Final Report Summary - ENNSATOX (Engineered Nanoparticle Impact on Aquatic Environments: Structure, Activity and Toxicology)
Executive Summary:
The ENNSATOX project focused on the testing and modelling of the biological activity of a discrete series of nanoparticles in the context of aqueous environments. These nanoparticles were amorphous SiO2, crystalline ZnO and crystalline TiO2. SiO2, ZnO and TiO2 were sourced commercially. Additional samples of ZnO were synthesised in-house. The particles were comprehensively physico-chemically characterised and dispersion and purification protocols developed. Subsequently the activity of the particles was tested through a tiered system of increasingly complex levels of biological organisation. The simplest systems employed were bio membrane models of supported phospholipid monolayers/bilayers and vesicles. More complex systems used in the project included: cell membranes, cell cultures, algae, crustaceans e.g. Daphnia, and Zebra fish. The tests were configured to address the general hypothesis that the activity of nanoparticles is mostly mediated through the biological membrane. To integrate data to answer this question there was a constant feedback of results between the different experiments (workpackages) to see whether the hypothesis held. At the same time, particularly with the simpler models, the activity of particles of different shape, size and functionality was analysed. Concurrently with the testing programme an extensive series of experiments was carried out examining the behaviour of the particles in environmental aqueous systems with particular focus on their (a) charge properties, (b) tendency to aggregate and (c) solubility and to establish how these processes affected their biological activity. A key feature of the ENNSATOX programme was the integration of the environmental aqueous behaviour of the particles and their bioaccumulation in single cells into a global simulation model. The ENNSATOX programme achieved all of its promised targets. The key outputs from ENNSATOX were the following. (1) Using the supported membrane models, a nanosensor has been developed which measures the biomembrane activity of particle suspensions in a high throughput (10 minutes per sample), on-line configuration. Its success is evidenced by the fact that it has been used by other projects within the EU NanoSafety Cluster for this purpose. Using the supported biomembrane models, a series of papers has very exactly established the relation between SiO2 particle size and the biomembrane activity of the particles. This has been extended to the other particles: ZnO and TiO2 to show a commonality in this mechanism. (2) ZnO and TiO2 particles have been very extensively characterised and their strong tendency to aggregate has been established. A very extensive study was carried out on the solubility of ZnO particles. This was also modelled and results of model and experiment agreed very closely. The smaller ZnO particles are more soluble even in aggregated form. (3) A comprehensive model was developed to simulate: (a) aggregation, (b) sedimentation and, (c) biouptake rates of particles into model cells. The simulation results from this model agreed with experimental results, (4) The in-vitro and in-vivo experiments provided a fundamental mechanistic study into the biological activity and toxicity of nanoparticles and the role of the biomembrane/membrane proteins in this. The individual effects of SiO2 and ZnO activity are very different at the lipid-bilayer and membrane protein levels, ZnO interacts with protein while SiO2 appears to coat biomembranes. As far as acute effects are concerned: SiO2 interacts with DNA in cultured cells to cause DNA damage though ZnO effects are clearly more toxic. In most cases, EM and phagocytosis assays showed that particle coating of biomembrane and penetration was found. In relation to TiO2, aggregates were rapidly formed in biological solutions and no toxic effects were seen. The data indicates that acute effects of SiO2 on organisms are unlikely although the selective action of ZnO on a critical protein ion channel was observed. (5) ZnO biological activity was such that at the biomembrane level particulate ZnO interacts in its uncoated form when it's particle size is small. However its propensity to aggregate and become coated in biological media means that particulate ZnO is not the significant toxic species. Its significant solubility to give Zn2+ ion and the weight per weight similar toxicity of Zn2+ to multicellular organisms indicates that it is the Zn2+ species which is the toxic agent. TiO2 has a strong tendency to aggregate so although the smaller size uncoated TiO2 is active at the biomembrane level, in biological media a low toxicity was observed to higher organisms.
Project Context and Objectives:
Synopsis, inspiration and ideas behind ENNSATOX
The use of engineered nanoparticles in cosmetics, pharmaceuticals, sensors and many other commercial applications has been growing exponentially over the past decade. EU and Member State research into the environmental impact of these materials, particularly in aquatic systems, is at an early stage. ENNSATOX has addressed this deficit through a comprehensive investigation relating the structure and functionality of well-characterised engineered nanoparticles to their biological activity in environmental aquatic systems. An integrated approach assessed the activity of the particles in a series of biological models of increasing complexity. Parallel environmental studies were carried out on the behaviour of the nanoparticles in natural waters and how they modified the particles' chemical reactivity, physical form and biological activity. A comprehensive theoretical model was also developed describing the environmental system as a series of biological compartments where particles transport between a) compartments by advection-diffusion and b) between phases by a transfer function. Following optimisation of the transfer functions a generic predictive model was derived for the environmental impact of each class of nanoparticle in aqueous systems. A generalised understanding of the dependence of the nanoparticle biological activity on its structure and functionality was obtained including the role and interaction of the biological membranes within organisms. ENNSATOX generated: 1) exploitable IP (nanosensor and ecotoxicology predictive software package); 2) set of standard protocols for assay of nanoparticle biological activity which can be later accredited; 3) global dissemination of results; 4) creation of an EU laboratory service; 5) tools and data to inform EU Regulation and the EC's code of conduct for responsible nanosciences and nanotechnologies research, ftp://ftp.cordis.europa.eu/pub/nanotechnology/docs/nanocode recommendationpe0894c08424_en.pdf.
Scientific, economic and societal context of ENNSATOX
Nanomaterials are becoming increasingly important in their applications and uses in many industries, consumer products and healthcare systems (The Nanotech Report, 6th Edition, Lux Inc, New York, 2008). Current worldwide sales of products incorporating nanomaterials are €1.1trillion and are expected to rise to €4.1 trillion by 2015. Engineered nanoparticles represent a major part of this growth. However, an understanding of their toxicological properties has not kept pace with the exponential rate of increase of research into their synthesis, characterisation and application. Research into their behaviour, impact and fate in aquatic environments is at a very early stage. Out of 14 funded FP5/FP6 nanotoxicology projects only one is dedicated fully to this area (”EU Nanotechnology R&D in Health and Environmental Impact of Nanoparticles”, report, http://cordis.europa.eu/nanotechnology/home.html Jan 2008). The aforementioned report details the member states with the largest number of nanotoxicity research projects as follows: UK (46), Switzerland (24) and Denmark (12) of which the numbers dedicated to the fate of nanoparticles and their impact in the aquatic environment are UK (6), Switzerland (2) and Denmark (1) respectively. The majority of risk studies are concentrated on airborne particulates. A similar situation is also seen in the recently updated US National Nanotechnology Initiative (NNI) Strategy for Nanotech-related Research for the Environment, Health and Safety Research, Feb 2008 http://www.nano.gov/NNI EHS Research Strategy.pdf where the focus for aquatic environmental research is into environmental transport mechanisms and standardisation of nanoparticles rather than their ecotoxicological effects. The toxic effects of nanomaterials are poorly understood and their effects on aquatic wildlife are largely unknown. In the absence of such basic toxicological information, it is difficult to set environmental quality standards or perform risk assessments for these materials. As a result two EU Member States have recently recommended a voluntary moratorium on the release of engineered nanoparticles into the environment backed by a voluntary reporting system. These Member States are Germany (Nanotechnology: Health and Environmental Risk of Nanoparticles, Joint Working Party Report, Aug 2006, http://www.baua.de/nn 49456/en/Topics-from-A-to-Z/Hazardous-Substances/Nanotechnology/pdf/draft-research-strategy.pdf and the UK (Council for Science & Technology Review of Government Progress of its Action Plan for Nanoscience and Technology, March 2007, http://www.baua.de/nn 49456/en/Topics-from-A-to-Z/Hazardous-Substances/Nanotechnology/pdf/draft-research-strategy.pdf) to be administered by the Department of Environment, Food and Rural Affairs (DEFRA). On 17 January 2008 the UK's Soil Association, the national organic food certification body, issued a complete moratorium on the use of engineered nanoparticles for organic food production (http://www.soilassociation.org). More recently, Poland et al. on 20 May 2008, described important findings relating the dimensional characteristics of carbon nanotube and inorganic fibres to the inability of macrophages to prevent mesothelioma risks in rat lungs. http://www.nature.com/nnano/journal/v3/n7/abs/nnano.2008.111.html. ENNSATOX addressed these critical uncertainties by seeking to relate the structure and functionality of a well known class of nanoparticles of varying morphology to their biological activity at successive levels of molecular, cellular and organism organisation. Its research focused in particular on the impact of nanoparticles on these biological systems in aqueous environments with relevance to the interpretation of their effects on ecosytems. The work programmes examined the importance of the biological membrane in the toxicology, interactions and bioaccumulation of nanoparticles in aquatic organisms. The study operated at a series of levels and took into account not only the responses of the individual organism to the specific agent but also related this to the mechanism of activity of the agent. This goal was achieved by engaging in a multidisciplinary approach and integrating the results into a multi component model. In so doing it filled an important knowledge gap and informed the EU's code of conduct for responsible nanosciences and nanotechnologies research,
ftp://ftp.cordis.europa.eu/pub/nanotechnology/docs/nanocoderecommendation-pe0894c08424en.pdf for the purpose of future regulation by the EU (REACH Directive) and Member states.
The underlying concept of the proposed research was to address the current uncertainty of nanoparticle toxicity and environmental impact using an integrated multidisciplinary approach. The philosophy of ENNSATOX's work plan was to initially produce and thoroughly characterise different morphologies and sizes of a model nanoparticle (ZnO) using the most advanced state-of-the-art methods in physical chemistry and microscopy. This was extended to additional classes of nanoparticles in particular SiO2 and TiO2. At the same time, the programme looked at the nanoparticles' activity in a series of biological models of increasing complexity and organisation. Next, the behaviour of the nanoparticles in environmentally relevant aquatic systems was examined see whether the environment altered the chemical and/or structural nature of these particles. Throughout the study an integrative model was used to plan the activities and at the end of the programme, a predictive mathematical model was developed incorporating all of the elucidated parameters.
The ENNSATOX hypothesis has been:
The biological activity and environmental impact of nanoparticles is directly dependent on their structure and functionality. By evaluating these relationships we can develop predictive models which can be deployed for statutory controls of nanoparticle use.
Toxicity assays were performed using in-vitro models of cell and tissue culture and in-vivo models of several different aquatic species of key indicator organisms. As part of this project, all the procedures for toxicity testing were selectively developed and optimised for nanoparticles. This meant that streamlined protocols for nanoparticle toxicity testing could formulated which can later be exploited as routine tests for nanomaterials. The biological membrane and its dependent mechanisms play important roles in nanoparticle toxicity for two reasons. Firstly the biological membrane forms the boundary of the living cell which nanoparticles will need to cross and, secondly, the biological membrane hosts many of the physiological processes such as respiration and nerve conduction and any disruption in its structure will lead to a disruption in the function of the incumbent processes. The effect of nanoparticles on biological membrane structure is entirely unknown as is the permeability of nanoparticles in cell membranes. This study therefore allocated considerable resources to look at the interaction of nanoparticles with biological membranes by using highly novel supported membrane models, real biological membranes (liposomes and cells) and whole cell and tissues (successive complexity). The model membranes represent the most basic model for nanoparticle interaction and delivered important preliminary structure-activity relationships which were used to guide the more complex in-vitro and in-vivo studies. Already one of the model membrane tests being deployed in this study is in the has been patented and licensed as a generalised toxicity testing procedure which can be applied to investigate the activity of nanoparticles (L.A.Nelson and A.Vakurov 10008A Nanoparticle Sensor PCT filed, published on 15/9/11 with ref: WO2011/110825, Due to enter national phase 9/10/12). A major outcome of this study has been the development of calibrated toxicity testing protocols for nanoparticle biological activity including the above patented procedure. A SETAC World Congress in Sydney (August 2008) had an extensive session on nanomaterials and it was apparent that there were many issues to be addressed concerning how the materials should be tested for biological activity and the mechanism of toxicity. ENNSATOX therefore has made advances which could be a significant asset commercially.
The objectives of ENNSATOX were configured as:
1) Sourcing and comprehensively characterising a representative group of nanoparticles: initially ZnO and later SiO2 and TiO2 and other metal oxides of varying morphology and dimension. In-house synthesis was limited to ZnO nanoparticles not easily obtainable commercially, or from other projects within the NanoSafety Cluster. Well defined production methods were used for particle synthesis. Characterisation proceeded as an iterative process throughout the programme. The outcome of this objective was the quality and quantity of the standardised particles produced.
2) Characterising the interaction of the nanoparticles with the following biological models: supported phospholipid membranes of increasing complexity, in-vitro models of cell and tissue culture, in-vivo models of several different species of key indicator organisms. A feature of this objective is the direct comparison of the effects in the different testing systems which leads to the general theory of nanoparticle biological activity.
3) Formulating direct and predictive structure-activity relationships between nanoparticle form and nanoparticle biological activity. Success in this objective was achieved following results from objectives 1 and 2 and was a central feature of ENNSATOX.
4) Analysing the behaviour and fate of nanoparticles and their impact on models of biota in environmental aquatic systems. This advanced on the initial structural-activity relationships by testing their application in the environmental aquatic situation.
5) Configuring a mathematical model for the behaviour of nanoparticles in aquatic environments taking account of their interactions with biota of increasing complexity. This objective quantified the interactions and served as a means of verifying and measuring objectives 1-4.
6) Drawing up standard procedures for the exploitation and dissemination of the results for statutory planning and accredited use. In order to accomplish the challenge ENNSATOX assembled a group of RTD performers of unprecedented excellence from across Europe.
Our consortium had outstanding capabilities and achievements in:
? Nanoparticle manipulation, synthesis and characterisation. (Leeds, Wageningen);
? Supported model membrane technology (Leeds, Naples, Wageningen);
? Environmental and molecular mathematical modelling (Lleida, Wageningen, Leeds, Antwerp);
? In vitro and in vivo biological models (Naples, Leeds, Antwerp, Wageningen, MBA);
? Surface and colloid chemistry (Leeds, Wageningen, Naples);
? Environmental impact assessment (Wageningen, Antwerp, MBA); and,
? Dissemination of best practice worldwide (MBA, SETAC).
The objectives directly address in an integrated manner the impact of the nanoparticles on the environment. Implicit in this is the approach towards understanding the environmental and biological fate, transport, and transformation of nanoparticles in various biological compartments in aquatic systems. It is clear that the above objectives incorporate investigations into the toxicokinetics, cellular and molecular mechanisms, behaviour and fate, bio-persistence and biokinetics of nanoparticles. This enabled a fundamental understanding of the exposure, behaviour, mechanisms, consequences and potential effects to various endpoints of nanoparticle-biological entities interactions to be completed.
Contained within the objectives the following important questions have been addressed:
? What is the dispersion and solubility of nanoparticles in water?
? What are the most likely routes of exposure for environmentally relevant species?
? Can nanoparticles interfere with critical physiological mechanisms in aquatic organisms?
? Can nanoparticles bioaccumulate in aquatic organisms?
? Can nanoparticles be metabolised to less toxic forms?
? What biomarkers are relevant for determining nanoparticle exposure levels?
? What end-points are significant for determining risk of nanoparticles?
? What are the mechanisms of toxicity of nanoparticles in environmentally relevant systems?
? Does the presence of nanoparticles in the environment affect the toxicity of other compounds and vice versa?
Finally and very importantly ENNSATOX addressed two of the most important ethical issues in modern technology:
1) The synthesis and use of any novel compound or material has repeatedly led to ethical problems in its use either related to human health or environmental quality or both. Two very well known examples are the use of DDT as an insecticide and the use of lead tetraethyl in petrol. It is essential therefore from an ethical point of view to know the precise environmental impact of any newly synthesised material before it can be deployed for commercial use. This project not only addresses precisely this issue regarding engineered nanoparticle deployment but it also sets a protocol for examining nanoparticles as and when they are developed.
2) ENNSATOX developed methods for evaluating the environmental and biological hazard of nanoparticles which implicitly decrease the use of animals in the bioassay. This is ethically preferable in accordance with the three Rs concept of reduction, refinement and replacement in animal testing. Eventually toxicity assays will be limited to rapid in-vitro techniques and the use of predictive mathematical models using parameters developed from structure-activity models.
Project Results:
Please note that the figures refer to the attached Summary Information document.
The scientific (RTD) activities are conducted within seven work packages (WP1-7), with two other work packages being specifically concerned with exploitation/IPR and pre-validation (WP7), and dissemination (WP8):
WP1: Synthesis and characterisation of a selected group of nanoparticles:
To keep the study focused, three groups of nanoparticles have been examined: silicon dioxide (SiO2), zinc oxide (ZnO) and titanium dioxide (TiO2), of different morphology and dimension. Although Leeds is responsible for the synthesis, sourcing and processing of the nanoparticles, their characterisation has been cross calibrated with Wageningen. Nanoparticle characterisation in the in vitro, in vivo and aquatic systems was carried out throughout the programme as and when appropriate (WPs 2, 3, 4 and 5) in order to follow their behaviour and fate in the respective systems. Figure 1 shows a characteristic image from this study of “home' synthesised ZnO nanoparticles. The following paragraphs list the most significant achievements of WP1.
We have assembled (sourced and in-house synthesis) a very useful set of industrially relevant well defined and thoroughly characterised OECD nanoparticles (silica, zinc oxide and titania) which vary in terms of size (5 – 700 nm), aspect ratio (e.g. zinc oxide needles), crystallinity (silica – amorphous, zinc oxide crystalline and titania- crystalline polymorphs) and chemistry (both nanoparticle type and surfactant chemistry). We have developed and implemented relatively simple cleaning methods for these particles. We have investigated their dispersion characteristics in a range of media relevant to toxicological testing using a combination of existing (DLS, Zeta Potential) and novel (TEM) techniques. Furthermore these sets of particles are postulated to have different mechanisms for inducing toxicological responses (ZnO – soluble zinc, titania – redox processes, silica – membrane disruption). Using our Nanoparticle Characterisation Protocol (NCP) we have undertaken a thorough investigation of a range of commercial ZnO samples and as a result, successfully synthesised ZnO nanoparticles with narrower size and shape distributions using a polyol mediated precipitation synthesis– an example image of which is shown below in figure 3.
Hydrothermal synthesis was also employed to produce ZnO nanoparticles which were also characterised using the NCP. The following reaction parameters were varied in order to control particle size and shape: zinc precursor type (acetate, chloride) and concentration; reaction temperature and time; starting pH and base species (sodium carbonate, sodium hydroxide, ammonium hydroxide). The physical characteristics of a selection of particles were presented to the consortium meeting in September 2011 for consideration by partners. Nanorods produced from aqueous zinc chloride and sodium carbonate were selected as the priority for circulation to all partners: EN-Z-7 (shown in figure 2 below), average dimensions ~ 80 nm (width) by ~ 700 nm (length), surface area 18 m2/g; wurtzite crystal structure; flocculate in distilled water (d90 < 1 mm) but can be deflocculated by ultrasonic agitation. Other shapes and sizes were supplied for comparative toxicity tests in UNIVLEEDS. These included: EN- Z- 8, plate-like particles, 35 nm by 270 nm from zinc acetate and sodium hydroxide; EN- Z-9, micro-rods ~ 1?m by ~ 6 ?m, from zinc acetate and ammonium hydroxide solutions. Additional research using high resolution TEM has been undertaken to investigate ZnO particle growth mechanisms. This has illustrated that rods grow from supersaturated solution by a sequential nucleation and growth mechanism. These new insights provide an understanding of why specific reaction variables, for example base-type and pH, have such a dramatic effect on particle morphology. This information will enable more accurate control of both size and shape of particles in future nanosafety research and a journal paper is currently near submission.
Revised handling procedures for ZnO powders have been formulated based on wet and dry ageing studies. These recommendations are designed to minimise physical and chemical changes between WP1 and subsequent WPs. In addition, Control protocols have been defined to distinguish solubilised zinc from NP ZnO in toxicity assays. See Deliverable 1.3.1 for both points.
We have successfully developed a range of new procedures to directly image nanoparticle dispersions using TEM via rapid plunge freezing and warming (and more recently gel formation, resin infiltration and sectioning). This will soon be in the open literature in ”Quantitative Characterization of Nanoparticle Agglomeration within Biological Media”, Nicole Hondow, Rik Brydson, Peiyi Wang, Mark D. Holton, M. Rowan Brown, Paul Rees, Huw D. Summers, and Andy Brown accepted for publication in Journal of Nanoparticle Research May 2012.
We have shown conclusively that a protein corona forms around ZnO nanoparticles following the addition of serum to cell growth media (see Figure 6c above) and that this aids dispersion (see Figure 7). This forms the basis of an accepted oral presentation at the European Microscopy Congress in September 2012 in Manchester (R. Wallace).
We have achieved considerable success at imaging and quantifying uptake of nanoparticles within cell sections. From the observation of uptake of silica nanoparticles at low temperatures we have obtained evidence that a novel mechanism of passive nanoparticle uptake could be in operation (i.e. not clathrin mediated endocytosis) Figure 7(a). Furthermore we have obtained rare evidence of the actual cellular uptake of ZnO nanoparticles within cells during the MTT assay (WP3) – this would imply that it is not necessarily just extracellular soluble zinc which is responsible for toxicity but the mechanism involves cellular contact and uptake of ZnO nanoparticles and then possible dissolution in low pH lysosomes Figure 7(b).
WP2: Interactions of different classes of nanoparticles with model membrane systems:
Leeds and Wageningen possess a whole suite of experimental model biological membrane systems of increasing levels of complexity (Figures 8 & 9). Wageningen have considerable expertise in surface and colloid chemistry and extensive expertise modelling membrane interactions, and were responsible therefore for correlating the model membrane-nanoparticle interactions with theoretical mathematical models using self consistent mean field theory. The form, structure and functionality of the particles has been related to their activity towards the model membrane systems. The NAPLES lab has examined the effects of nanoparticles at the level of single channels (HERG K+ channels). The principle is to understand how nanoparticles affect the organisation and fluidity of the biological membrane, how they influence the functioning of ion channels and enzymes located in the membrane environment and whether the nanoparticles are themselves permeable in the membrane structure. Figure 10 shows an image of SiO2 nanoparticles adsorbed on to a cyanobacterium membrane.
To test the biomembrane activity of SiO2 nanoparticles a custom built high throughput sensor has been developed, which is displayed in Figure 11. This device can assess the biomembrane activity of a nanoparticle dispersion in 10 minutes.
Interesting results have been found using this sensor. For instance, SiO2 nanoparticles have been shown to adsorb on the surface of phospholipid membranes, as seen in SEM images of silica nanoparticles on phospholipid monolayers on electrode surfaces (see Figure 12). The extent of contact of the SiO2 particles' surface with the phospholipid membrane surface determines the effect on the membrane's properties and is dependent on the particle size. This is very clearly displayed in Figure 13.
The most significant achievements of WP2 can be summarised in the following:
For three nanoparticles studied with the DOPC monolayer system: particle diameter is the predominant parameter which controls the interaction between the uncoated, disaggregated particle and the lipid layer. It was shown for SiO2 that this was solely a geometric effect relating to the packing of the particles on the phospholipid surface and was identical whether particles interacted with bilayers or supported monolayers. In the case of ZnO and TiO2 which are prone to aggregation, the interaction of the aggregates is less than that of the disaggregated particles but is also related to the primary particle size.
Charged NPs, such as the negatively-charged silica NPs, affect the structure and dynamics of lipid bilayers by a reorientation of the phospholipid head groups
Strongly charged lipid membranes repel charged particles in solution with physiological relevant ionic strength due to ion double layers that form around the lipid bilayers and nanoparticles.
NPs can make lipid vesicles leak when they strongly bind to the membrane. When the particles are in excess to the lipid bilayer, the disruption on interaction can be transient and might be due to a mechanism by which the vesicles release strain that is formed by the binding NP. When the lipids are in excess compared to the NP, leakage might occur if the interaction is very strong and the lipid folds around the NP. The latter is only possible if the interaction is very strong as is observed for SiO2.
A correlation has been found between vesicle leakage and the surface area ratio of particles and vesicles, indicating that size of NP plays an important role in this potential toxic interaction.
Nanoparticles can disrupt ion gradients across membrane without creating “large' pores.
WP3: Interactions with in-vitro models:
These studies are directed to nanoparticle interactions at both the cellular level and the tissue level. The test systems will be established on in-vitro models. The cellular level will include test systems ranging from tissues and cultured cells to DNA (Figure 14). The tissue level includes nerve axons from the squid consisting of a single axon and glia, and ascidian embryos (rapidly developing chordate embryos to 12 hrs). The principle is to understand how the nanoparticles affect the structure and function of these systems using both real time assays and electron microscopy. The in vitro work is led by Naples and is spread between Naples and Leeds (WP 3). Naples has extensive facilities in electron microscopy and biophysical and molecular biological techniques and considerable world expertise in electrophysiology. The effect of SiO2 nanoparticles on the development of ascidian larvae is shown in Figure 15.
Some of the most exciting recent work carried out by WP3 has been on the effect of ZnO particles on membrane proteins. NPs provided by WP2 were tested directly on HEK cells that heterologously express the hERG K+ channel. This gave us the opportunity of assessing the impact of the NPs on defined membrane proteins directly. The range of concentrations used was 0.1-10 ?g ml-1 for both SiO2 (dialyzed and non -dialyzed) and ZnO. Cells were held at -70 mV under voltage clamp and hERG K+ channels were activated by patterns of voltage steps which produced outward ionic currents which were subject to biophysical analysis (Figure 16).
The channel activity was stable for at least an hour without run-down although experiments were normally carried out in the first 20 minutes. Examination of the hERG current kinetics (activation /inactivation and peak currents revealed no effect of SiO2 up to 10 ?g mL-1 but a notable selective effect of ZnO on channel kinetics (Figure 17). To establish if this effect was due to release of Zn2+ ions from the NPs, we carried out experiments where increasing concentrations of ZnCl2 were added and the peak currents measured. As can be seen in Figure 16, increasing the concentration of Zn2+ begins to block the channel only in the mM range. The effect of the NPs in Figure 17 is the opposite to this, i.e. they increase the current. Therefore the NP effect cannot be due to residual Zn2+.
Although we were unable to demonstrate that there are predictable metrics for NPs our finding of a specific effect of ZnO on hERG allows us to predict a possible model for why ZnO interacts with this channel. hERG is one of the best characterized of membrane ion channels and it is well known that the inactivation gate of the channel resides in the outer loop that connects the fifth and sixth membrane spanning domains the channel.
Our analysis of the data reveals that ZnO alters the inactivation gate of hERG and thus the most likely explanation for our data is a model that predicts a charge interaction between the ZnO NP and this outer portion of the membrane protein (Figure 18).This also indicates strongly that there is a very close sub –nm distance between the NP and the protein and that this has to be occurring on the external face of the bilayer.
The consensus of WP3 underlines the differences between two very different aspects of “nano toxicity' true nano effects and the intrinsic toxicity generated by the breakdown/dynamic activity of NPs in solution. True nano effects can only be clearly identified in tests which have a maximum duration of not more than one hour. In this case action on cell physiology was noted only in the case of ZnO on biological membrane ion channels and in the case of SiO2 on phagocytosis. No effects were noted for other NPs. NPs undoubtedly bind to biological membranes and influence phagocytosis but we failed to note significant effects other than these mentioned above. With respect to longer time periods we can highlight the fact that ZnO is also the most active /toxic of the NPs tested. It causes more significantly toxic effects than the other NPs in cell viability and Comet assays, whereas TiO2 is relatively non- toxic. Equally a similar pattern was noted in the bacterial gene reporter assays. Neural network activity in C intestinalis was unaltered by the NP panel. In the former three tests we could not clearly establish a true nano effect from a nano breakdown effect or other action.
To summarise: The individual effects of action of SiO2 and ZnO are very different at the biomembrane and protein levels, ZnO interacts with protein while SiO2 appears to coat biomembranes but no interaction with biomembrane proteins was noted. As far as acute effects are concerned: SiO2 interacts with DNA in cultured cells to cause DNA damage though ZnO effects are clearly more toxic in most cases. The question remains as to how NP's access the DNA (directly or indirectly as the result of activation of secondary metabolites) and, when, and how these substances cross the cell membrane. This question has been addressed by EM and phagocytosis assays, where biomembrane coating and penetration was found but no damage was registered in the short term. In relation to TiO2, aggregates were rapidly formed in biological solutions and no toxic effects were seen. The data indicates that acute effects of SiO2 on organisms are unlikely which is actually the case as seen above, although the selective action of ZnO on the hERG channel (which is after all the major cardiovascular potassium channel) is notable especially as the dosage used is relatively low.
WP4: Interactions with in vivo models:
In vivo testing has been performed on at least eight different species to allow the construction of species sensitivity distributions for the selected nanoparticles. This also included three standard toxicity species of which the acute and chronic toxicity is well documented and characterised for a variety of toxicants (e.g. Chlorella, Daphnia, and Danio). The in vivo experiments addressed three main issues: namely bioavailability, accumulation and toxicity. A series of chronic experiments were erformed in which effects on growth and reproduction were determined. This work was led by Antwerp with an input from Anton Dohrn. Antwerp is one of the world leaders in molecular, cellular and whole-organism toxicology, and both experimental and predictive modelling. During Period 2 studies were concentrated on looking at the chronic toxicity and internalisation routes of nanoparticles. In a chronic exposure scenario over 21 days, the ZnO nanodispersion (Alpha Aesar NanoTek) is toxic to Daphnia magna at low concentrations (around 0.02 mg Zn/L), whereby especially the reproduction was affected. As it has already been observed in studies carried out in WP3, toxicity of the ZnO nanodispersion cannot be solely due to the Zn or Zn2+ toxicity (see Figure 19). Results also showed the uptake and internalisation of the ZnO nanoparticles in Ciona intestinalis (see Figure 20). However, under the tested conditions, algae and daphnids did not take up the particles. Figure 21 shows a dose response curve of the effect of SM30 SiO2 on a culture of freshwater algae.
Chronic toxicity was determined with a standardized test using Daphnia magna. Experiments with ZnO nano dispersion (40 wt.% in water colloidal dispersion, NanoTek, Alfa Aesar) revealed an EC50 for D. magna mortality of 0.02 mg Zn/L. Compared to literature data for Zn, which are ranging around an EC50 of 0.2 mg Zn/L, the nanoparticles seem to be more toxic than the metal itself.
A second chronic toxicity test revealed that for ZnO NanoSun, the EC50 was similar to the one for ZnCl. Both substances showed an EC50 around 0.1 mg Zn/L (Figure 22). Additionally, it came out that most of the nanoparticles dissolved to form Zn2+ ions. Just as the salt ZnCl2 did. However, a small fraction of nanoparticles appeared to aggregate and form particles of a size between 100 and 450 nm.
In order to determine and visualize the uptake and internalisation of the nanoparticles, the selected model species Pseudokirchneriella subcapitata (WP4/UA), Daphnia magna (WP4/UA) and Ciona intestinalis (WP3/SZN) were exposed to the ZnO nanodispersion during acute (P. subcapitata, D. magna, C. intestinalis) and chronic (D. magna) experiments. Exponentially growing algae, daphnids and tunicates were exposed at the acute level to the nanoparticles spiked in water. At the chronic level the effect of exposure route and environmental conditions (pH) on the uptake and internalisation of the particles in the daphnids was tested as well. Results showed that the ZnO nanoparticles were taken up by the species C. intestinalis and were able to penetrate cell structures. However, in P. subcapitata and D. magna no nanoparticles could be visualised with transmission electronic microscopy (TEM) techniques, neither at the acute, nor at the chronic level under different exposure route and environmental conditions.
WP5: Nanoparticle environmental impact:
The biophysicochemical behaviour of nanoparticles, and their ensuing bioavailability and toxicity characteristics, strongly depends on the nature and the extent of molecular interactions with organic and inorganic materials in the environment. Wageningen together with an input from Antwerp are responsible for analysing the influence of chemical conditions and binding of particular species on the biointeraction and bioaccumulation of nanoparticles. Wageningen has extensive experience in the relationship between the chemical speciation of dissolved and particulate material in “natural' waters and its bioavailability. They have studied how the nanoparticles and the nanoparticle-water interface is modified when they enter a typical aqueous environmental system such as river, estuarine and sea water and how this affects their biological activity. Experiments have been carried out in laboratory controlled and relevant microcosms. The rate of the actual transfer of oxide nanoparticles across the cell membrane of a few selected aquatic organisms (microorganisms, invertebrates and fish); in relation to their local speciation and the physicochemical conditions at the outer side of the biointerface has also been investigated. The alteration of the nanoparticles during the in vivo experiments described in (WP4) has been investigated and related to their effects. In the first half of the programme, studies have mainly concentrated on the surface chemistry of SiO2 particles and their interaction with the soluble heavy metal ion Pb2+.
From the work in WP5 and the corresponding deliverables some general conclusions can be drawn with respect to the impact of the NPs in the aquatic environment.
Characterisation of SiO2, ZnO and TiO2 particles using titration and electrokinetic data generally confirmed that with increasing salt concentration, the charge density of nanoparticles increases and the zeta-potential decreases. In contrast to SiO2 and TiO2, ZnO is susceptible to dissolution and ZnO dispersions are colloidally highly unstable, but adsorption of organic matter stabilizes against (further) aggregation.
Since SiO2 particles are negatively charged over the wide range of practically significant pH values, it may act as a transport carrier of positively charged compounds, in particular (toxic) divalent metal ions (like Pb2+ and Zn2+). A significant result is that Pb2+ ions form labile complexes with silica NPs, which implies that they can be important in the facilitated transport of these ions to bio-interfaces. The lability of the metal ion-NP complex decreases with increasing pH and decreasing ionic strengths. With respect to the transport of ions by NPs to biointerfaces, the kinetic model (predicting the normalized flux) can be used to extract relevant parameters from experimental data.
The charge of nanoparticles is important for their interactions with biointerfaces, such as biomembranes, cell walls and biofilms, which are generally negatively charged. Silica NPs are negatively charged, ZnO NPs carry a positive charge below pH 8-9 (i.e. under most environmental conditions), and TiO2 are positively charged below pH 5.5 and negatively charged above that pH. In WP2 is described how charged NPs, such as the negatively-charged silica NPs, affect the structure and dynamics of lipid bilayers by a reorientation of the phospholipid head groups and can make lipid bilayers leak.
Silica NPs form stable dispersions at not too high ionic strengths. We showed that these NPs can penetrate and accumulate in model biogels at environmental pH values (pH 5.5) both at high and low ionic strengths, despite the unfavourable electrostatic potential in the gel. Therefore, these NPs are available for uptake by organisms living in biofilm structures and/or surrounded by a cell wall. ZnO NPs, however, form aggregates under all conditions. Therefore, bioavailability and biouptake of these particles as such is very low, which seems to be corroborated by uptake and internalisation studies with a few selected aquatic organisms. In contrast to SiO2 and TiO2, however, ZnO particles dissolve in the aqueous medium very readily, especially at pH values of 7 and higher, and the dissolved Zn2+ ions can cause toxic effects.
WP6: Integrated Modelling:
No environmental toxicological study is complete unless the various parts are integrated together in a theoretical model. This is essential not only for planning the study but also for assessing the final transfer parameters. Such a process is continuously iterative throughout the investigation until towards the end of the study, when the parameters are completely optimised. As a result predictions as to the impact of the nanoparticles on the aquatic environmental ecological models which predicted the transport and fate of soluble contaminants can be determined. A compartmental model is being used where the compartments are represented by the cell membrane, total cell, cell organelles, tissue and model aquatic organisms.
WP6 initially examined both the rate of dissolution and the extent of dissolution of ZnO nanoparticles with relation to particle size and solution pH. The model took account of the Gibbs-Thornton effect which predicts that particles of smaller size are more soluble. The model was validated against experimental systems which used the AGNES technique to measure Zn2+ concentrations in ZnO particle dispersions and hence ZnO solubility. Figure 23 displays the results of these experiments.
Next WP6 looked at sedimentation and transport models of nanoparticles. The model of the sedimentation of nanoparticles is depicted in Figure 24.
This model was validated against DLS and UV-vis spectroscopy measurements in centrifugation/settling experiments using stable aggregates prepared as described in the previous section. These experiments proved that the effective size and density of the NP aggregates (due to their fractal structure) is determinant for their transport properties. The fitted size distributions agreed satisfactorily with DLS measurements (see Figure 25).
A biomembrane model shown in Figure 26 was constructed so that the transport of nanoparticles to cells and their internalisation could be simulated.
The model was tested against experimental uptake data (see Figure 27). Several conclusions were extracted in the case studies analysed, namely: i) the uptake in well dispersed NP systems (i.e. with relatively high diffusion coefficients) may not be limited by transport of the NPs through the diffusion layer, but NP aggregation is expected to decrease the flux of particles available to microorganisms in the case of large aggregates (control by diffusion); ii) adsorption to the membrane reaches a steady state relatively fast compared to internalization; iii) a plateau in the uptake can be observed for long times when the internalization and efflux fluxes equilibrate. The application of this model requires very well controlled experimental conditions and detailed information of the system (e.g. in situ measurement of NP aggregate sizes). On the other hand, the model provides a theoretically sound framework for the comparison of uptake tests in different systems (type of NPs and microorganism). It also allows an estimation of the relative amounts of particles adsorbed on the membrane and present in the intracellular compartments. This would be useful in the development of more involved models aiming at predicting toxic effects, under the assumption that this effect is proportional to the amount of NPs taken up.
To summarise the most important findings from WP6 are the following.
ZnO solubility studies carried out in conjunction with WP5 showed that the smaller ZnO particles with lower primary size are more soluble in water. In this analysis the theory was based on the Gibbs-Thornton effect and entirely agreed with experiment.
The reaction-diffusion problem of the transport of stable NPs (and/or the conveyed free ions) from bulk solution up to a given interface, coupled with possible chemical reactions in the diffusion layer, was solved numerically for different geometries and diffusion domains.
Models for the prediction of the kinetic and equilibrium solubility of ZnO NPs were developed as a function of environmental variables (pH, ionic strength) and particle structure (size).
The time evolution of the concentration profiles of NPs while sedimenting in aqueous dispersions as a function of aggregate size and density can be satisfactorily predicted from the diffusion-sedimentation model. This model can be used to estimate the fate and behaviour of NPs in natural waters.
The description of the NP transport was integrated into uptake models to describe the amount of particles internalized by a microorganism or cell culture as a function of membrane adsorption and permeation rate constants.
Multiparameter regression analysis was carried out using data from the Consortium to obtain information on the influence of physicochemical parameters such as size, dose or time of exposure on NP toxicity.
WP7: Exploitation and pre-validation:
The Marine Biological Association of the UK a leading environmental charitable organisation is leading this activity. MBA has a track record of co-ordinating EU contracts and of carrying out bioassays for developing environmental quality objectives, with expertise in transferring analytical technology and significant regulatory experience. This includes considering all the above issues as well as developing accredited toxicity tests and assays for NPs in the aquatic situation. An important output will be aiding environmental legislation on these materials. Another important outcome is guidance on as to an effective means of calibrating and accrediting the toxicity testing procedures being developed.
One of the most significant contributions of the WP6 to ENNSATOX was in the following activity. The MBA Developed and pre-validated cost-effective, rugged toxicity tests for engineered nanomaterials: A key development has been that of a microplate-based assay to measure the impact of NP on growth of marine phytoplankton. This has been completed fully on a range of SiO2, ZnO and TiO2 nanoparticles. A further development has been the adoption of a microplate- based assay to measure the impact of NP on Tisbe battagliai using lethality as an endpoint. This has been completed fully on a range of SiO2, ZnO and TiO2 nanoparticles below. In the third of this suite of standardized tests, the impact of NP on development of oyster larvae is quantified. This assay is also being used to compare activity of a range of SiO2, ZnO and TiO2 nanoparticles. These tests fulfil the rigorous conditions for toxicity testing as set out by the statutory agencies and are complementary to tests used by them for Direct Toxicity Assessment (DTA). The rationale for choosing to evaluate three separate tests is that it is unlikely that any single organism will prove to be a universal indicator. By selecting three different taxa and endpoints (lethality, growth, development) a comprehensive coverage of likely effects has been gained.
Effects of NP on growth (algae)
Experiments carried out with the unicellular micro-alga Isochrysis galbana to demonstrate that the assessment of NPs on algal growth may be achieved in microplate assays using absorbance at 440 nm or 680nm (normalised to a background absorbance at 750nm) as a surrogate measure of cell density. This assumption has been validated in a number of tests against actual counts of cell densities. Trials were performed to validate the microplate assay by determining the effect of a standard metal toxicant, Zn2+ (as zinc sulphate), on growth of Isochrysis galbana. Typical results show reduction in growth to be dose dependent with growth reduced by ~50% at 1µg l-1 Zn2+. Zn2+ (zinc sulphate) has been used as a positive control in each subsequent test with NP including: SiO2 NP (Ludox SM30, particle size 13-14nm, dialysed and undialysed, and other particles provided by the Consortium). Results for non-dialysed Ludox were comparable with those of dialysed samples, indicating that effects were largely due to NP. Overall, results for SiO2 suggest a hormesis-type trend - with the growth of algae being stimulated at low doses (up to 20 µg ml-1) and thereafter decreasing with increasing dose (see Figure 28).
The reduction in growth was highly significant at 100 µg ludox ml-1, relative to controls, and was comparable in scale to the suppression caused by the standard toxicant 1µg ml-1 Zn (as ZnS04). Hormesis is not uncommon in ecotoxicological studies although the explanation and mechanism in this particular example has yet to be discovered. It may be that nanoparticulate SiO2 is behaving as a nutrient (or carrier of nutrients) at low dose but exhibits toxicity above a threshold of ~ 40 µg ml-1. This overall non-linear response makes the calculation of EC50 complex and we recommend deriving EC50 levels only from the linear, inhibitory region of the dose-response curve. Ludox SM-30, purified by gel-filtration, gave similar results to those described above for dialysed and undialysed material. Somewhat unusual response behaviour was observed in trials with larger particle size SiO2 (150-200, mean 170 nm; Fibre Optics – AngstromSphere). At the highest dose tested (100µg ml-1), after a prolonged lag phase, hormesis was observed (days 4-5), but by day 7 any growth promotion had disappeared, suggesting an ability to destabilise “normal growth' patterns. ZnO nanoparticles tested show toxicity increasing linearly with Zn concentration in the exposure media and may be a function of dissolved Zn. Dialysis appears to have little effect on toxicity of these particles (see also Period 1 report). TiO2: Work on algal growth effects during Period 2 of the ENNSATOX project has focused on TiO2, as characterised and supplied by the Consortium. Tests have been performed on five different forms (EN-T-1 to EN-T-5). Because of the light absorbing characteristics of many of the Ti particles used, caution has to be applied in separating these artefacts from absorbances due to algal growth (by subtracting the contribution due to particles from that of the algal cells).
Effects of NP on mortality (copepods)
Much of the latter half of the project has also been devoted to developing and quality-assuring this standard assay. As with the Isochrysis growth assay this test has proved to be robust and reliable provided that cultures are well maintained and careful attention given to quality control issues. The cultures can be maintained for more than one year meaning that apart from this initial outlay, costs are relatively minimal and large numbers of individuals can be provided for a constant throughput of tests.
Highlights of the tests with T. battagliai were: the virtual absence of toxic effects during exposure to SiO2 nanoparticles (see Figure 29) (and low responses to TiO2 (see Figure 30)) at suspension concentrations of up to 100 µg/ml (LC50>100 µg/ml as Si). Exposures at concentrations higher than this were not reliable due to the optical density of the suspensions and inability to see copepods in the test suspensions. However, it was noticeable that at concentrations >40 µg/ml Si or Ti, T. battagliai appeared to produce more mucus, possibly indicative that the particles were irritant rather than toxic. Toxicity tests with a range of ZnO nanoparticulates showed much greater evidence of toxicity. Typical mortality curves for ZnO NP are shown in Figure 31 and contrast with the lack of mortality in Si and Ti NP exposures. Mean LC50 values for different ZnO particles tested fell within the range 0.036 to 0.47 µg/ml (as Zn).These data are to be included in the overall assessment of structure-activity relationships.
Effects of NP on development (oyster larvae Crassostrea gigas)
The technique of fertilising conditioned oyster eggs and producing synchronised, high quality 32-cell embryos for testing activity of nanoparticles has been completed successfully. Optimal densities of up to 500 embryos per 5ml microplate well have been used to compare the effect of nanoparticulate (EN-Z-4) and dissolved ionic Zn2+ on development to the D-larval stage over 24hr. The test measures the percentages of normally- and abnormally- developed larvae over this period. The calculated EC50 for EN-Z-4 ZnO and Zn2+, (based on percentage net response, PNR, relative to controls) were 0.31 and 0.236 µg/ml, respectively, expressed as Zn. Once again the fact that ZnO particles exhibit EC50s that are comparable to ionic zinc, suggest that the nanoparticles dissolve and exert their activity in seawater. Effects concentrations for Zn NP in oyster larvae were not dissimilar to those described for Tisbe battagliai but are lower than thresholds affecting growth in Isochrysis galbana suggesting that invertebrates exhibit greater sensitivity to Zn.
To conclude all three primary assays investigated present robust and reliable methods for assessing and comparing biological activity of ENP and this WP has successfully achieved its aim of prevalidation of practical techniques for aquatic toxicity testing. WP7 has collated all data from the toxicity tests throughout the consortium and compared it with their own measurements. Generally ZnO, SiO2 and TiO2 nanoparticle dispersions show contrasting toxicities with the alga Isochrysis galabana. The nanoparticles can be ranked in decreasing order of toxicity: ZnO >TiO2>> SiO2 (based on mean EC50 concentrations of 2.59 7.53 and 170 mg L-1 as Zn respectively). The toxicity of ZnO was similar in most examples to that of the positive control Zn2+ (0.8 mg L-1) implying that the toxicity of Zn is largely due to dissolved Zn2+ as shown by others (e.g. WP4). Dialysis/purification of Si and Zn samples had little influence on toxicity. These were the general consensus of the order of results of tests with organisms found throughout the consortium.
Potential Impact:
The tests, model programs, standards and knowledge created by ENNSATOX's unique interdisciplinary research have had substantial societal and economic impact through: 1) its relevance to the call; 2) enhancing the state-of-the-art of nanotoxicology science and 3) informing policy and regulation. This impact has been delivered by: a) a robust dissemination programme and public engagement; b) Its IP strategy and c) an exploitation management plan.
Relevance to the original call: FP7-NMP-2008-SMALL-2 (1.3.2)
(i) Better in vitro or in vivo methodologies for the regulatory demands for the safety assessment of nanotechnology products. In WPs 2, 3, 4, 5 and 7, model membrane, in vitro and in vivo tests have been used to assay nanoparticle biological activity and relate it to the nanoparticles' structure, activity and functionality. The results have been successfully cross-correlated with each other to assess the relevance of the model membrane assays for measuring biomembrane activity. Executing these assays in concert allowed the group to characterise the most cost-effective protocol for screening nanoparticles and nanomaterials. This protocol has represented a key deliverable of this study and will be subject to European and international accreditation after exhaustive calibration and standardisation trials. The validation of the results of all assays through the standard procedures in WP7 substantiated and confirmed the relevance of the procedures and reinforced their impact on regulatory controls.
The research has maintained a continuous and positive interaction with the development of REACH regulation. It has informed on the precise hazard of the nanomaterials tested at all levels of the assays and at all stages of the programme. The consortium has been in continuous contact with the European Chemicals Agency (ECHA), Helsinki, which maintains the database for the REACH system. The results of the research has supported the work of several key International Standardisation Organisation (ISO) Committees (see section (vi) below). ENNSATOX has maintained a key role in the NanoSafety Cluster (NSC). The co-ordinator attended and contributed to most of the NSC meetings during the life of the project. ENNSATOX also actively collaborated with other projects within the NSC including NanoFATE, ENPRA and NanoReTox.
(ii) Better understanding of the impact of the nanoparticles on health, safety and the environment. WP5 elucidated the effect of the specific nanoparticle sizes and morphologies on representative compartments in a representative aquatic environment. The impact of the nanoparticles has been assessed through their change in form and speciation as they were discharged into an aqueous system and their consequent effect on biological membrane structure and function. These studies were integrated with those on the influence of the nanoparticles on cell and tissue structure and function and on the whole organism. The quantitative relationship between the structure and functionality of the nanoparticles and their biological activity has been successfully determined. The most important structural factors in the activity are particle size and the nature of the particle surface. The resulting parameters have been inputted into the integrated model quantitatively describing the impact of the particles on the aqueous environment. The model now predicts the impact of a class of nanoparticles and/or nanomaterials on the aquatic environment if both the characteristics of the materials and the properties of the environment are known. The greater understanding and prediction of the impact of nanoparticles on the biota in the environment decreases the requirement for expensive and politically sensitive bioassays at all levels. WP2 has developed a test model membrane system, the NANOSENSOR, which has advanced an understanding of the health effects of NP. WP2 has significantly advanced the state-of-the-art and substituted tests for standard in vivo and in vitro toxicity assays for NP. Validation of WP2 toxicity assays has been a key impact in this project. The results of medium throughput testing in WP 3 show that membrane ion channels (proteins) can be a significant target for ZnO NPs. As the hERG channel is the major cardiac K+ channel that stablizes the heartbeat of mammals, these results will be followed up to see if this could be a suitable screen for NP-protein interactions and potential deterimental interaction of NPs at the level of cell signalling.
(iii) Future definition of appropriate measures, where needed. WP6 has delivered predictive models of the effect of nanoparticles on key biota in riverine, estuarine and seawater environments. An underlying theme of the investigation has been to relate structure and function to activity. These predictive models will enable future environmental quality objectives to be set and met and statutory controls to be introduced on a firm scientific basis. These predictive models relate to the discharge, both accidental and intended, of classes of engineered particles into the environment. The results and findings of the studies are indispensable to Member States environmental protection agencies, the European Environmental Agency (EEA) and internationally (e.g. the US Environmental Protection Agency).
(iv) Safe and cost-effective minimisation of the exposure of workers and consumers. The coordinating beneficiary (Leeds) through its research in nanoparticle manufacturing is a reference centre for the UK's National Health & Safety Executive for monitoring and safe handling of nanoparticles. It has supplied regular guidance to all beneficiaries. In addition WP6 has identified critical compartments and pathways by which biologically active nanoparticles represent significant hazard to human health and wildlife and thus steps can be identified to minimise exposure via the environment. If biologically active nanoparticles are present in river water abstracted for potable use we can now identify, through WPs 5 and 6, processes which could be used (e.g. nanoparticle flocculation and filtration) for use in the subsequent water treatment to eliminate this hazard.
The consortium has worked closely with Lang Tran from the Institute of Occupational Medicine (IOM), UK continuously informing them of new findings obtained on the impact of nanoparticles on the "health" of the aquatic environment. These results will be incorporated into the IOM's database and promoted through its ”Safenano” website – the world's most visited nanotoxicity E, H & S site.
(v) Sustainable and responsible development. Three aspects of sustainable and responsible development have been addressed by this project. These are: (a) living within environmental limits, (b) using sound science responsibly and (c) achieving a sustainable economy. In the context of this work the manufacture of nanoparticles should be carried out in a responsible and cost effective manner with a minimum of waste. This entails using nanoparticle synthesis in an energy efficient manner and minimising the manufacture of toxic material which can damage the environment. WP7 has enabled statutory policy to be developed regarding the environmentally safe, responsible and sustainable use of these materials and their subsequent secure disposal. The outcome of the study will ensure that the societal and environmental costs of nanoparticle use fall on those who impose them. The results of this study and the predictive models which are generated from them has greatly facilitated the reduction in animal testing of nanomaterials.
(vi) Support to research and regulation. The ENNSATOX research has identified key approaches to be followed regarding the biological activity of nanoparticles. WPs 2, 3, 4, 5 and 7 have carried out generic investigations on the activity of a selected group of nanoparticles towards elements of biological organisation of increasing complexity in an aquatic environment. Critical areas of interactions and modes of action have been identified for study in future research projects. Of equal importance has been the crucial information gained on the toxicology of different classes of nanoparticles in the aquatic environment. This information can be used to assist in the construction of statutory controls, risk assessments and regulations for the use of nanoparticle materials. The consortium's continuous engagement with the European Chemicals Agency (ECHA), (see sub-section (i) above) will inform this process and will enable any new hazards associated with nanoparticles and nanomaterials to be transferred to the REACH database. In particular the findings of the study are critically important to the REGULATION (EC) No 1907/2006 OF THE EUROPEAN PARLIAMENT AND OF THE COUNCIL. The following ISO Technical Committees (TC): 147 water Quality, 190 Soil Quality, 217 Cosmetics and 243 Consumer Product Safety will have direct interests in the results of this project. In particular the work will have a direct bearing on the considerations of ISO/TC 229 on nanotechnology safety
(vii) Implementation of the European Commission's Action Plan for Nanotechnology. The ENNSATOX project directly addresses Point-6 in the European Commission's Action Plan: i.e. "All applications and use of nanoscience and nanotechnologies must comply with the high level of public health, safety, consumers' and workers' protection, and environmental protection chosen by the Community". WP7 has directly provided data and tools, to assist in the implementation of the Action Plan, for identifying safety concerns regarding the use of nanomaterials and minimising exposure to workers in the environment. It has also contributed to the development of risk assessment procedures for the use of nanoparticles and their effect on the environment and examined and proposed adaptations of EU regulations regarding nanoparticle use. These new contributions to risk assessment procedures will be published on the following EU web-sites: http://cordis.europa.eu/nanotechnology/ src/publication_events.htm and http://cordis.europa.eu/ nanotechnology/actionplan.htm to enable their maximum dissemination and impact.
(viii) Reinforcement of the international dimension of European research within the 7th Framework Programme. The relationships between the structure and functionality and biological and chemical reactivity of nanoparticles in the environment is at very early stage. There is a clear need for rigorous studies and the findings to be made available internationally to achieve international consensus. From the results obtained by WPs 2, 3, 4, 5 and 6, WP7 has informed evidence-based regulation development for the safe use and transport of classes of nanoparticles within and across borders. SETAC has provided an important global forum for achieving this aim. It has very strong representation from both the EU and US.
Impact of the research on the state-of-the-art and key stakeholders
(i) Previous state-of-the-art: The state-of-the-art on nanotoxicology before the start of ENNSATOX was rather fragmentary. Many studies had been initiated but they tended to be carried out by specialist groups rather than interdisciplinary consortia. Much of the work was phenomological and tended to be contradictory. These studies lacked a complete understanding of the underlying mechanisms involved. Some of the problems arose due to a lack of communication between groups who manufacture and characterise nanoparticles and those who carry out the necessary bioassays to investigate nanoparticle biological activity. There was also a lack of communication between environmental scientists and toxicologists and environmental managers.
The methodologies used in the individual work packages in ENNSATOX have all been state-of-the-art. For example, the model membrane systems have been used to understand toxicological mechanisms with respect to dissolved compounds and also as sensors for toxicity. Mathematical models previously used to understand the transport, fate and impact of dissolved compounds in the environment, have now been employed to analyse the equivalent processes for nanoparticles. (The latter have been shown to behave in an intermediate way between dissolved and particulate species). This interdisciplinary approach is itself a new paradigm and a step-change in the state-of-the-art
(ii) Significant results from project:
ENNSATOX has delivered -
• comprehensive and physically meaningful structure-activity relationships on categorical groups of engineered nanoparticles,
• protocols for examining the short and long term risks of nanoparticles to aquatic biota,
• Evaluation and adaption of existing tests (e.g. Comet, cell death) and application of new tests (hERG assay, phagocytosis assay, developmental assay, species distributions).
• prototype demonstrators for in-situ toxicity testing of nanoparticles in particular the NANOSENSOR, and hERG assay.
• generic mathematical models which can be used as predictive tools to understand impact of nanoparticles introduced into the environment.
(iii) Impact of results on state-of-the-art:
The development of the structure-activity relationships has impacted strongly on the current nanotoxicological field by improving the fundamental understanding of toxicological mechanisms and uniting the current fragmentary studies, by providing a coherent theory of nanoparticle biological activity in aquatic systems.
Protocols have been developed which extend the impact of current methods of toxicity testing assays. In particular, ENNSATOX has extended the understanding of how model biomembrane systems interact with nanoparticles and how nanoparticles are transported through biomembranes. ENNSATOX has extended the capability of toxicity assays from dissolved compound activity to particle activity. The impact of the latter is potentially enormous. Their manipulability, ease of interrogation and low cost, will enhance future research, knowledge and support regulation.
Finally a major outcome of ENNSATOX has been the computer simulation models developed to predict the transport, fate and impact of nanoparticles in the aquatic environment. The application of these models to the behaviour of nanoparticles is particularly novel, will accelerate knowledge generation and will impact strongly on the state-of-the-art in all areas.
(iv) Lead Users of the results:
The lead users of the aforementioned ENNSATOX results are extensive and are listed as follows -
Water industry needs to know the hazard presented by engineered nanoparticles present in potable waters at all stages of treatment. Protocols to assay the toxicity of nanoparticles present in water will be important associated with the increased production of these materials.
Environmental and marine agencies need to know also the hazards presented by different classes of nanomaterials which might be discharged into river, estuarine and marine environments. In this context, all four categories of aforementioned results will have major potential usage by these bodies. The predictive mathematical models developed by ENNSATOX will have particular application.
Pharmaceutical and cosmetic companies will be especially concerned with the structure-activity relationships and also the protocols developed to determine these.
Government agencies and statutory bodies throughout the EU will need to know hazards presented by nanoparticles in aqueous environments in order to develop risk assessments for the nanomaterials' use. The results will also be used to help set discharge limits and environmental quality controls on nanoparticle use. The results will be extremely useful to these bodies in formulating policy to protect the environment. Protocols for nanoparticle toxicity assay will be accredited by these bodies for general use.
Commercial and academic nanomaterial and nanoparticle producers will use results of studies to formulate risk assessment procedures and disposal methods for the materials. Results will be incorporated into workers' protocol for nanomaterial handling.
The Defence industry throughout the EU including NATO is very interested in the toxicity of nanomaterials and nanoparticles where these are manufactured with terrorist ambitions. The defence industry is also interested into ways to detect hazardous materials used as weapons.
(v) Steps taken to ensure impact:
Results have and are being disseminated to the aforementioned bodies in (iv) through:
• conference participation by the principal investigators;
• seminars by participant PIs in universities and leading companies throughout the EU;
• communication via SETAC, an ENNSATOX beneficiary, specifically funded to promote the communication of the results;
• the ENNSATOX web-site: http://www.ennsatox.eu/?contentid=260;
• participation in ENNSATOX of the MBA, a leading international independent environmental research organisation, who have pre-validated the toxicity assays;
• publication in top quality peer-reviewed journals;
• through the ENNSATOX Lead User group committee;
• regular presentations and participation at the NST meetings and EU-US workshops.
(vi) Relevance of the results to the Lead User:
Water industry will use toxicity assays developed by consortium as standard tests for water at all stages of treatment where nanoparticle contamination is suspected. These toxicity assays have been developed rapid, effective and relevant and will save considerable time and money for the water companies throughout EU.
Environmental and marine agencies will use results on short and long term risks of nanomaterials and nanoparticles to set discharge limits of these materials into rivers, estuaries and seawaters and consequent environmental quality controls. These agencies will also use the mathematical models developed in WP6 to carry out predictions on the impact of nanoparticles on the aquatic environment and to advise commercial organisations and governments within the EU on this.
Pharmaceutical and cosmetic companies will use results on structure-activity relationships to predict hazard and risk from new nanomaterials and nanoparticles being synthesised. The companies will also use toxicity assays to characterise the activity of their own products. Current methods used by pharmaceutical and cosmetic companies to assess toxicity of dissolved compounds and nanomaterials are time consuming and expensive and often involve animal testing.
Government agencies and statutory bodies throughout the EU. These bodies will be especially interested in the results of the study for the purpose of setting statutory discharge limits and statutory handling procedures for the materials.
Commercial and academic nanomaterial and nanoparticle producers will use results of studies to formulate risk assessment procedures and disposal methods for the materials. Results will be incorporated into workers' protocol for nanomaterial handling.
Defence industry can use the results of the investigations to commission new, faster and simpler toxicity tests to assess any security risks. The predictive structure-activity theory and the global environmental mathematical model will be used to assess risks from a terrorist attack from toxic nanomaterials.
(vii) The results are being delivered to the Lead User in the following way:
Workshops and seminars have been held in participant laboratories with potential lead users. ENNSATOX has already held a one day KT workshop in September 2011 and a four day International Winter School in Plymouth in January 2012 in association with NanoFATE. SETAC has been the main contact point for the transfer of results to government, commercial and industrial sectors. New results and protocols arising from the programme has been transmitted to interested lead users via SETAC without infringing IP restrictions.
(viii) The transfer of the results will be funded
All the activities listed in (vii) have been covered by the dissemination funds. As well as SETAC's (beneficiary 7) budget, all beneficiaries have a small portion of budget set aside for dissemination.
(ix) Rationale for European participation in project
It would be impossible to have run this project at a national level. Its extensive interdisciplinary nature involving nanoparticle manufacture and characterisation, model membrane studies, in vitro and in vivo studies and experimental and theoretical environmental modelling will never be available in one European country. Such interdisciplinary studies have to call upon the best possible expertise from several European member states. This lack of collaboration previously has led to only limited and contradictory studies in nanotoxicology. The cross-border complimentarity of this study ensured a global generic approach which provided a step change to the understanding of toxicological mechanisms of nanoparticles. The spread of the beneficiaries through different EU countries enabled a maximum dissemination of the results to be made since the results can be used by lead users in all of the countries which are participating. This will ultimately lead to an increase in environmental quality, environmental health and safety of people in these countries.
(x) Basis for long term collaboration: ENNSATOX has reinforced existing links between beneficiaries who all individually have a broad based interest in the aquatic toxicology and biological activity of nanoparticles. The meetings, joint publications and joint inventions/patents have ensured a desire to continue work in this scientifically very interesting and high impact area. The impact of the results on the consortium itself has therefore also been an exploration of mechanisms of securing continuing funding for this work. Because of the strong knowledge transfer element associated with the study the future work could involve the commercialisation of the some of the deliverables from this study. These include: a toxicity sensing protocol/demonstrator for nanoparticles, simulation models for predicting transport, fate and impact of nanoparticles in aquatic environments and structure-activity models of nanoparticle interaction with biota. The co-ordinator and one of the ENNSATOX partners are now working on a fresh EU proposal to commercialise an environmental toxicity sensor for submission later this year.
Political, societal and economic impact
(i) Enhancing competitiveness of European industry
Nanotechnology and the development of new nanomaterials is the most exciting new area since the biotechnology explosion of the 1960 s and 1970s. It promises to bring new and extensive economic growth to world society as well as solving many of the existing problems of providing sustainable energy and eliminating CO2 build-up in the atmosphere. It is anticipated also that nanomaterials and nanoparticles will be increasingly used in health care and environmental control. However all new technologies can bring enormous risks to bear on both the public and the environment. Most of the risks associated with past new technologies in the last century were not anticipated and the risks and problems which were later found led to a detrimental effect on the technology in some cases leading to its withdrawal. Two typical examples of this are the nuclear energy industry and the use of chlorinated pesticides. Whatever the outcome, the late discovery of these risks decreased the competitiveness of these new technologies on the world playing field. A major political impact of this current project is to precisely deduce the environmental health risks of engineered nanoparticles in a generic manner so that steps can be introduced within Europe to decrease or eliminate these risks. This will allow the nanotechnology industries in Europe to deal in a responsible way with the environmental and health risk controls firmly in place. The results will also help in setting up a Joint Technology Initiative (JTI): (http://europa.eu/rapid/pressReleasesAction.do?reference=MEMO/07/570) in environmental nanotoxicology.
(ii) To increase European wide S & T collaboration
This project has brought together outstanding scientists in complimentary areas of expertise to work towards an objective of immense strategic importance. The impact of the programme has served to bind together the scientists more strongly leading to their joint involvement on working groups and committees regulating the use of nanoparticles and nanomaterials. The programme has also disseminated its results throughout Europe by regular presentations at and active involvement with the NST. Important collaborations with other EU funded projects have arisen from this increasing the level of integration of nanotoxicology within Europe.
(iii) To contribute to an increase in level of research investment
The EU, Member States and industries are making a huge investment in nanotechnology. To maintain confidence in this investment and harvesting the benefits, the knowledge produced by ENNSATOX will be essential for earning the confidence and support of all stakeholders, including citizens. In this way it will contribute towards the goal of a 3% annual increase in research funding (http://ec.europa.eu/invest-in-research/index_en.htm).
(iv) To improve the co-ordination of European, national and regional research policies
The rigour of ENNSATOX's research for understanding and characterising in a generic way the risk associated with the production of nanoparticles and their introduction into the environment represents a glue achieving European harmonisation in E, H & S nanotoxicological risk assessment strategies. Nanotoxicology has been noted as a major initiative for the EU Joint Research Commission (http://ec.europa.eu/dgs/jrc/index.cfm) and the results of this study can only help to consolidate this initiative.
(v) To strengthen the scientific excellence of basic research in Europe
ENNSATOX involved world leaders in their respective fields applying themselves to a highly strategic problem. All RTD performers carry out world leading fundamental research at the forefront of their areas. Cross-European collaboration can only enhance and strengthen their own research areas by introducing newer complimentary approaches. The unique feature of this project has been that the individual contributions from each beneficiary are very fundamental whereas the combination of the contributions forms a highly strategic and eventually applied objective. The objective is also greatly sustained by the combination of such important research areas and itself is at the forefront in the competition regarding scientific quality, adventure and impact. Such a multidisciplinary yet scientifically excellent study is a quantum leap ahead of other single or bi-disciplinary nanotoxicological studies.
(vi) To promote the development of European research careers
The ENNSATOX multidisciplinary research has attracted researchers to work in the best scientific centres. This programme has stimulated young researchers to work in fields of excellence within a multidisciplinary framework essential in the present climate of research and technology. Research workers have been trained at the highest level in nanoparticle manufacturing and characterisation, biophysical and environmental mathematical modelling, surface and colloid chemistry, supported biomembranes and in vitro and in vivo bioassays. All these disciplines have been brought together to improve an understanding into the biological activity of nanoparticles. The diversity of the disciplines with this clear common objective has had a powerful impact on young researchers at the beginning of their careers. It has transformed the researchers into highly flexible scientists with a wide ranging knowledge but a capacity to excel in their own fields. They now have competitive career advantages over young researchers. ENNSATOX has been very successful at not only training its funded staff and students in this interesting area but also at bringing in other participants not funded by the project to work on associated problems.
(vii) To provide the knowledge-base to support key Community policies
The enlargement of the knowledge-base in nanotoxicology remains one of the key priority areas of the EU science policy (see http://projects-2007.jrc.ec.europa.eu/show.gx? Object.object_id=PROJECTS0000000003007403). The impact of the results from ENNSATOX have facilitated a step increase to the EU nanotoxicology knowledge-base. ENNSATOX has delivered analytical protocols, technical demonstrators e.g. the NANOSENSOR and predictive mathematical simulations for coping with the increased regulations and risk assessments generated by this increased knowledge. This in its turn has led to a reassessment of the current state-of-the-art concerns about nanoparticle and nanomaterials safety.
(viii) To increase availability, co-ordination and access in relation to top-level European scientific and technological infrastructure
This programme has brought the global approach to nanotoxicology into the European scientific theatre. Through the generation of interesting findings it has enabled the scientists and research workers to communicate their findings to other workers in Europe outside the consortium especially within the NST framework. This has stimulated new collaborations within the EU and allowed access to the highest level scientific and technical infrastructure where this becomes available.
(ix) Refinement and restructuring of risk assessment models concerning nanoparticle and nanomaterial safety
The widened understanding and predictive capability developed from this programme has enabled protocols on nanomaterial and nanoparticle safety to be renewed. Where generalities have been found through straightforward structure-activity models (such as the inverse relationship between particle size and biomembrane activity), risk assessment procedures can be simplified leading to cost savings in nanomaterial and nanoparticle production.
(x) Societal concern over use of new materials and chemicals
European society has become progressively disillusioned with the promoted use of new technologies. In all cases there are often un-researched hazards associated with the new technologies which cause the public to lose confidence in their proposed benefits. One very good example is the emergence of GM crop production to solve world food shortages which has later been shown to be linked to many potential hazards. The ENNSATOX research has been timely and coincides with the emergence of nanotechnology and nanomanufacturing as the promoted "panacea" of the 21st century. The research has generated results which specify exactly the hazards of new nanomaterials and nanoparticles. This will increase the public confidence in the use of these materials by society and in the long term ensure the success of the technology. To summarise:
The major technical and scientific impact are from:
• The combination of the most advanced techniques for the characterisation of nanoparticles with the most comprehensive and novel in vitro and in vivo assays of biological activity to develop structure-activity relationships which can guide future research activities in this area.
• The development of calibrated protocols for assaying nanoparticle biological activity which can be accredited for use by environmental agencies and the water industry.
• The characterisation of the importance of the biological membrane in nanoparticle toxicology in environmental aquatic systems.
• The development of generic theory which taking account of the nanoparticle structure and functionality and the biogeochemical characteristics of the impacted environment will predict the overall impact of the material on the said environment.
• The construction of structure-activity relationships of nanomaterial and nanoparticles in their biological action.
• The development of newer, more efficient and faster toxicity testing procedures which drastically reduce the need for the animal testing of nanoparticle biological activity.
The main environmental impact can be derived from:
• The assessment of the impact of discharges of nanoparticles into the aquatic environment so that future statutory environmental quality objectives can be met.
• The development of holistic calibrated mathematical models on the transport, behaviour, toxicology and fate of nanoparticles discharged into aquatic environments. These will be used by environmental agencies to predict the impact of specified groups of nanoparticles on the aquatic environment.
The statutory impact is from:
• The ability to develop risk assessment protocols on the environmental hazards of engineered nanoparticles of specified form and composition. This will have particular impact in that it will lead to decreased dependence on animal experimentation procedures to develop the said protocols.
• The precise description of the relative hazard and risk associated with the major classes of nanoparticles. This risk is especially important and significant to the configuration of this programme of work in that it describes the long term hazard of the material. Thus a particular material may appear quite safe when handled in its original form. After having passed through the various aquatic environmental and biological compartments it may have some unforeseen effect on the environment/human health which only the integrated and generic nature of this project can discover.
The political and economic impact can:
• Establish the EU as a responsible region of excellence in nanotoxicology, a discipline which has its roots in the diverse areas of fundamental science represented in the consortium. The results will facilitate the rapid development of nanotechnology unhindered by uninformed concerns over the putative health and environmental risks associated with it. The results will aid insurance actuaries to accurately evaluate the risks associated with working with nanomaterials and to set informed premiums
The societal impact can:
• Ensure that full public confidence is maintained at all times in Europe in the development and use of nanomaterials and nanoparticles. This will be done by continuous and objective dissemination of the results to the public throughout the programme of research.
The ethical impact can:
• address the potential long term public health hazard of a new technology in this case engineered nanoparticle deployment and to set a protocol for examining nanoparticles as and when they are developed.
• develop methods for evaluating the environmental and biological hazard of nanoparticles which implicitly decreases the use of animals in the bioassay. This is ethically preferable and meets the three Rs protocol. Eventually toxicity assays will be limited to rapid and relevant in-vitro techniques and the use of predictive mathematical models using parameters developed from structure-activity relationships.
NB: The coordinating beneficiary (Leeds) is associated with a unique Interdisciplinary Applied Ethics Research Centre skilled in the ethics associated with nanotechnology.
List of Websites:
ENgineered Nanoparticle Impact on Aquatic Environments: Structure, Activity and Toxicology
Contract Agreement: NMP4-SL-2009-229244
Website: http://www.ennsatox.eu
Coordinator:
Professor Andrew Nelson
School of Chemistry
University of Leeds
Leeds LS2 9JT, UK
Telephone: +44(0)113 343 6409
Email: a.l.nelson@leeds.ac.uk
The ENNSATOX project focused on the testing and modelling of the biological activity of a discrete series of nanoparticles in the context of aqueous environments. These nanoparticles were amorphous SiO2, crystalline ZnO and crystalline TiO2. SiO2, ZnO and TiO2 were sourced commercially. Additional samples of ZnO were synthesised in-house. The particles were comprehensively physico-chemically characterised and dispersion and purification protocols developed. Subsequently the activity of the particles was tested through a tiered system of increasingly complex levels of biological organisation. The simplest systems employed were bio membrane models of supported phospholipid monolayers/bilayers and vesicles. More complex systems used in the project included: cell membranes, cell cultures, algae, crustaceans e.g. Daphnia, and Zebra fish. The tests were configured to address the general hypothesis that the activity of nanoparticles is mostly mediated through the biological membrane. To integrate data to answer this question there was a constant feedback of results between the different experiments (workpackages) to see whether the hypothesis held. At the same time, particularly with the simpler models, the activity of particles of different shape, size and functionality was analysed. Concurrently with the testing programme an extensive series of experiments was carried out examining the behaviour of the particles in environmental aqueous systems with particular focus on their (a) charge properties, (b) tendency to aggregate and (c) solubility and to establish how these processes affected their biological activity. A key feature of the ENNSATOX programme was the integration of the environmental aqueous behaviour of the particles and their bioaccumulation in single cells into a global simulation model. The ENNSATOX programme achieved all of its promised targets. The key outputs from ENNSATOX were the following. (1) Using the supported membrane models, a nanosensor has been developed which measures the biomembrane activity of particle suspensions in a high throughput (10 minutes per sample), on-line configuration. Its success is evidenced by the fact that it has been used by other projects within the EU NanoSafety Cluster for this purpose. Using the supported biomembrane models, a series of papers has very exactly established the relation between SiO2 particle size and the biomembrane activity of the particles. This has been extended to the other particles: ZnO and TiO2 to show a commonality in this mechanism. (2) ZnO and TiO2 particles have been very extensively characterised and their strong tendency to aggregate has been established. A very extensive study was carried out on the solubility of ZnO particles. This was also modelled and results of model and experiment agreed very closely. The smaller ZnO particles are more soluble even in aggregated form. (3) A comprehensive model was developed to simulate: (a) aggregation, (b) sedimentation and, (c) biouptake rates of particles into model cells. The simulation results from this model agreed with experimental results, (4) The in-vitro and in-vivo experiments provided a fundamental mechanistic study into the biological activity and toxicity of nanoparticles and the role of the biomembrane/membrane proteins in this. The individual effects of SiO2 and ZnO activity are very different at the lipid-bilayer and membrane protein levels, ZnO interacts with protein while SiO2 appears to coat biomembranes. As far as acute effects are concerned: SiO2 interacts with DNA in cultured cells to cause DNA damage though ZnO effects are clearly more toxic. In most cases, EM and phagocytosis assays showed that particle coating of biomembrane and penetration was found. In relation to TiO2, aggregates were rapidly formed in biological solutions and no toxic effects were seen. The data indicates that acute effects of SiO2 on organisms are unlikely although the selective action of ZnO on a critical protein ion channel was observed. (5) ZnO biological activity was such that at the biomembrane level particulate ZnO interacts in its uncoated form when it's particle size is small. However its propensity to aggregate and become coated in biological media means that particulate ZnO is not the significant toxic species. Its significant solubility to give Zn2+ ion and the weight per weight similar toxicity of Zn2+ to multicellular organisms indicates that it is the Zn2+ species which is the toxic agent. TiO2 has a strong tendency to aggregate so although the smaller size uncoated TiO2 is active at the biomembrane level, in biological media a low toxicity was observed to higher organisms.
Project Context and Objectives:
Synopsis, inspiration and ideas behind ENNSATOX
The use of engineered nanoparticles in cosmetics, pharmaceuticals, sensors and many other commercial applications has been growing exponentially over the past decade. EU and Member State research into the environmental impact of these materials, particularly in aquatic systems, is at an early stage. ENNSATOX has addressed this deficit through a comprehensive investigation relating the structure and functionality of well-characterised engineered nanoparticles to their biological activity in environmental aquatic systems. An integrated approach assessed the activity of the particles in a series of biological models of increasing complexity. Parallel environmental studies were carried out on the behaviour of the nanoparticles in natural waters and how they modified the particles' chemical reactivity, physical form and biological activity. A comprehensive theoretical model was also developed describing the environmental system as a series of biological compartments where particles transport between a) compartments by advection-diffusion and b) between phases by a transfer function. Following optimisation of the transfer functions a generic predictive model was derived for the environmental impact of each class of nanoparticle in aqueous systems. A generalised understanding of the dependence of the nanoparticle biological activity on its structure and functionality was obtained including the role and interaction of the biological membranes within organisms. ENNSATOX generated: 1) exploitable IP (nanosensor and ecotoxicology predictive software package); 2) set of standard protocols for assay of nanoparticle biological activity which can be later accredited; 3) global dissemination of results; 4) creation of an EU laboratory service; 5) tools and data to inform EU Regulation and the EC's code of conduct for responsible nanosciences and nanotechnologies research, ftp://ftp.cordis.europa.eu/pub/nanotechnology/docs/nanocode recommendationpe0894c08424_en.pdf.
Scientific, economic and societal context of ENNSATOX
Nanomaterials are becoming increasingly important in their applications and uses in many industries, consumer products and healthcare systems (The Nanotech Report, 6th Edition, Lux Inc, New York, 2008). Current worldwide sales of products incorporating nanomaterials are €1.1trillion and are expected to rise to €4.1 trillion by 2015. Engineered nanoparticles represent a major part of this growth. However, an understanding of their toxicological properties has not kept pace with the exponential rate of increase of research into their synthesis, characterisation and application. Research into their behaviour, impact and fate in aquatic environments is at a very early stage. Out of 14 funded FP5/FP6 nanotoxicology projects only one is dedicated fully to this area (”EU Nanotechnology R&D in Health and Environmental Impact of Nanoparticles”, report, http://cordis.europa.eu/nanotechnology/home.html Jan 2008). The aforementioned report details the member states with the largest number of nanotoxicity research projects as follows: UK (46), Switzerland (24) and Denmark (12) of which the numbers dedicated to the fate of nanoparticles and their impact in the aquatic environment are UK (6), Switzerland (2) and Denmark (1) respectively. The majority of risk studies are concentrated on airborne particulates. A similar situation is also seen in the recently updated US National Nanotechnology Initiative (NNI) Strategy for Nanotech-related Research for the Environment, Health and Safety Research, Feb 2008 http://www.nano.gov/NNI EHS Research Strategy.pdf where the focus for aquatic environmental research is into environmental transport mechanisms and standardisation of nanoparticles rather than their ecotoxicological effects. The toxic effects of nanomaterials are poorly understood and their effects on aquatic wildlife are largely unknown. In the absence of such basic toxicological information, it is difficult to set environmental quality standards or perform risk assessments for these materials. As a result two EU Member States have recently recommended a voluntary moratorium on the release of engineered nanoparticles into the environment backed by a voluntary reporting system. These Member States are Germany (Nanotechnology: Health and Environmental Risk of Nanoparticles, Joint Working Party Report, Aug 2006, http://www.baua.de/nn 49456/en/Topics-from-A-to-Z/Hazardous-Substances/Nanotechnology/pdf/draft-research-strategy.pdf and the UK (Council for Science & Technology Review of Government Progress of its Action Plan for Nanoscience and Technology, March 2007, http://www.baua.de/nn 49456/en/Topics-from-A-to-Z/Hazardous-Substances/Nanotechnology/pdf/draft-research-strategy.pdf) to be administered by the Department of Environment, Food and Rural Affairs (DEFRA). On 17 January 2008 the UK's Soil Association, the national organic food certification body, issued a complete moratorium on the use of engineered nanoparticles for organic food production (http://www.soilassociation.org). More recently, Poland et al. on 20 May 2008, described important findings relating the dimensional characteristics of carbon nanotube and inorganic fibres to the inability of macrophages to prevent mesothelioma risks in rat lungs. http://www.nature.com/nnano/journal/v3/n7/abs/nnano.2008.111.html. ENNSATOX addressed these critical uncertainties by seeking to relate the structure and functionality of a well known class of nanoparticles of varying morphology to their biological activity at successive levels of molecular, cellular and organism organisation. Its research focused in particular on the impact of nanoparticles on these biological systems in aqueous environments with relevance to the interpretation of their effects on ecosytems. The work programmes examined the importance of the biological membrane in the toxicology, interactions and bioaccumulation of nanoparticles in aquatic organisms. The study operated at a series of levels and took into account not only the responses of the individual organism to the specific agent but also related this to the mechanism of activity of the agent. This goal was achieved by engaging in a multidisciplinary approach and integrating the results into a multi component model. In so doing it filled an important knowledge gap and informed the EU's code of conduct for responsible nanosciences and nanotechnologies research,
ftp://ftp.cordis.europa.eu/pub/nanotechnology/docs/nanocoderecommendation-pe0894c08424en.pdf for the purpose of future regulation by the EU (REACH Directive) and Member states.
The underlying concept of the proposed research was to address the current uncertainty of nanoparticle toxicity and environmental impact using an integrated multidisciplinary approach. The philosophy of ENNSATOX's work plan was to initially produce and thoroughly characterise different morphologies and sizes of a model nanoparticle (ZnO) using the most advanced state-of-the-art methods in physical chemistry and microscopy. This was extended to additional classes of nanoparticles in particular SiO2 and TiO2. At the same time, the programme looked at the nanoparticles' activity in a series of biological models of increasing complexity and organisation. Next, the behaviour of the nanoparticles in environmentally relevant aquatic systems was examined see whether the environment altered the chemical and/or structural nature of these particles. Throughout the study an integrative model was used to plan the activities and at the end of the programme, a predictive mathematical model was developed incorporating all of the elucidated parameters.
The ENNSATOX hypothesis has been:
The biological activity and environmental impact of nanoparticles is directly dependent on their structure and functionality. By evaluating these relationships we can develop predictive models which can be deployed for statutory controls of nanoparticle use.
Toxicity assays were performed using in-vitro models of cell and tissue culture and in-vivo models of several different aquatic species of key indicator organisms. As part of this project, all the procedures for toxicity testing were selectively developed and optimised for nanoparticles. This meant that streamlined protocols for nanoparticle toxicity testing could formulated which can later be exploited as routine tests for nanomaterials. The biological membrane and its dependent mechanisms play important roles in nanoparticle toxicity for two reasons. Firstly the biological membrane forms the boundary of the living cell which nanoparticles will need to cross and, secondly, the biological membrane hosts many of the physiological processes such as respiration and nerve conduction and any disruption in its structure will lead to a disruption in the function of the incumbent processes. The effect of nanoparticles on biological membrane structure is entirely unknown as is the permeability of nanoparticles in cell membranes. This study therefore allocated considerable resources to look at the interaction of nanoparticles with biological membranes by using highly novel supported membrane models, real biological membranes (liposomes and cells) and whole cell and tissues (successive complexity). The model membranes represent the most basic model for nanoparticle interaction and delivered important preliminary structure-activity relationships which were used to guide the more complex in-vitro and in-vivo studies. Already one of the model membrane tests being deployed in this study is in the has been patented and licensed as a generalised toxicity testing procedure which can be applied to investigate the activity of nanoparticles (L.A.Nelson and A.Vakurov 10008A Nanoparticle Sensor PCT filed, published on 15/9/11 with ref: WO2011/110825, Due to enter national phase 9/10/12). A major outcome of this study has been the development of calibrated toxicity testing protocols for nanoparticle biological activity including the above patented procedure. A SETAC World Congress in Sydney (August 2008) had an extensive session on nanomaterials and it was apparent that there were many issues to be addressed concerning how the materials should be tested for biological activity and the mechanism of toxicity. ENNSATOX therefore has made advances which could be a significant asset commercially.
The objectives of ENNSATOX were configured as:
1) Sourcing and comprehensively characterising a representative group of nanoparticles: initially ZnO and later SiO2 and TiO2 and other metal oxides of varying morphology and dimension. In-house synthesis was limited to ZnO nanoparticles not easily obtainable commercially, or from other projects within the NanoSafety Cluster. Well defined production methods were used for particle synthesis. Characterisation proceeded as an iterative process throughout the programme. The outcome of this objective was the quality and quantity of the standardised particles produced.
2) Characterising the interaction of the nanoparticles with the following biological models: supported phospholipid membranes of increasing complexity, in-vitro models of cell and tissue culture, in-vivo models of several different species of key indicator organisms. A feature of this objective is the direct comparison of the effects in the different testing systems which leads to the general theory of nanoparticle biological activity.
3) Formulating direct and predictive structure-activity relationships between nanoparticle form and nanoparticle biological activity. Success in this objective was achieved following results from objectives 1 and 2 and was a central feature of ENNSATOX.
4) Analysing the behaviour and fate of nanoparticles and their impact on models of biota in environmental aquatic systems. This advanced on the initial structural-activity relationships by testing their application in the environmental aquatic situation.
5) Configuring a mathematical model for the behaviour of nanoparticles in aquatic environments taking account of their interactions with biota of increasing complexity. This objective quantified the interactions and served as a means of verifying and measuring objectives 1-4.
6) Drawing up standard procedures for the exploitation and dissemination of the results for statutory planning and accredited use. In order to accomplish the challenge ENNSATOX assembled a group of RTD performers of unprecedented excellence from across Europe.
Our consortium had outstanding capabilities and achievements in:
? Nanoparticle manipulation, synthesis and characterisation. (Leeds, Wageningen);
? Supported model membrane technology (Leeds, Naples, Wageningen);
? Environmental and molecular mathematical modelling (Lleida, Wageningen, Leeds, Antwerp);
? In vitro and in vivo biological models (Naples, Leeds, Antwerp, Wageningen, MBA);
? Surface and colloid chemistry (Leeds, Wageningen, Naples);
? Environmental impact assessment (Wageningen, Antwerp, MBA); and,
? Dissemination of best practice worldwide (MBA, SETAC).
The objectives directly address in an integrated manner the impact of the nanoparticles on the environment. Implicit in this is the approach towards understanding the environmental and biological fate, transport, and transformation of nanoparticles in various biological compartments in aquatic systems. It is clear that the above objectives incorporate investigations into the toxicokinetics, cellular and molecular mechanisms, behaviour and fate, bio-persistence and biokinetics of nanoparticles. This enabled a fundamental understanding of the exposure, behaviour, mechanisms, consequences and potential effects to various endpoints of nanoparticle-biological entities interactions to be completed.
Contained within the objectives the following important questions have been addressed:
? What is the dispersion and solubility of nanoparticles in water?
? What are the most likely routes of exposure for environmentally relevant species?
? Can nanoparticles interfere with critical physiological mechanisms in aquatic organisms?
? Can nanoparticles bioaccumulate in aquatic organisms?
? Can nanoparticles be metabolised to less toxic forms?
? What biomarkers are relevant for determining nanoparticle exposure levels?
? What end-points are significant for determining risk of nanoparticles?
? What are the mechanisms of toxicity of nanoparticles in environmentally relevant systems?
? Does the presence of nanoparticles in the environment affect the toxicity of other compounds and vice versa?
Finally and very importantly ENNSATOX addressed two of the most important ethical issues in modern technology:
1) The synthesis and use of any novel compound or material has repeatedly led to ethical problems in its use either related to human health or environmental quality or both. Two very well known examples are the use of DDT as an insecticide and the use of lead tetraethyl in petrol. It is essential therefore from an ethical point of view to know the precise environmental impact of any newly synthesised material before it can be deployed for commercial use. This project not only addresses precisely this issue regarding engineered nanoparticle deployment but it also sets a protocol for examining nanoparticles as and when they are developed.
2) ENNSATOX developed methods for evaluating the environmental and biological hazard of nanoparticles which implicitly decrease the use of animals in the bioassay. This is ethically preferable in accordance with the three Rs concept of reduction, refinement and replacement in animal testing. Eventually toxicity assays will be limited to rapid in-vitro techniques and the use of predictive mathematical models using parameters developed from structure-activity models.
Project Results:
Please note that the figures refer to the attached Summary Information document.
The scientific (RTD) activities are conducted within seven work packages (WP1-7), with two other work packages being specifically concerned with exploitation/IPR and pre-validation (WP7), and dissemination (WP8):
WP1: Synthesis and characterisation of a selected group of nanoparticles:
To keep the study focused, three groups of nanoparticles have been examined: silicon dioxide (SiO2), zinc oxide (ZnO) and titanium dioxide (TiO2), of different morphology and dimension. Although Leeds is responsible for the synthesis, sourcing and processing of the nanoparticles, their characterisation has been cross calibrated with Wageningen. Nanoparticle characterisation in the in vitro, in vivo and aquatic systems was carried out throughout the programme as and when appropriate (WPs 2, 3, 4 and 5) in order to follow their behaviour and fate in the respective systems. Figure 1 shows a characteristic image from this study of “home' synthesised ZnO nanoparticles. The following paragraphs list the most significant achievements of WP1.
We have assembled (sourced and in-house synthesis) a very useful set of industrially relevant well defined and thoroughly characterised OECD nanoparticles (silica, zinc oxide and titania) which vary in terms of size (5 – 700 nm), aspect ratio (e.g. zinc oxide needles), crystallinity (silica – amorphous, zinc oxide crystalline and titania- crystalline polymorphs) and chemistry (both nanoparticle type and surfactant chemistry). We have developed and implemented relatively simple cleaning methods for these particles. We have investigated their dispersion characteristics in a range of media relevant to toxicological testing using a combination of existing (DLS, Zeta Potential) and novel (TEM) techniques. Furthermore these sets of particles are postulated to have different mechanisms for inducing toxicological responses (ZnO – soluble zinc, titania – redox processes, silica – membrane disruption). Using our Nanoparticle Characterisation Protocol (NCP) we have undertaken a thorough investigation of a range of commercial ZnO samples and as a result, successfully synthesised ZnO nanoparticles with narrower size and shape distributions using a polyol mediated precipitation synthesis– an example image of which is shown below in figure 3.
Hydrothermal synthesis was also employed to produce ZnO nanoparticles which were also characterised using the NCP. The following reaction parameters were varied in order to control particle size and shape: zinc precursor type (acetate, chloride) and concentration; reaction temperature and time; starting pH and base species (sodium carbonate, sodium hydroxide, ammonium hydroxide). The physical characteristics of a selection of particles were presented to the consortium meeting in September 2011 for consideration by partners. Nanorods produced from aqueous zinc chloride and sodium carbonate were selected as the priority for circulation to all partners: EN-Z-7 (shown in figure 2 below), average dimensions ~ 80 nm (width) by ~ 700 nm (length), surface area 18 m2/g; wurtzite crystal structure; flocculate in distilled water (d90 < 1 mm) but can be deflocculated by ultrasonic agitation. Other shapes and sizes were supplied for comparative toxicity tests in UNIVLEEDS. These included: EN- Z- 8, plate-like particles, 35 nm by 270 nm from zinc acetate and sodium hydroxide; EN- Z-9, micro-rods ~ 1?m by ~ 6 ?m, from zinc acetate and ammonium hydroxide solutions. Additional research using high resolution TEM has been undertaken to investigate ZnO particle growth mechanisms. This has illustrated that rods grow from supersaturated solution by a sequential nucleation and growth mechanism. These new insights provide an understanding of why specific reaction variables, for example base-type and pH, have such a dramatic effect on particle morphology. This information will enable more accurate control of both size and shape of particles in future nanosafety research and a journal paper is currently near submission.
Revised handling procedures for ZnO powders have been formulated based on wet and dry ageing studies. These recommendations are designed to minimise physical and chemical changes between WP1 and subsequent WPs. In addition, Control protocols have been defined to distinguish solubilised zinc from NP ZnO in toxicity assays. See Deliverable 1.3.1 for both points.
We have successfully developed a range of new procedures to directly image nanoparticle dispersions using TEM via rapid plunge freezing and warming (and more recently gel formation, resin infiltration and sectioning). This will soon be in the open literature in ”Quantitative Characterization of Nanoparticle Agglomeration within Biological Media”, Nicole Hondow, Rik Brydson, Peiyi Wang, Mark D. Holton, M. Rowan Brown, Paul Rees, Huw D. Summers, and Andy Brown accepted for publication in Journal of Nanoparticle Research May 2012.
We have shown conclusively that a protein corona forms around ZnO nanoparticles following the addition of serum to cell growth media (see Figure 6c above) and that this aids dispersion (see Figure 7). This forms the basis of an accepted oral presentation at the European Microscopy Congress in September 2012 in Manchester (R. Wallace).
We have achieved considerable success at imaging and quantifying uptake of nanoparticles within cell sections. From the observation of uptake of silica nanoparticles at low temperatures we have obtained evidence that a novel mechanism of passive nanoparticle uptake could be in operation (i.e. not clathrin mediated endocytosis) Figure 7(a). Furthermore we have obtained rare evidence of the actual cellular uptake of ZnO nanoparticles within cells during the MTT assay (WP3) – this would imply that it is not necessarily just extracellular soluble zinc which is responsible for toxicity but the mechanism involves cellular contact and uptake of ZnO nanoparticles and then possible dissolution in low pH lysosomes Figure 7(b).
WP2: Interactions of different classes of nanoparticles with model membrane systems:
Leeds and Wageningen possess a whole suite of experimental model biological membrane systems of increasing levels of complexity (Figures 8 & 9). Wageningen have considerable expertise in surface and colloid chemistry and extensive expertise modelling membrane interactions, and were responsible therefore for correlating the model membrane-nanoparticle interactions with theoretical mathematical models using self consistent mean field theory. The form, structure and functionality of the particles has been related to their activity towards the model membrane systems. The NAPLES lab has examined the effects of nanoparticles at the level of single channels (HERG K+ channels). The principle is to understand how nanoparticles affect the organisation and fluidity of the biological membrane, how they influence the functioning of ion channels and enzymes located in the membrane environment and whether the nanoparticles are themselves permeable in the membrane structure. Figure 10 shows an image of SiO2 nanoparticles adsorbed on to a cyanobacterium membrane.
To test the biomembrane activity of SiO2 nanoparticles a custom built high throughput sensor has been developed, which is displayed in Figure 11. This device can assess the biomembrane activity of a nanoparticle dispersion in 10 minutes.
Interesting results have been found using this sensor. For instance, SiO2 nanoparticles have been shown to adsorb on the surface of phospholipid membranes, as seen in SEM images of silica nanoparticles on phospholipid monolayers on electrode surfaces (see Figure 12). The extent of contact of the SiO2 particles' surface with the phospholipid membrane surface determines the effect on the membrane's properties and is dependent on the particle size. This is very clearly displayed in Figure 13.
The most significant achievements of WP2 can be summarised in the following:
For three nanoparticles studied with the DOPC monolayer system: particle diameter is the predominant parameter which controls the interaction between the uncoated, disaggregated particle and the lipid layer. It was shown for SiO2 that this was solely a geometric effect relating to the packing of the particles on the phospholipid surface and was identical whether particles interacted with bilayers or supported monolayers. In the case of ZnO and TiO2 which are prone to aggregation, the interaction of the aggregates is less than that of the disaggregated particles but is also related to the primary particle size.
Charged NPs, such as the negatively-charged silica NPs, affect the structure and dynamics of lipid bilayers by a reorientation of the phospholipid head groups
Strongly charged lipid membranes repel charged particles in solution with physiological relevant ionic strength due to ion double layers that form around the lipid bilayers and nanoparticles.
NPs can make lipid vesicles leak when they strongly bind to the membrane. When the particles are in excess to the lipid bilayer, the disruption on interaction can be transient and might be due to a mechanism by which the vesicles release strain that is formed by the binding NP. When the lipids are in excess compared to the NP, leakage might occur if the interaction is very strong and the lipid folds around the NP. The latter is only possible if the interaction is very strong as is observed for SiO2.
A correlation has been found between vesicle leakage and the surface area ratio of particles and vesicles, indicating that size of NP plays an important role in this potential toxic interaction.
Nanoparticles can disrupt ion gradients across membrane without creating “large' pores.
WP3: Interactions with in-vitro models:
These studies are directed to nanoparticle interactions at both the cellular level and the tissue level. The test systems will be established on in-vitro models. The cellular level will include test systems ranging from tissues and cultured cells to DNA (Figure 14). The tissue level includes nerve axons from the squid consisting of a single axon and glia, and ascidian embryos (rapidly developing chordate embryos to 12 hrs). The principle is to understand how the nanoparticles affect the structure and function of these systems using both real time assays and electron microscopy. The in vitro work is led by Naples and is spread between Naples and Leeds (WP 3). Naples has extensive facilities in electron microscopy and biophysical and molecular biological techniques and considerable world expertise in electrophysiology. The effect of SiO2 nanoparticles on the development of ascidian larvae is shown in Figure 15.
Some of the most exciting recent work carried out by WP3 has been on the effect of ZnO particles on membrane proteins. NPs provided by WP2 were tested directly on HEK cells that heterologously express the hERG K+ channel. This gave us the opportunity of assessing the impact of the NPs on defined membrane proteins directly. The range of concentrations used was 0.1-10 ?g ml-1 for both SiO2 (dialyzed and non -dialyzed) and ZnO. Cells were held at -70 mV under voltage clamp and hERG K+ channels were activated by patterns of voltage steps which produced outward ionic currents which were subject to biophysical analysis (Figure 16).
The channel activity was stable for at least an hour without run-down although experiments were normally carried out in the first 20 minutes. Examination of the hERG current kinetics (activation /inactivation and peak currents revealed no effect of SiO2 up to 10 ?g mL-1 but a notable selective effect of ZnO on channel kinetics (Figure 17). To establish if this effect was due to release of Zn2+ ions from the NPs, we carried out experiments where increasing concentrations of ZnCl2 were added and the peak currents measured. As can be seen in Figure 16, increasing the concentration of Zn2+ begins to block the channel only in the mM range. The effect of the NPs in Figure 17 is the opposite to this, i.e. they increase the current. Therefore the NP effect cannot be due to residual Zn2+.
Although we were unable to demonstrate that there are predictable metrics for NPs our finding of a specific effect of ZnO on hERG allows us to predict a possible model for why ZnO interacts with this channel. hERG is one of the best characterized of membrane ion channels and it is well known that the inactivation gate of the channel resides in the outer loop that connects the fifth and sixth membrane spanning domains the channel.
Our analysis of the data reveals that ZnO alters the inactivation gate of hERG and thus the most likely explanation for our data is a model that predicts a charge interaction between the ZnO NP and this outer portion of the membrane protein (Figure 18).This also indicates strongly that there is a very close sub –nm distance between the NP and the protein and that this has to be occurring on the external face of the bilayer.
The consensus of WP3 underlines the differences between two very different aspects of “nano toxicity' true nano effects and the intrinsic toxicity generated by the breakdown/dynamic activity of NPs in solution. True nano effects can only be clearly identified in tests which have a maximum duration of not more than one hour. In this case action on cell physiology was noted only in the case of ZnO on biological membrane ion channels and in the case of SiO2 on phagocytosis. No effects were noted for other NPs. NPs undoubtedly bind to biological membranes and influence phagocytosis but we failed to note significant effects other than these mentioned above. With respect to longer time periods we can highlight the fact that ZnO is also the most active /toxic of the NPs tested. It causes more significantly toxic effects than the other NPs in cell viability and Comet assays, whereas TiO2 is relatively non- toxic. Equally a similar pattern was noted in the bacterial gene reporter assays. Neural network activity in C intestinalis was unaltered by the NP panel. In the former three tests we could not clearly establish a true nano effect from a nano breakdown effect or other action.
To summarise: The individual effects of action of SiO2 and ZnO are very different at the biomembrane and protein levels, ZnO interacts with protein while SiO2 appears to coat biomembranes but no interaction with biomembrane proteins was noted. As far as acute effects are concerned: SiO2 interacts with DNA in cultured cells to cause DNA damage though ZnO effects are clearly more toxic in most cases. The question remains as to how NP's access the DNA (directly or indirectly as the result of activation of secondary metabolites) and, when, and how these substances cross the cell membrane. This question has been addressed by EM and phagocytosis assays, where biomembrane coating and penetration was found but no damage was registered in the short term. In relation to TiO2, aggregates were rapidly formed in biological solutions and no toxic effects were seen. The data indicates that acute effects of SiO2 on organisms are unlikely which is actually the case as seen above, although the selective action of ZnO on the hERG channel (which is after all the major cardiovascular potassium channel) is notable especially as the dosage used is relatively low.
WP4: Interactions with in vivo models:
In vivo testing has been performed on at least eight different species to allow the construction of species sensitivity distributions for the selected nanoparticles. This also included three standard toxicity species of which the acute and chronic toxicity is well documented and characterised for a variety of toxicants (e.g. Chlorella, Daphnia, and Danio). The in vivo experiments addressed three main issues: namely bioavailability, accumulation and toxicity. A series of chronic experiments were erformed in which effects on growth and reproduction were determined. This work was led by Antwerp with an input from Anton Dohrn. Antwerp is one of the world leaders in molecular, cellular and whole-organism toxicology, and both experimental and predictive modelling. During Period 2 studies were concentrated on looking at the chronic toxicity and internalisation routes of nanoparticles. In a chronic exposure scenario over 21 days, the ZnO nanodispersion (Alpha Aesar NanoTek) is toxic to Daphnia magna at low concentrations (around 0.02 mg Zn/L), whereby especially the reproduction was affected. As it has already been observed in studies carried out in WP3, toxicity of the ZnO nanodispersion cannot be solely due to the Zn or Zn2+ toxicity (see Figure 19). Results also showed the uptake and internalisation of the ZnO nanoparticles in Ciona intestinalis (see Figure 20). However, under the tested conditions, algae and daphnids did not take up the particles. Figure 21 shows a dose response curve of the effect of SM30 SiO2 on a culture of freshwater algae.
Chronic toxicity was determined with a standardized test using Daphnia magna. Experiments with ZnO nano dispersion (40 wt.% in water colloidal dispersion, NanoTek, Alfa Aesar) revealed an EC50 for D. magna mortality of 0.02 mg Zn/L. Compared to literature data for Zn, which are ranging around an EC50 of 0.2 mg Zn/L, the nanoparticles seem to be more toxic than the metal itself.
A second chronic toxicity test revealed that for ZnO NanoSun, the EC50 was similar to the one for ZnCl. Both substances showed an EC50 around 0.1 mg Zn/L (Figure 22). Additionally, it came out that most of the nanoparticles dissolved to form Zn2+ ions. Just as the salt ZnCl2 did. However, a small fraction of nanoparticles appeared to aggregate and form particles of a size between 100 and 450 nm.
In order to determine and visualize the uptake and internalisation of the nanoparticles, the selected model species Pseudokirchneriella subcapitata (WP4/UA), Daphnia magna (WP4/UA) and Ciona intestinalis (WP3/SZN) were exposed to the ZnO nanodispersion during acute (P. subcapitata, D. magna, C. intestinalis) and chronic (D. magna) experiments. Exponentially growing algae, daphnids and tunicates were exposed at the acute level to the nanoparticles spiked in water. At the chronic level the effect of exposure route and environmental conditions (pH) on the uptake and internalisation of the particles in the daphnids was tested as well. Results showed that the ZnO nanoparticles were taken up by the species C. intestinalis and were able to penetrate cell structures. However, in P. subcapitata and D. magna no nanoparticles could be visualised with transmission electronic microscopy (TEM) techniques, neither at the acute, nor at the chronic level under different exposure route and environmental conditions.
WP5: Nanoparticle environmental impact:
The biophysicochemical behaviour of nanoparticles, and their ensuing bioavailability and toxicity characteristics, strongly depends on the nature and the extent of molecular interactions with organic and inorganic materials in the environment. Wageningen together with an input from Antwerp are responsible for analysing the influence of chemical conditions and binding of particular species on the biointeraction and bioaccumulation of nanoparticles. Wageningen has extensive experience in the relationship between the chemical speciation of dissolved and particulate material in “natural' waters and its bioavailability. They have studied how the nanoparticles and the nanoparticle-water interface is modified when they enter a typical aqueous environmental system such as river, estuarine and sea water and how this affects their biological activity. Experiments have been carried out in laboratory controlled and relevant microcosms. The rate of the actual transfer of oxide nanoparticles across the cell membrane of a few selected aquatic organisms (microorganisms, invertebrates and fish); in relation to their local speciation and the physicochemical conditions at the outer side of the biointerface has also been investigated. The alteration of the nanoparticles during the in vivo experiments described in (WP4) has been investigated and related to their effects. In the first half of the programme, studies have mainly concentrated on the surface chemistry of SiO2 particles and their interaction with the soluble heavy metal ion Pb2+.
From the work in WP5 and the corresponding deliverables some general conclusions can be drawn with respect to the impact of the NPs in the aquatic environment.
Characterisation of SiO2, ZnO and TiO2 particles using titration and electrokinetic data generally confirmed that with increasing salt concentration, the charge density of nanoparticles increases and the zeta-potential decreases. In contrast to SiO2 and TiO2, ZnO is susceptible to dissolution and ZnO dispersions are colloidally highly unstable, but adsorption of organic matter stabilizes against (further) aggregation.
Since SiO2 particles are negatively charged over the wide range of practically significant pH values, it may act as a transport carrier of positively charged compounds, in particular (toxic) divalent metal ions (like Pb2+ and Zn2+). A significant result is that Pb2+ ions form labile complexes with silica NPs, which implies that they can be important in the facilitated transport of these ions to bio-interfaces. The lability of the metal ion-NP complex decreases with increasing pH and decreasing ionic strengths. With respect to the transport of ions by NPs to biointerfaces, the kinetic model (predicting the normalized flux) can be used to extract relevant parameters from experimental data.
The charge of nanoparticles is important for their interactions with biointerfaces, such as biomembranes, cell walls and biofilms, which are generally negatively charged. Silica NPs are negatively charged, ZnO NPs carry a positive charge below pH 8-9 (i.e. under most environmental conditions), and TiO2 are positively charged below pH 5.5 and negatively charged above that pH. In WP2 is described how charged NPs, such as the negatively-charged silica NPs, affect the structure and dynamics of lipid bilayers by a reorientation of the phospholipid head groups and can make lipid bilayers leak.
Silica NPs form stable dispersions at not too high ionic strengths. We showed that these NPs can penetrate and accumulate in model biogels at environmental pH values (pH 5.5) both at high and low ionic strengths, despite the unfavourable electrostatic potential in the gel. Therefore, these NPs are available for uptake by organisms living in biofilm structures and/or surrounded by a cell wall. ZnO NPs, however, form aggregates under all conditions. Therefore, bioavailability and biouptake of these particles as such is very low, which seems to be corroborated by uptake and internalisation studies with a few selected aquatic organisms. In contrast to SiO2 and TiO2, however, ZnO particles dissolve in the aqueous medium very readily, especially at pH values of 7 and higher, and the dissolved Zn2+ ions can cause toxic effects.
WP6: Integrated Modelling:
No environmental toxicological study is complete unless the various parts are integrated together in a theoretical model. This is essential not only for planning the study but also for assessing the final transfer parameters. Such a process is continuously iterative throughout the investigation until towards the end of the study, when the parameters are completely optimised. As a result predictions as to the impact of the nanoparticles on the aquatic environmental ecological models which predicted the transport and fate of soluble contaminants can be determined. A compartmental model is being used where the compartments are represented by the cell membrane, total cell, cell organelles, tissue and model aquatic organisms.
WP6 initially examined both the rate of dissolution and the extent of dissolution of ZnO nanoparticles with relation to particle size and solution pH. The model took account of the Gibbs-Thornton effect which predicts that particles of smaller size are more soluble. The model was validated against experimental systems which used the AGNES technique to measure Zn2+ concentrations in ZnO particle dispersions and hence ZnO solubility. Figure 23 displays the results of these experiments.
Next WP6 looked at sedimentation and transport models of nanoparticles. The model of the sedimentation of nanoparticles is depicted in Figure 24.
This model was validated against DLS and UV-vis spectroscopy measurements in centrifugation/settling experiments using stable aggregates prepared as described in the previous section. These experiments proved that the effective size and density of the NP aggregates (due to their fractal structure) is determinant for their transport properties. The fitted size distributions agreed satisfactorily with DLS measurements (see Figure 25).
A biomembrane model shown in Figure 26 was constructed so that the transport of nanoparticles to cells and their internalisation could be simulated.
The model was tested against experimental uptake data (see Figure 27). Several conclusions were extracted in the case studies analysed, namely: i) the uptake in well dispersed NP systems (i.e. with relatively high diffusion coefficients) may not be limited by transport of the NPs through the diffusion layer, but NP aggregation is expected to decrease the flux of particles available to microorganisms in the case of large aggregates (control by diffusion); ii) adsorption to the membrane reaches a steady state relatively fast compared to internalization; iii) a plateau in the uptake can be observed for long times when the internalization and efflux fluxes equilibrate. The application of this model requires very well controlled experimental conditions and detailed information of the system (e.g. in situ measurement of NP aggregate sizes). On the other hand, the model provides a theoretically sound framework for the comparison of uptake tests in different systems (type of NPs and microorganism). It also allows an estimation of the relative amounts of particles adsorbed on the membrane and present in the intracellular compartments. This would be useful in the development of more involved models aiming at predicting toxic effects, under the assumption that this effect is proportional to the amount of NPs taken up.
To summarise the most important findings from WP6 are the following.
ZnO solubility studies carried out in conjunction with WP5 showed that the smaller ZnO particles with lower primary size are more soluble in water. In this analysis the theory was based on the Gibbs-Thornton effect and entirely agreed with experiment.
The reaction-diffusion problem of the transport of stable NPs (and/or the conveyed free ions) from bulk solution up to a given interface, coupled with possible chemical reactions in the diffusion layer, was solved numerically for different geometries and diffusion domains.
Models for the prediction of the kinetic and equilibrium solubility of ZnO NPs were developed as a function of environmental variables (pH, ionic strength) and particle structure (size).
The time evolution of the concentration profiles of NPs while sedimenting in aqueous dispersions as a function of aggregate size and density can be satisfactorily predicted from the diffusion-sedimentation model. This model can be used to estimate the fate and behaviour of NPs in natural waters.
The description of the NP transport was integrated into uptake models to describe the amount of particles internalized by a microorganism or cell culture as a function of membrane adsorption and permeation rate constants.
Multiparameter regression analysis was carried out using data from the Consortium to obtain information on the influence of physicochemical parameters such as size, dose or time of exposure on NP toxicity.
WP7: Exploitation and pre-validation:
The Marine Biological Association of the UK a leading environmental charitable organisation is leading this activity. MBA has a track record of co-ordinating EU contracts and of carrying out bioassays for developing environmental quality objectives, with expertise in transferring analytical technology and significant regulatory experience. This includes considering all the above issues as well as developing accredited toxicity tests and assays for NPs in the aquatic situation. An important output will be aiding environmental legislation on these materials. Another important outcome is guidance on as to an effective means of calibrating and accrediting the toxicity testing procedures being developed.
One of the most significant contributions of the WP6 to ENNSATOX was in the following activity. The MBA Developed and pre-validated cost-effective, rugged toxicity tests for engineered nanomaterials: A key development has been that of a microplate-based assay to measure the impact of NP on growth of marine phytoplankton. This has been completed fully on a range of SiO2, ZnO and TiO2 nanoparticles. A further development has been the adoption of a microplate- based assay to measure the impact of NP on Tisbe battagliai using lethality as an endpoint. This has been completed fully on a range of SiO2, ZnO and TiO2 nanoparticles below. In the third of this suite of standardized tests, the impact of NP on development of oyster larvae is quantified. This assay is also being used to compare activity of a range of SiO2, ZnO and TiO2 nanoparticles. These tests fulfil the rigorous conditions for toxicity testing as set out by the statutory agencies and are complementary to tests used by them for Direct Toxicity Assessment (DTA). The rationale for choosing to evaluate three separate tests is that it is unlikely that any single organism will prove to be a universal indicator. By selecting three different taxa and endpoints (lethality, growth, development) a comprehensive coverage of likely effects has been gained.
Effects of NP on growth (algae)
Experiments carried out with the unicellular micro-alga Isochrysis galbana to demonstrate that the assessment of NPs on algal growth may be achieved in microplate assays using absorbance at 440 nm or 680nm (normalised to a background absorbance at 750nm) as a surrogate measure of cell density. This assumption has been validated in a number of tests against actual counts of cell densities. Trials were performed to validate the microplate assay by determining the effect of a standard metal toxicant, Zn2+ (as zinc sulphate), on growth of Isochrysis galbana. Typical results show reduction in growth to be dose dependent with growth reduced by ~50% at 1µg l-1 Zn2+. Zn2+ (zinc sulphate) has been used as a positive control in each subsequent test with NP including: SiO2 NP (Ludox SM30, particle size 13-14nm, dialysed and undialysed, and other particles provided by the Consortium). Results for non-dialysed Ludox were comparable with those of dialysed samples, indicating that effects were largely due to NP. Overall, results for SiO2 suggest a hormesis-type trend - with the growth of algae being stimulated at low doses (up to 20 µg ml-1) and thereafter decreasing with increasing dose (see Figure 28).
The reduction in growth was highly significant at 100 µg ludox ml-1, relative to controls, and was comparable in scale to the suppression caused by the standard toxicant 1µg ml-1 Zn (as ZnS04). Hormesis is not uncommon in ecotoxicological studies although the explanation and mechanism in this particular example has yet to be discovered. It may be that nanoparticulate SiO2 is behaving as a nutrient (or carrier of nutrients) at low dose but exhibits toxicity above a threshold of ~ 40 µg ml-1. This overall non-linear response makes the calculation of EC50 complex and we recommend deriving EC50 levels only from the linear, inhibitory region of the dose-response curve. Ludox SM-30, purified by gel-filtration, gave similar results to those described above for dialysed and undialysed material. Somewhat unusual response behaviour was observed in trials with larger particle size SiO2 (150-200, mean 170 nm; Fibre Optics – AngstromSphere). At the highest dose tested (100µg ml-1), after a prolonged lag phase, hormesis was observed (days 4-5), but by day 7 any growth promotion had disappeared, suggesting an ability to destabilise “normal growth' patterns. ZnO nanoparticles tested show toxicity increasing linearly with Zn concentration in the exposure media and may be a function of dissolved Zn. Dialysis appears to have little effect on toxicity of these particles (see also Period 1 report). TiO2: Work on algal growth effects during Period 2 of the ENNSATOX project has focused on TiO2, as characterised and supplied by the Consortium. Tests have been performed on five different forms (EN-T-1 to EN-T-5). Because of the light absorbing characteristics of many of the Ti particles used, caution has to be applied in separating these artefacts from absorbances due to algal growth (by subtracting the contribution due to particles from that of the algal cells).
Effects of NP on mortality (copepods)
Much of the latter half of the project has also been devoted to developing and quality-assuring this standard assay. As with the Isochrysis growth assay this test has proved to be robust and reliable provided that cultures are well maintained and careful attention given to quality control issues. The cultures can be maintained for more than one year meaning that apart from this initial outlay, costs are relatively minimal and large numbers of individuals can be provided for a constant throughput of tests.
Highlights of the tests with T. battagliai were: the virtual absence of toxic effects during exposure to SiO2 nanoparticles (see Figure 29) (and low responses to TiO2 (see Figure 30)) at suspension concentrations of up to 100 µg/ml (LC50>100 µg/ml as Si). Exposures at concentrations higher than this were not reliable due to the optical density of the suspensions and inability to see copepods in the test suspensions. However, it was noticeable that at concentrations >40 µg/ml Si or Ti, T. battagliai appeared to produce more mucus, possibly indicative that the particles were irritant rather than toxic. Toxicity tests with a range of ZnO nanoparticulates showed much greater evidence of toxicity. Typical mortality curves for ZnO NP are shown in Figure 31 and contrast with the lack of mortality in Si and Ti NP exposures. Mean LC50 values for different ZnO particles tested fell within the range 0.036 to 0.47 µg/ml (as Zn).These data are to be included in the overall assessment of structure-activity relationships.
Effects of NP on development (oyster larvae Crassostrea gigas)
The technique of fertilising conditioned oyster eggs and producing synchronised, high quality 32-cell embryos for testing activity of nanoparticles has been completed successfully. Optimal densities of up to 500 embryos per 5ml microplate well have been used to compare the effect of nanoparticulate (EN-Z-4) and dissolved ionic Zn2+ on development to the D-larval stage over 24hr. The test measures the percentages of normally- and abnormally- developed larvae over this period. The calculated EC50 for EN-Z-4 ZnO and Zn2+, (based on percentage net response, PNR, relative to controls) were 0.31 and 0.236 µg/ml, respectively, expressed as Zn. Once again the fact that ZnO particles exhibit EC50s that are comparable to ionic zinc, suggest that the nanoparticles dissolve and exert their activity in seawater. Effects concentrations for Zn NP in oyster larvae were not dissimilar to those described for Tisbe battagliai but are lower than thresholds affecting growth in Isochrysis galbana suggesting that invertebrates exhibit greater sensitivity to Zn.
To conclude all three primary assays investigated present robust and reliable methods for assessing and comparing biological activity of ENP and this WP has successfully achieved its aim of prevalidation of practical techniques for aquatic toxicity testing. WP7 has collated all data from the toxicity tests throughout the consortium and compared it with their own measurements. Generally ZnO, SiO2 and TiO2 nanoparticle dispersions show contrasting toxicities with the alga Isochrysis galabana. The nanoparticles can be ranked in decreasing order of toxicity: ZnO >TiO2>> SiO2 (based on mean EC50 concentrations of 2.59 7.53 and 170 mg L-1 as Zn respectively). The toxicity of ZnO was similar in most examples to that of the positive control Zn2+ (0.8 mg L-1) implying that the toxicity of Zn is largely due to dissolved Zn2+ as shown by others (e.g. WP4). Dialysis/purification of Si and Zn samples had little influence on toxicity. These were the general consensus of the order of results of tests with organisms found throughout the consortium.
Potential Impact:
The tests, model programs, standards and knowledge created by ENNSATOX's unique interdisciplinary research have had substantial societal and economic impact through: 1) its relevance to the call; 2) enhancing the state-of-the-art of nanotoxicology science and 3) informing policy and regulation. This impact has been delivered by: a) a robust dissemination programme and public engagement; b) Its IP strategy and c) an exploitation management plan.
Relevance to the original call: FP7-NMP-2008-SMALL-2 (1.3.2)
(i) Better in vitro or in vivo methodologies for the regulatory demands for the safety assessment of nanotechnology products. In WPs 2, 3, 4, 5 and 7, model membrane, in vitro and in vivo tests have been used to assay nanoparticle biological activity and relate it to the nanoparticles' structure, activity and functionality. The results have been successfully cross-correlated with each other to assess the relevance of the model membrane assays for measuring biomembrane activity. Executing these assays in concert allowed the group to characterise the most cost-effective protocol for screening nanoparticles and nanomaterials. This protocol has represented a key deliverable of this study and will be subject to European and international accreditation after exhaustive calibration and standardisation trials. The validation of the results of all assays through the standard procedures in WP7 substantiated and confirmed the relevance of the procedures and reinforced their impact on regulatory controls.
The research has maintained a continuous and positive interaction with the development of REACH regulation. It has informed on the precise hazard of the nanomaterials tested at all levels of the assays and at all stages of the programme. The consortium has been in continuous contact with the European Chemicals Agency (ECHA), Helsinki, which maintains the database for the REACH system. The results of the research has supported the work of several key International Standardisation Organisation (ISO) Committees (see section (vi) below). ENNSATOX has maintained a key role in the NanoSafety Cluster (NSC). The co-ordinator attended and contributed to most of the NSC meetings during the life of the project. ENNSATOX also actively collaborated with other projects within the NSC including NanoFATE, ENPRA and NanoReTox.
(ii) Better understanding of the impact of the nanoparticles on health, safety and the environment. WP5 elucidated the effect of the specific nanoparticle sizes and morphologies on representative compartments in a representative aquatic environment. The impact of the nanoparticles has been assessed through their change in form and speciation as they were discharged into an aqueous system and their consequent effect on biological membrane structure and function. These studies were integrated with those on the influence of the nanoparticles on cell and tissue structure and function and on the whole organism. The quantitative relationship between the structure and functionality of the nanoparticles and their biological activity has been successfully determined. The most important structural factors in the activity are particle size and the nature of the particle surface. The resulting parameters have been inputted into the integrated model quantitatively describing the impact of the particles on the aqueous environment. The model now predicts the impact of a class of nanoparticles and/or nanomaterials on the aquatic environment if both the characteristics of the materials and the properties of the environment are known. The greater understanding and prediction of the impact of nanoparticles on the biota in the environment decreases the requirement for expensive and politically sensitive bioassays at all levels. WP2 has developed a test model membrane system, the NANOSENSOR, which has advanced an understanding of the health effects of NP. WP2 has significantly advanced the state-of-the-art and substituted tests for standard in vivo and in vitro toxicity assays for NP. Validation of WP2 toxicity assays has been a key impact in this project. The results of medium throughput testing in WP 3 show that membrane ion channels (proteins) can be a significant target for ZnO NPs. As the hERG channel is the major cardiac K+ channel that stablizes the heartbeat of mammals, these results will be followed up to see if this could be a suitable screen for NP-protein interactions and potential deterimental interaction of NPs at the level of cell signalling.
(iii) Future definition of appropriate measures, where needed. WP6 has delivered predictive models of the effect of nanoparticles on key biota in riverine, estuarine and seawater environments. An underlying theme of the investigation has been to relate structure and function to activity. These predictive models will enable future environmental quality objectives to be set and met and statutory controls to be introduced on a firm scientific basis. These predictive models relate to the discharge, both accidental and intended, of classes of engineered particles into the environment. The results and findings of the studies are indispensable to Member States environmental protection agencies, the European Environmental Agency (EEA) and internationally (e.g. the US Environmental Protection Agency).
(iv) Safe and cost-effective minimisation of the exposure of workers and consumers. The coordinating beneficiary (Leeds) through its research in nanoparticle manufacturing is a reference centre for the UK's National Health & Safety Executive for monitoring and safe handling of nanoparticles. It has supplied regular guidance to all beneficiaries. In addition WP6 has identified critical compartments and pathways by which biologically active nanoparticles represent significant hazard to human health and wildlife and thus steps can be identified to minimise exposure via the environment. If biologically active nanoparticles are present in river water abstracted for potable use we can now identify, through WPs 5 and 6, processes which could be used (e.g. nanoparticle flocculation and filtration) for use in the subsequent water treatment to eliminate this hazard.
The consortium has worked closely with Lang Tran from the Institute of Occupational Medicine (IOM), UK continuously informing them of new findings obtained on the impact of nanoparticles on the "health" of the aquatic environment. These results will be incorporated into the IOM's database and promoted through its ”Safenano” website – the world's most visited nanotoxicity E, H & S site.
(v) Sustainable and responsible development. Three aspects of sustainable and responsible development have been addressed by this project. These are: (a) living within environmental limits, (b) using sound science responsibly and (c) achieving a sustainable economy. In the context of this work the manufacture of nanoparticles should be carried out in a responsible and cost effective manner with a minimum of waste. This entails using nanoparticle synthesis in an energy efficient manner and minimising the manufacture of toxic material which can damage the environment. WP7 has enabled statutory policy to be developed regarding the environmentally safe, responsible and sustainable use of these materials and their subsequent secure disposal. The outcome of the study will ensure that the societal and environmental costs of nanoparticle use fall on those who impose them. The results of this study and the predictive models which are generated from them has greatly facilitated the reduction in animal testing of nanomaterials.
(vi) Support to research and regulation. The ENNSATOX research has identified key approaches to be followed regarding the biological activity of nanoparticles. WPs 2, 3, 4, 5 and 7 have carried out generic investigations on the activity of a selected group of nanoparticles towards elements of biological organisation of increasing complexity in an aquatic environment. Critical areas of interactions and modes of action have been identified for study in future research projects. Of equal importance has been the crucial information gained on the toxicology of different classes of nanoparticles in the aquatic environment. This information can be used to assist in the construction of statutory controls, risk assessments and regulations for the use of nanoparticle materials. The consortium's continuous engagement with the European Chemicals Agency (ECHA), (see sub-section (i) above) will inform this process and will enable any new hazards associated with nanoparticles and nanomaterials to be transferred to the REACH database. In particular the findings of the study are critically important to the REGULATION (EC) No 1907/2006 OF THE EUROPEAN PARLIAMENT AND OF THE COUNCIL. The following ISO Technical Committees (TC): 147 water Quality, 190 Soil Quality, 217 Cosmetics and 243 Consumer Product Safety will have direct interests in the results of this project. In particular the work will have a direct bearing on the considerations of ISO/TC 229 on nanotechnology safety
(vii) Implementation of the European Commission's Action Plan for Nanotechnology. The ENNSATOX project directly addresses Point-6 in the European Commission's Action Plan: i.e. "All applications and use of nanoscience and nanotechnologies must comply with the high level of public health, safety, consumers' and workers' protection, and environmental protection chosen by the Community". WP7 has directly provided data and tools, to assist in the implementation of the Action Plan, for identifying safety concerns regarding the use of nanomaterials and minimising exposure to workers in the environment. It has also contributed to the development of risk assessment procedures for the use of nanoparticles and their effect on the environment and examined and proposed adaptations of EU regulations regarding nanoparticle use. These new contributions to risk assessment procedures will be published on the following EU web-sites: http://cordis.europa.eu/nanotechnology/ src/publication_events.htm and http://cordis.europa.eu/ nanotechnology/actionplan.htm to enable their maximum dissemination and impact.
(viii) Reinforcement of the international dimension of European research within the 7th Framework Programme. The relationships between the structure and functionality and biological and chemical reactivity of nanoparticles in the environment is at very early stage. There is a clear need for rigorous studies and the findings to be made available internationally to achieve international consensus. From the results obtained by WPs 2, 3, 4, 5 and 6, WP7 has informed evidence-based regulation development for the safe use and transport of classes of nanoparticles within and across borders. SETAC has provided an important global forum for achieving this aim. It has very strong representation from both the EU and US.
Impact of the research on the state-of-the-art and key stakeholders
(i) Previous state-of-the-art: The state-of-the-art on nanotoxicology before the start of ENNSATOX was rather fragmentary. Many studies had been initiated but they tended to be carried out by specialist groups rather than interdisciplinary consortia. Much of the work was phenomological and tended to be contradictory. These studies lacked a complete understanding of the underlying mechanisms involved. Some of the problems arose due to a lack of communication between groups who manufacture and characterise nanoparticles and those who carry out the necessary bioassays to investigate nanoparticle biological activity. There was also a lack of communication between environmental scientists and toxicologists and environmental managers.
The methodologies used in the individual work packages in ENNSATOX have all been state-of-the-art. For example, the model membrane systems have been used to understand toxicological mechanisms with respect to dissolved compounds and also as sensors for toxicity. Mathematical models previously used to understand the transport, fate and impact of dissolved compounds in the environment, have now been employed to analyse the equivalent processes for nanoparticles. (The latter have been shown to behave in an intermediate way between dissolved and particulate species). This interdisciplinary approach is itself a new paradigm and a step-change in the state-of-the-art
(ii) Significant results from project:
ENNSATOX has delivered -
• comprehensive and physically meaningful structure-activity relationships on categorical groups of engineered nanoparticles,
• protocols for examining the short and long term risks of nanoparticles to aquatic biota,
• Evaluation and adaption of existing tests (e.g. Comet, cell death) and application of new tests (hERG assay, phagocytosis assay, developmental assay, species distributions).
• prototype demonstrators for in-situ toxicity testing of nanoparticles in particular the NANOSENSOR, and hERG assay.
• generic mathematical models which can be used as predictive tools to understand impact of nanoparticles introduced into the environment.
(iii) Impact of results on state-of-the-art:
The development of the structure-activity relationships has impacted strongly on the current nanotoxicological field by improving the fundamental understanding of toxicological mechanisms and uniting the current fragmentary studies, by providing a coherent theory of nanoparticle biological activity in aquatic systems.
Protocols have been developed which extend the impact of current methods of toxicity testing assays. In particular, ENNSATOX has extended the understanding of how model biomembrane systems interact with nanoparticles and how nanoparticles are transported through biomembranes. ENNSATOX has extended the capability of toxicity assays from dissolved compound activity to particle activity. The impact of the latter is potentially enormous. Their manipulability, ease of interrogation and low cost, will enhance future research, knowledge and support regulation.
Finally a major outcome of ENNSATOX has been the computer simulation models developed to predict the transport, fate and impact of nanoparticles in the aquatic environment. The application of these models to the behaviour of nanoparticles is particularly novel, will accelerate knowledge generation and will impact strongly on the state-of-the-art in all areas.
(iv) Lead Users of the results:
The lead users of the aforementioned ENNSATOX results are extensive and are listed as follows -
Water industry needs to know the hazard presented by engineered nanoparticles present in potable waters at all stages of treatment. Protocols to assay the toxicity of nanoparticles present in water will be important associated with the increased production of these materials.
Environmental and marine agencies need to know also the hazards presented by different classes of nanomaterials which might be discharged into river, estuarine and marine environments. In this context, all four categories of aforementioned results will have major potential usage by these bodies. The predictive mathematical models developed by ENNSATOX will have particular application.
Pharmaceutical and cosmetic companies will be especially concerned with the structure-activity relationships and also the protocols developed to determine these.
Government agencies and statutory bodies throughout the EU will need to know hazards presented by nanoparticles in aqueous environments in order to develop risk assessments for the nanomaterials' use. The results will also be used to help set discharge limits and environmental quality controls on nanoparticle use. The results will be extremely useful to these bodies in formulating policy to protect the environment. Protocols for nanoparticle toxicity assay will be accredited by these bodies for general use.
Commercial and academic nanomaterial and nanoparticle producers will use results of studies to formulate risk assessment procedures and disposal methods for the materials. Results will be incorporated into workers' protocol for nanomaterial handling.
The Defence industry throughout the EU including NATO is very interested in the toxicity of nanomaterials and nanoparticles where these are manufactured with terrorist ambitions. The defence industry is also interested into ways to detect hazardous materials used as weapons.
(v) Steps taken to ensure impact:
Results have and are being disseminated to the aforementioned bodies in (iv) through:
• conference participation by the principal investigators;
• seminars by participant PIs in universities and leading companies throughout the EU;
• communication via SETAC, an ENNSATOX beneficiary, specifically funded to promote the communication of the results;
• the ENNSATOX web-site: http://www.ennsatox.eu/?contentid=260;
• participation in ENNSATOX of the MBA, a leading international independent environmental research organisation, who have pre-validated the toxicity assays;
• publication in top quality peer-reviewed journals;
• through the ENNSATOX Lead User group committee;
• regular presentations and participation at the NST meetings and EU-US workshops.
(vi) Relevance of the results to the Lead User:
Water industry will use toxicity assays developed by consortium as standard tests for water at all stages of treatment where nanoparticle contamination is suspected. These toxicity assays have been developed rapid, effective and relevant and will save considerable time and money for the water companies throughout EU.
Environmental and marine agencies will use results on short and long term risks of nanomaterials and nanoparticles to set discharge limits of these materials into rivers, estuaries and seawaters and consequent environmental quality controls. These agencies will also use the mathematical models developed in WP6 to carry out predictions on the impact of nanoparticles on the aquatic environment and to advise commercial organisations and governments within the EU on this.
Pharmaceutical and cosmetic companies will use results on structure-activity relationships to predict hazard and risk from new nanomaterials and nanoparticles being synthesised. The companies will also use toxicity assays to characterise the activity of their own products. Current methods used by pharmaceutical and cosmetic companies to assess toxicity of dissolved compounds and nanomaterials are time consuming and expensive and often involve animal testing.
Government agencies and statutory bodies throughout the EU. These bodies will be especially interested in the results of the study for the purpose of setting statutory discharge limits and statutory handling procedures for the materials.
Commercial and academic nanomaterial and nanoparticle producers will use results of studies to formulate risk assessment procedures and disposal methods for the materials. Results will be incorporated into workers' protocol for nanomaterial handling.
Defence industry can use the results of the investigations to commission new, faster and simpler toxicity tests to assess any security risks. The predictive structure-activity theory and the global environmental mathematical model will be used to assess risks from a terrorist attack from toxic nanomaterials.
(vii) The results are being delivered to the Lead User in the following way:
Workshops and seminars have been held in participant laboratories with potential lead users. ENNSATOX has already held a one day KT workshop in September 2011 and a four day International Winter School in Plymouth in January 2012 in association with NanoFATE. SETAC has been the main contact point for the transfer of results to government, commercial and industrial sectors. New results and protocols arising from the programme has been transmitted to interested lead users via SETAC without infringing IP restrictions.
(viii) The transfer of the results will be funded
All the activities listed in (vii) have been covered by the dissemination funds. As well as SETAC's (beneficiary 7) budget, all beneficiaries have a small portion of budget set aside for dissemination.
(ix) Rationale for European participation in project
It would be impossible to have run this project at a national level. Its extensive interdisciplinary nature involving nanoparticle manufacture and characterisation, model membrane studies, in vitro and in vivo studies and experimental and theoretical environmental modelling will never be available in one European country. Such interdisciplinary studies have to call upon the best possible expertise from several European member states. This lack of collaboration previously has led to only limited and contradictory studies in nanotoxicology. The cross-border complimentarity of this study ensured a global generic approach which provided a step change to the understanding of toxicological mechanisms of nanoparticles. The spread of the beneficiaries through different EU countries enabled a maximum dissemination of the results to be made since the results can be used by lead users in all of the countries which are participating. This will ultimately lead to an increase in environmental quality, environmental health and safety of people in these countries.
(x) Basis for long term collaboration: ENNSATOX has reinforced existing links between beneficiaries who all individually have a broad based interest in the aquatic toxicology and biological activity of nanoparticles. The meetings, joint publications and joint inventions/patents have ensured a desire to continue work in this scientifically very interesting and high impact area. The impact of the results on the consortium itself has therefore also been an exploration of mechanisms of securing continuing funding for this work. Because of the strong knowledge transfer element associated with the study the future work could involve the commercialisation of the some of the deliverables from this study. These include: a toxicity sensing protocol/demonstrator for nanoparticles, simulation models for predicting transport, fate and impact of nanoparticles in aquatic environments and structure-activity models of nanoparticle interaction with biota. The co-ordinator and one of the ENNSATOX partners are now working on a fresh EU proposal to commercialise an environmental toxicity sensor for submission later this year.
Political, societal and economic impact
(i) Enhancing competitiveness of European industry
Nanotechnology and the development of new nanomaterials is the most exciting new area since the biotechnology explosion of the 1960 s and 1970s. It promises to bring new and extensive economic growth to world society as well as solving many of the existing problems of providing sustainable energy and eliminating CO2 build-up in the atmosphere. It is anticipated also that nanomaterials and nanoparticles will be increasingly used in health care and environmental control. However all new technologies can bring enormous risks to bear on both the public and the environment. Most of the risks associated with past new technologies in the last century were not anticipated and the risks and problems which were later found led to a detrimental effect on the technology in some cases leading to its withdrawal. Two typical examples of this are the nuclear energy industry and the use of chlorinated pesticides. Whatever the outcome, the late discovery of these risks decreased the competitiveness of these new technologies on the world playing field. A major political impact of this current project is to precisely deduce the environmental health risks of engineered nanoparticles in a generic manner so that steps can be introduced within Europe to decrease or eliminate these risks. This will allow the nanotechnology industries in Europe to deal in a responsible way with the environmental and health risk controls firmly in place. The results will also help in setting up a Joint Technology Initiative (JTI): (http://europa.eu/rapid/pressReleasesAction.do?reference=MEMO/07/570) in environmental nanotoxicology.
(ii) To increase European wide S & T collaboration
This project has brought together outstanding scientists in complimentary areas of expertise to work towards an objective of immense strategic importance. The impact of the programme has served to bind together the scientists more strongly leading to their joint involvement on working groups and committees regulating the use of nanoparticles and nanomaterials. The programme has also disseminated its results throughout Europe by regular presentations at and active involvement with the NST. Important collaborations with other EU funded projects have arisen from this increasing the level of integration of nanotoxicology within Europe.
(iii) To contribute to an increase in level of research investment
The EU, Member States and industries are making a huge investment in nanotechnology. To maintain confidence in this investment and harvesting the benefits, the knowledge produced by ENNSATOX will be essential for earning the confidence and support of all stakeholders, including citizens. In this way it will contribute towards the goal of a 3% annual increase in research funding (http://ec.europa.eu/invest-in-research/index_en.htm).
(iv) To improve the co-ordination of European, national and regional research policies
The rigour of ENNSATOX's research for understanding and characterising in a generic way the risk associated with the production of nanoparticles and their introduction into the environment represents a glue achieving European harmonisation in E, H & S nanotoxicological risk assessment strategies. Nanotoxicology has been noted as a major initiative for the EU Joint Research Commission (http://ec.europa.eu/dgs/jrc/index.cfm) and the results of this study can only help to consolidate this initiative.
(v) To strengthen the scientific excellence of basic research in Europe
ENNSATOX involved world leaders in their respective fields applying themselves to a highly strategic problem. All RTD performers carry out world leading fundamental research at the forefront of their areas. Cross-European collaboration can only enhance and strengthen their own research areas by introducing newer complimentary approaches. The unique feature of this project has been that the individual contributions from each beneficiary are very fundamental whereas the combination of the contributions forms a highly strategic and eventually applied objective. The objective is also greatly sustained by the combination of such important research areas and itself is at the forefront in the competition regarding scientific quality, adventure and impact. Such a multidisciplinary yet scientifically excellent study is a quantum leap ahead of other single or bi-disciplinary nanotoxicological studies.
(vi) To promote the development of European research careers
The ENNSATOX multidisciplinary research has attracted researchers to work in the best scientific centres. This programme has stimulated young researchers to work in fields of excellence within a multidisciplinary framework essential in the present climate of research and technology. Research workers have been trained at the highest level in nanoparticle manufacturing and characterisation, biophysical and environmental mathematical modelling, surface and colloid chemistry, supported biomembranes and in vitro and in vivo bioassays. All these disciplines have been brought together to improve an understanding into the biological activity of nanoparticles. The diversity of the disciplines with this clear common objective has had a powerful impact on young researchers at the beginning of their careers. It has transformed the researchers into highly flexible scientists with a wide ranging knowledge but a capacity to excel in their own fields. They now have competitive career advantages over young researchers. ENNSATOX has been very successful at not only training its funded staff and students in this interesting area but also at bringing in other participants not funded by the project to work on associated problems.
(vii) To provide the knowledge-base to support key Community policies
The enlargement of the knowledge-base in nanotoxicology remains one of the key priority areas of the EU science policy (see http://projects-2007.jrc.ec.europa.eu/show.gx? Object.object_id=PROJECTS0000000003007403). The impact of the results from ENNSATOX have facilitated a step increase to the EU nanotoxicology knowledge-base. ENNSATOX has delivered analytical protocols, technical demonstrators e.g. the NANOSENSOR and predictive mathematical simulations for coping with the increased regulations and risk assessments generated by this increased knowledge. This in its turn has led to a reassessment of the current state-of-the-art concerns about nanoparticle and nanomaterials safety.
(viii) To increase availability, co-ordination and access in relation to top-level European scientific and technological infrastructure
This programme has brought the global approach to nanotoxicology into the European scientific theatre. Through the generation of interesting findings it has enabled the scientists and research workers to communicate their findings to other workers in Europe outside the consortium especially within the NST framework. This has stimulated new collaborations within the EU and allowed access to the highest level scientific and technical infrastructure where this becomes available.
(ix) Refinement and restructuring of risk assessment models concerning nanoparticle and nanomaterial safety
The widened understanding and predictive capability developed from this programme has enabled protocols on nanomaterial and nanoparticle safety to be renewed. Where generalities have been found through straightforward structure-activity models (such as the inverse relationship between particle size and biomembrane activity), risk assessment procedures can be simplified leading to cost savings in nanomaterial and nanoparticle production.
(x) Societal concern over use of new materials and chemicals
European society has become progressively disillusioned with the promoted use of new technologies. In all cases there are often un-researched hazards associated with the new technologies which cause the public to lose confidence in their proposed benefits. One very good example is the emergence of GM crop production to solve world food shortages which has later been shown to be linked to many potential hazards. The ENNSATOX research has been timely and coincides with the emergence of nanotechnology and nanomanufacturing as the promoted "panacea" of the 21st century. The research has generated results which specify exactly the hazards of new nanomaterials and nanoparticles. This will increase the public confidence in the use of these materials by society and in the long term ensure the success of the technology. To summarise:
The major technical and scientific impact are from:
• The combination of the most advanced techniques for the characterisation of nanoparticles with the most comprehensive and novel in vitro and in vivo assays of biological activity to develop structure-activity relationships which can guide future research activities in this area.
• The development of calibrated protocols for assaying nanoparticle biological activity which can be accredited for use by environmental agencies and the water industry.
• The characterisation of the importance of the biological membrane in nanoparticle toxicology in environmental aquatic systems.
• The development of generic theory which taking account of the nanoparticle structure and functionality and the biogeochemical characteristics of the impacted environment will predict the overall impact of the material on the said environment.
• The construction of structure-activity relationships of nanomaterial and nanoparticles in their biological action.
• The development of newer, more efficient and faster toxicity testing procedures which drastically reduce the need for the animal testing of nanoparticle biological activity.
The main environmental impact can be derived from:
• The assessment of the impact of discharges of nanoparticles into the aquatic environment so that future statutory environmental quality objectives can be met.
• The development of holistic calibrated mathematical models on the transport, behaviour, toxicology and fate of nanoparticles discharged into aquatic environments. These will be used by environmental agencies to predict the impact of specified groups of nanoparticles on the aquatic environment.
The statutory impact is from:
• The ability to develop risk assessment protocols on the environmental hazards of engineered nanoparticles of specified form and composition. This will have particular impact in that it will lead to decreased dependence on animal experimentation procedures to develop the said protocols.
• The precise description of the relative hazard and risk associated with the major classes of nanoparticles. This risk is especially important and significant to the configuration of this programme of work in that it describes the long term hazard of the material. Thus a particular material may appear quite safe when handled in its original form. After having passed through the various aquatic environmental and biological compartments it may have some unforeseen effect on the environment/human health which only the integrated and generic nature of this project can discover.
The political and economic impact can:
• Establish the EU as a responsible region of excellence in nanotoxicology, a discipline which has its roots in the diverse areas of fundamental science represented in the consortium. The results will facilitate the rapid development of nanotechnology unhindered by uninformed concerns over the putative health and environmental risks associated with it. The results will aid insurance actuaries to accurately evaluate the risks associated with working with nanomaterials and to set informed premiums
The societal impact can:
• Ensure that full public confidence is maintained at all times in Europe in the development and use of nanomaterials and nanoparticles. This will be done by continuous and objective dissemination of the results to the public throughout the programme of research.
The ethical impact can:
• address the potential long term public health hazard of a new technology in this case engineered nanoparticle deployment and to set a protocol for examining nanoparticles as and when they are developed.
• develop methods for evaluating the environmental and biological hazard of nanoparticles which implicitly decreases the use of animals in the bioassay. This is ethically preferable and meets the three Rs protocol. Eventually toxicity assays will be limited to rapid and relevant in-vitro techniques and the use of predictive mathematical models using parameters developed from structure-activity relationships.
NB: The coordinating beneficiary (Leeds) is associated with a unique Interdisciplinary Applied Ethics Research Centre skilled in the ethics associated with nanotechnology.
List of Websites:
ENgineered Nanoparticle Impact on Aquatic Environments: Structure, Activity and Toxicology
Contract Agreement: NMP4-SL-2009-229244
Website: http://www.ennsatox.eu
Coordinator:
Professor Andrew Nelson
School of Chemistry
University of Leeds
Leeds LS2 9JT, UK
Telephone: +44(0)113 343 6409
Email: a.l.nelson@leeds.ac.uk
