to larvae. Critically, this was the opposite to the life stage effect of CuSO4. The marine mussels showed some limited accumulation of either Cu or Ti from the relevant ENM exposures, and it is likely that the ionic strength of the seawater as well as the ability of the animals to secrete mucous materials leading to sloughing of biodeposits containing the ENMs prevented larger accumulation. The D. magna studies examined apparent body burdens over 24 h uptake or clearance experiments. Some apparent increases and losses were observed, but may be best explained by rapid adsorption/desorption phenomena rather than in true internalisation of the materials in the tissues. Nonetheless, the apparent uptake plots for most organisms fitted a rectangular hyperbola and therefore showed the expected features of uptake kinetics curves. Overall, the bioaccumulation experiments demonstrated that modified OECD methods provide data that can be fitted to uptake and excretion curves, and identify parameters that are analogous to the bioaccumulation factors for soluble chemicals, although the basis of the observations here are from colloid chemistry. In conclusion, the WP7 in vivo experiments on a range of model organism was achieved, with the analysis of data from proteomics and RNA sequencing for the Nanosafety Classifier in other WPs. It was possible to identify ENMs of toxicological concern, the modes of action and the bioaccumulation potential; but there were no clear coating-dependent effect across all the organisms or all materials.
WP7 therefore brings a significant amount of in vivo animal data to the subsequent modelling and predictions.